WO2019018662A1

WO2019018662A1 - Tgf-beta inhibition to treat hematologic symptoms of shwachman-diamond syndrome

Info

Publication number: WO2019018662A1
Application number: PCT/US2018/042913
Authority: WO
Inventors: Carl NOVINA; Cailin JOYCE; Akiko SHIMAMURA
Original assignee: Boston Childrens Hospital; Dana Farber Cancer Institute Inc
Current assignee: Boston Childrens Hospital; Dana Farber Cancer Institute Inc
Priority date: 2017-07-19
Filing date: 2018-07-19
Publication date: 2019-01-24
Anticipated expiration: 2020-01-19

Abstract

La présente invention concerne des compositions et des méthodes de traitement des symptômes du syndrome de Shwachman-Diamond.The present invention provides compositions and methods for treating the symptoms of Shwachman-Diamond syndrome.

Description

TGF-BETA INHIBITION TO TREAT HEMATOLOGIC SYMPTOMS OF

SHWACHMAN-DIAMOND SYNDROME

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.

Provisional Application No: 62/534,414, filed July 19, 2017, which is incorporated herein by reference in its entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under grant number W81XWH-14-1- 0124 awarded by the Department of Defense, under grant number 1 ROl DK 102165 awarded by the National Institutes of Health, under grant number T32 CA070083 awarded by the National Institutes of Health, under grant number R24 DK099808 awarded by the National Institutes of Health, and under grant number F32 HL124941 awarded by the National Heart, Lung, and Blood Institute. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Shwachman-Diamond Syndrome (SDS) patients suffer from bone marrow failure, exocrine pancreatic dysfunction, skeletal anomalies, and increased risk of acute myeloid leukemia. The birth-prevalence of SDS is estimated at around 1 out of every 76,000 live births. Prior to the invention described herein, the only curative treatment for the hematologic complications of SDS was a hematopoietic stem cell transplant, but outcomes were limited by the high risk of regimen-related toxicities. Thus, prior to the invention described herein, there was a pressing need to develop additional therapeutic modalities for SDS.

SUMMARY OF THE INVENTION

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.

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).

In some cases, the bone marrow failure comprises (or manifests as) SDS. Preferably, 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/mm³), exocrine pancreatic dysfunction, growth retardation, chronic steatorrhea, metaphyseal dysplasia, myelodysplasia, megakaryocyte dysplasia, erythroid dysplasia, acute myeloid leukemia (AML), and generalized osteopenia.

For example, 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%.

In some cases, the TGFp inhibitor reduces hematological symptoms in the subject. For example, 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%.

In other cases, 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%.

Exemplary TGFP inhibitors for use in the methods described herein include AVID200, SD208, LY2157299, TEW-7197, bortezomib, P144, pirfenidone, LY333531, and LY2109761. In some cases, 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 (μΜ), about 1 μΜ to about 1 millimolar (mM), about 1 mM to about 1 M.

In one aspect, 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

administered for a duration of over one year, e.g., two years, three years, four years, or five years.

Preferably, 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%.

In one example, the TGFP inhibitor is administered orally. In another example, the TGFP inhibitor is administered systemically.

In some cases, the method further comprises administering existing therapeutic modalities in combination with the therapies described herein. For example, in one aspect, the method further comprises performing an allogeneic bone marrow transplant or an allogeneic hematopoietic stem cell transplant in the subject. In other cases, the method further comprises administering a granulocyte-colony stimulating factor (G-CSF) polypeptide to the subject. In another aspect, the method further comprises administering a Shwachman-Bodian-Diamond Syndrome (SBDS) polypeptide to the subject. For example, these polypeptides are administered at a dose of from 0.1 mg/kg body weight to about 10 mg/kg body weight.

Also provided are methods for regenerative medicine, i.e., the process of replacing, engineering, or regenerating human cells, tissues, or organs to restore or establish normal function. For example, in some cases, 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. In some cases, the CD34+ HSPC is isolated from the subject prior to contacting the TGFP inhibitor. For example, the CD34⁺ HSPC is isolated from the bone marrow of the subject and subsequently contacted with the TGFP inhibitor. In one aspect, 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. For example, TGFB family ligands (TGFP3, growth/differentiation factor 15 (GDF15), and bone morphogenic protein 1 (BMPl)) are elevated in SDS patient plasma. Accordingly, in some cases, the inhibitor of the nucleotide in the TGFP signaling pathway inhibits the expression or activity of TGFP3, GDF15, or BMPl .

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. For example, the gene comprises TGFP3, GDF15, or BMPl . Exemplary test samples are those obtained from blood, serum, or plasma.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term "about."

The phrase "aberrant expression" is used to refer to an expression level that deviates from (i.e., an increased or decreased expression level) the normal reference expression level of the gene. By "agent" is meant any small compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By "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. As used 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. For example, 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.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "control" or "reference" is meant a standard of comparison. In one aspect, as used herein, "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, β-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.

By "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. For example, 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. When the fluorescently labeled molecule is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used 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). 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.

By the terms "effective amount" and "therapeutically effective amount" of a formulation or formulation component is meant a sufficient amount of the formulation or component, alone or in a combination, to provide the desired effect. For example, by "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.

The terms "isolated," "purified, " or "biologically pure" refer 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. For a protein that can be subjected to

modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

Similarly, by "substantially pure" is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, 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.

By "hematopoietic stem cell" is meant an immature cell that can develop into all types of blood cells, including white blood cells, red blood cells, and platelets. -

By "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 protein. 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. For example, the isolated nucleic acid is a purified cDNA or RNA polynucleotide. Isolated nucleic acid molecules also include messenger ribonucleic acid

(mRNA) molecules.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, 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. Preferably, 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.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By "modulate" is meant alter (increase or decrease). Such alterations are detected by standard art-known methods such as those described herein.

The term, "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. In some aspects, 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. Relative to a control level, the level that is determined may be an increased level. As used herein, the term "increased" with respect to level (e.g., expression level, biological activity level, etc.) 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.

Relative to a control level, the level that is determined may be a decreased level. As used herein, the term "decreased" with respect to level (e.g., expression level, biological activity level, etc.) 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.

The phrase "pharmaceutically acceptable carrier" is art recognized and 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. Some examples of 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.

By "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.

"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. It is also understood that throughout the application, 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. For example, 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. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, 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.

By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

The term "sample" as used herein refers to a biological sample obtained for the purpose of evaluation in vitro. With regard to the methods disclosed herein, the sample or patient sample preferably may comprise any body fluid or tissue. In some embodiments, 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. In some aspects, the sample is a composite panel of at least two of a blood sample, a plasma sample, a serum sample, and a urine sample. In exemplary aspects, 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.

By "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). Preferably, 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.

The term "subject" as used herein includes all members of the animal kingdom prone to suffering from the indicated disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is a human. The methods are also applicable to companion animals such as dogs and cats as well as livestock such as cows, horses, sheep, goats, pigs, and other

domesticated and wild animals.

A subject "suffering from or suspected of suffering from" a specific disease, condition, or syndrome, e.g., SDS, has a sufficient number of risk factors or presents with a sufficient number or combination of signs or symptoms of the disease, condition, or syndrome such that a competent individual would diagnose or suspect that the subject was suffering from the disease, condition, or syndrome. Subjects suffering from, and suspected of suffering from, a specific disease, condition, or syndrome are not necessarily two distinct groups.

As used herein, "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.

The terms "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. In some cases, 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. The term "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 (subcutaneous or intravenous) 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).

Pharmaceutical compositions 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.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

The 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. By contrast, the transitional phrase "consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference.

Genbank and NCBI submissions indicated by accession number cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

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 are cells from four normal donors (n^N1=70, n^N2=58, n^N1=69, n^N1=59, n^total=256). FIG. 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. Color indicates 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. 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. 2A-FIG. 2B is a tSNE plot and histogram representing the alteration of cellular architecture of early hematopoiesis in SDS. FIG. 2A is a tSNE plot of hematopoietic lineage commitment showing cells from normal donors as in FIG. 1 A, untreated SDS patients (_nSDSi.i_{=72j n}SDSi.2_{=62j n}SDS2.i₌₇₈^ _ntotai_{=2 12}^ _and an SDS patient who was being treated with

4.2ug/kg/day G-CSF (n^{SDS2 2}=71). Clusters were determined using 'partitioning around medoids' version of k-means clustering (k=5), and labeled based on the enrichment of index sorted hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), multilymphoid progenitor cells (MLP), common myloid progenitor cells (CMP), granulocyte-monocyte progenitor cells (GMP), and megakaryocyte-erythroid progenitor cells (MEP) as shown in FIG. ID. The sum of normal cells and SDS cells in each cluster is significantly changed using the chi-squared (X²) test. FIG. 2B is a histogram showing the mean relative frequencies of normal, untreated SDS, and G-CSF-treated SDS cells in each cluster. No significant differences were detected by two-way analysis of variance (ANOVA) of averaged data due to high inter- individual variability. Error bars=SEM.

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) >| 1 | in at least one cluster. Plotted are the number of genes that were either up- or down-regulated in one, two, three or four clusters. GMP was excluded due to the paucity of SDS GMP. Inset pie chart shows the proportion of differentially expressed genes in each cluster. FIG. 3B is a Venn diagram of differentially expressed genes in each cluster that were annotated to the "Inflammatory Response" function in Ingenuity Pathway Analysis. The shaded region shows the area of maximal enrichment of TGFP targets (p=4.03xl0^"15). 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. 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. 4B is a box and whisker plot showing the mean intensity of phospho-SMAD2 staining in individual CD34⁺ nuclei from samples depicted in FIG. 4A. Significance was determined by two-way ANOVA, with Holm-Sidak's multiple comparisons test. Error bars= minimum and maximum values, excluding outliers that exceed median+1.5*IQR, **p<0.01, ***p<0.001. FIG. 4C is a box and whisker plot showing the mean intensity of phospho-SMAD2 staining in individual CD34⁺ nuclei in two additional pairs of SDS and normal donor bone marrow samples. Error bars= minimum and maximum values, excluding outliers that exceed median+1.5*IQR, **p<0.01,****p<0.0001. FIG. 4D is a whisker plot showing the number of colonies formed by normal donor and SDS patient bone marrow derived mononuclear cells with increasing concentrations of AVID200, normalized to the 0 uM treatment. Significance was determined relative to the 0 uM treatment by two-way ANOVA, with Holm-Sidak's multiple comparisons test. Error bars=SEM. *p<0.05, **p<0.01. 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. The data suggest that 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). Increased TGFpRl signaling leads to increased concentrations of nuclear phospho-SMAD2 and transcription of inflammatory response genes, which impairs HSC/MPP function. This model predicts that therapeutic inhibition of TGFP signaling in HSC/MPP improves hematopoietic function in SDS patients. 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. 5B is a series of area graphs that show a representative gating scheme used to purify CD34⁺ subsets in human bone marrow. Percentages = % of CD34⁺ cells. 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. 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^midCD45RA^"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

ANOVA, with HolmSidak's multiple comparisons test. *p<0.05, **p<0.01.

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

differentially expressed proteins using Ingenuity Pathway Analysis.

DETAILED DESCRIPTION OF THE INVENTION

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;

Ganapathi et al. 2007 Blood, 110: 1458-1465; Finch et al., 2011 Genes & Development, 25:917- 929; Burwick et al., 2012 Blood, 120:5143-5152; Austin et al., 2008 J Clin. Invest, 118: 1511- 1518), certain cell types are more sensitive to SBDS mutations than others. Bone marrow failure typically manifests first in the myeloid lineage, but erythroid and megakaryocyte dysfunction may occur to varying degrees (Huang, J.N. & Shimamura, A., 2010 Current Opinion in

Hematology, 18:30-35; Myers et al., 2014 The Journal of Pediatrics, 164:866-870). The development of rational therapies requires a deeper understanding of the cell type-specific responses to SBDS mutations.

To address this, as described herein, single cell RNA (ribonucleic acid) sequencing (scRNA-seq) was used to profile rare and heterogeneous hematopoietic stem and progenitor cells (HSPC) from SDS patients. As described in detail below, a single cell map of early lineage commitment was generated, and it was found that SDS hematopoiesis left-shifted with selective loss of granulocyte-monocyte progenitors (GMPs). Transcriptional targets of TGFp were dysregulated in SDS hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), but not in lineage-committed progenitors. TGFP inhibitors (AVID200 and SD208) increased hematopoietic colony formation of SDS patient BM. Finally, 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.

Although SDS was reported over 50 years ago and progress has been made using animal and cellular models (Finch et al., 2011 Genes & Development, 25:917-929; Tourlakis et al., 2012 Gastroenterology; Zambetti et al., 2015 Haematologica, 100: 1285-1293; Tulpule et al., 2013 Cell Stem Cell, 12:727-736), prior to the invention described herein, the molecular mechanisms leading to BM failure were unclear.

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. The data adds to an emerging body of evidence linking inflammation to BM dysfunction, including Fanconi Anemia (FA), where the pathogenic mechanism of TGFP is thought to be suppression of homologous recombination repair (Zhang et al., 2016 Cell Stem Cell, 18:668-681; Zhou et al., 2011 Cancer Res., 71 :955-963). 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.

Also described herein is the utilization of single cell 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. Moreover, inhibition of TGFP signaling extracellularly (e.g., by trapping TGFpi/3 ligands with AVID200) or intracellularly (e.g., by blocking TGFpRl kinase activity with SD-208)in SDS patient primary cells improves hematopoietic colony formation in vitro. Finally, as described herein, 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. Prior to the invention described herein, 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. Finally, 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

Bone marrow failure (BMF) 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.

Inherited bone marrow failure (IBMF) is a result of germline mutations passed down from parents or arising de novo. In addition to symptoms associated with aplastic anemia such as fatigue, hemorrhage, and recurrent bacterial infections, patients often present with additional indications unique to each syndrome.

The most common inherited bone marrow failure syndromes (IBMFSs) are Fanconi anemia (FA), dyskeratosis congenita (DC), Shwachman-Diamond syndrome (SDS), congenital amegakaryocytic thrombocytopenia (CAMT), Blackfan-Diamond anemia (BDA), and reticular dysgenesis (RD).

Shwachman-Diamond Syndrome (SDS)

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. 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.

Prior to the invention described herein, the only curative treatment for BM failure in SDS patients was hematopoietic stem cell transplant, but outcomes were limited by the inability to predict which patients would develop complications that outweigh significant transplant risks. Accordingly, prior to the invention described herein, there was an urgent need for rational therapies to supplant or delay transplant. SDS manifests with a wide range of abnormalities and symptoms, even within families, and generally presents during infancy or early childhood. The main characteristics of the syndrome include exocrine pancreatic dysfunction, hematologic abnormalities, and growth retardation. Since SDS affects multiple body systems, symptoms can vary from chronic diarrhea to low white cell blood count, pale skin, fatigue, and bruising.

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. In patients with SDS, 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.

Failure to thrive, growth retardation, and chronic steatorrhea are often associated with exocrine pancreatic insufficiency in SDS patients. Delayed bone age and maturation with metaphyseal dysplasia in SDS patients results in short stature, pectus carinatum, and generalized osteopenia. More than 50% of patients with SDS are below the third percentile for height, and short stature appears to be unrelated to nutritional status.

Other characteristics of SDS include cutaneous (e.g., eczema or ichthyosis) and dental anomalies, and 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.

SDS patients are also at risk for myelodysplastic syndrome (MDS), aplastic anemia, and acute myeloid leukemia (AML) due to the abnormalities in the BM. In addition to

malfunctioning bone marrow, SDS patients suffer from pancreatic abnormalities. These occur due to a lack of acinar cells that produce the necessary enzymes required for digestion, resulting in fatty stool, malnutrition, and weight gain (Shwachman-Diamond syndrome, ghr.nlm.nih.gov).

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/mm³), moderate anemia, and a rise in fetal hemoglobin. Additionally, 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.

There is no cure for SDS, and treatment is generally targeted at mitigating symptoms. Depending on the symptom an SDS patient manifests, 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). For example, pancreatic exocrine dysfunction may be treated with pancreatic enzyme supplementation, while neutropenia may be treated with granulocyte-colony stimulating factor (G-CSF) to boost peripheral neutrophil counts. Severe hematological complications require hematopoietic stem cell transplantation.

Hematopoietic Stem Cells

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

populations of 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). The process of 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).

Gene Expression Profiling

In general, 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), and reverse transcription polymerase chain reaction (RT-PCR). Alternatively, 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). For example, 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.

In some cases, 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. For example, 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.

By "Transforming Growth Factor β (TGFP) nucleic acid molecule" is meant 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):

1 acctccctcc gcggagcagc cagacagcga gggccccggc cgggggcagg ggggacgccc

61 cgtccggggc accccccccg gctctgagcc gcccgcgggg ccggcctcgg cccggagcgg 121 aggaaggagt cgccgaggag cagcctgagg ccccagagtc tgagacgagc cgccgccgcc 181 cccgccactg cggggaggag ggggaggagg agcgggagga gggacgagct ggtcgggaga 241 agaggaaaaa aacttttgag acttttccgt tgccgctggg agccggaggc gcggggacct 301 cttggcgcga cgctgccccg cgaggaggca ggacttgggg accccagacc gcctcccttt 361 gccgccgggg acgcttgctc cctccctgcc ccctacacgg cgtccctcag gcgcccccat 421 tccggaccag ccctcgggag tcgccgaccc ggcctcccgc aaagactttt ccccagacct 481 cgggcgcacc ccctgcacgc cgccttcatc cccggcctgt ctcctgagcc cccgcgcatc 541 ctagaccctt tctcctccag gagacggatc tctctccgac ctgccacaga tcccctattc 601 aagaccaccc accttctggt accagatcgc gcccatctag gttatttccg tgggatactg 661 agacaccccc ggtccaagcc tcccctccac cactgcgccc ttctccctga ggagcctcag 721 ctttccctcg aggccctcct accttttgcc gggagacccc cagcccctgc aggggcgggg 781 cctccccacc acaccagccc tgttcgcgct ctcggcagtg ccggggggcg ccgcctcccc 841 catgccgccc tccgggctgc ggctgctgcc gctgctgcta ccgctgctgt ggctactggt 901 gctgacgcct ggcccgccgg ccgcgggact atccacctgc aagactatcg acatggagct 961 ggtgaagcgg aagcgcatcg aggccatccg cggccagatc ctgtccaagc tgcggctcgc 1021 cagccccccg agccaggggg aggtgccgcc cggcccgctg cccgaggccg tgctcgccct 1081 gtacaacagc acccgcgacc gggtggccgg ggagagtgca gaaccggagc ccgagcctga 1141 ggccgactac tacgccaagg aggtcacccg cgtgctaatg gtggaaaccc acaacgaaat 1201 ctatgacaag ttcaagcaga gtacacacag catatatatg ttcttcaaca catcagagct 1261 ccgagaagcg gtacctgaac ccgtgttgct ctcccgggca gagctgcgtc tgctgaggag 1321 gctcaagtta aaagtggagc agcacgtgga gctgtaccag aaatacagca acaattcctg 1381 gcgatacctc agcaaccggc tgctggcacc cagcgactcg ccagagtggt tatcttttga 1441 tgtcaccgga gttgtgcggc agtggttgag ccgtggaggg gaaattgagg gctttcgcct 1501 tagcgcccac tgctcctgtg acagcaggga taacacactg caagtggaca tcaacgggtt 1561 cactaccggc cgccgaggtg acctggccac cattcatggc atgaaccggc ctttcctgct 1621 tctcatggcc accccgctgg agagggccca gcatctgcaa agctcccggc accgccgagc 1681 cctggacacc aactattgct tcagctccac ggagaagaac tgctgcgtgc ggcagctgta 1741 cattgacttc cgcaaggacc tcggctggaa gtggatccac gagcccaagg gctaccatgc 1801 caacttctgc ctcgggccct gcccctacat ttggagcctg gacacgcagt acagcaaggt 1861 cctggccctg tacaaccagc ataacccggg cgcctcggcg gcgccgtgct gcgtgccgca 1921 ggcgctggag ccgctgccca tcgtgtacta cgtgggccgc aagcccaagg tggagcagct 1981 gtccaacatg atcgtgcgct cctgcaagtg cagctgaggt cccgccccgc cccgccccgc 2041 cccggcaggc ccggccccac cccgccccgc ccccgctgcc ttgcccatgg gggctgtatt 2101 taaggacacc gtgccccaag cccacctggg gccccattaa agatggagag aggactgcgg 2161 atctctgtgt cattgggcgc ctgcctgggg tctccatccc tgacgttccc ccactcccac 2221 tccctctctc tccctctctg cctcctcctg cctgtctgca ctattccttt gcccggcatc 2281 aaggcacagg ggaccagtgg ggaacactac tgtagttaga tctatttatt gagcaccttg 2341 ggcactgttg aagtgcctta cattaatgaa ctcattcagt caccatagca acactctgag 2401 atggcaggga ctctgataac acccatttta aaggttgagg aaacaagccc agagaggtta 2461 agggaggagt tcctgcccac caggaacctg ctttagtggg ggatagtgaa gaagacaata 2521 aaagatagta gttcaggcca ggcggggtgc tcacgcctgt aatcctagca cttttgggag 2581 gcagagatgg gaggatactt gaatccaggc atttgagacc agcctgggta acatagtgag 2641 accctatctc tacaaaacac ttttaaaaaa tgtacacctg tggtcccagc tactctggag 2701 gctaaggtgg gaggatcact tgatcctggg aggtcaaggc tgcag

By "Transforming Growth Factor β (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):

1 mhvrslraaa phsfvalwap lfllrsalad fsldnevhss fihrrlrsqe rremqreils 61 ilglphrprp hlqgkhnsap mfmldlynam aveegggpgg qgfsypykav fstqgpplas 121 lqdshfltda dmvmsfvnlv ehdkeffhpr yhhrefrfdl skipegeavt aaefriykdy 181 irerfdnetf risvyqvlqe hlgresdlfl ldsrtlwase egwlvfdita tsnhw vnpr 241 hnlglqlsve tldgqsinpk lagligrhgp qnkqpfmvaf fkatevhfrs irstgskqrs 301 qnrsktpknq ealrmanvae nsssdqrqac kkhelyvsfr dlgwqdwiia pegyaayyce 361 gecafplnsy mnatnhaivq tlvhfinpet vpkpccaptq Inaisvlyfd dssnvilkky 421 rnmvvracgc h

By "Transforming Growth Factor β 3 (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):

1 aaaatttcag cagagagaaa tagagaaagc agtgtgtgtg catgtgtgtg tgtgtgagag

61 agagagggag aggagcgaga gggagaggga gagggagaga gagaaaggga gggaagcaga 121 gagtcaagtc caagggaatg agcgagagag gcagagacag gggaagaggc gtgcgagaga 181 aggaataaca gctttccgga gcaggcgtgc cgtgaactgg cttctatttt attttatttt 241 tttctccttt ttatttttta aagagaagca ggggacagaa gcaatggccg aggcagaaga 301 caagccgagg tgctggtgac cctgggcgtc tgagtggatg attggggctg ctgcgctcag 361 aggcctgcct ccctgccttc caatgcatat aaccccacac cccagccaat gaagacgaga 421 ggcagcgtga acaaagtcat ttagaaagcc cccgaggaag tgtaaacaaa agagaaagca 481 tgaatggagt gcctgagaga caagtgtgtc ctgtactgcc cccaccttta gctgggccag 541 caactgcccg gccctgcttc tccccaccta ctcactggtg atcttttttt ttttactttt 601 ttttcccttt tcttttccat tctcttttct tattttcttt caaggcaagg caaggatttt 661 gattttggga cccagccatg gtccttctgc ttcttcttta aaatacccac tttctcccca 721 tcgccaagcg gcgtttggca atatcagata tccactctat ttatttttac ctaaggaaaa 781 actccagctc ccttcccact cccagctgcc ttgccacccc tcccagccct ctgcttgccc 841 tccacctggc ctgctgggag tcagagccca gcaaaacctg tttagacaca tggacaagaa 901 tcccagcgct acaaggcaca cagtccgctt cttcgtcctc agggttgcca gcgcttcctg 961 gaagtcctga agctctcgca gtgcagtgag ttcatgcacc ttcttgccaa gcctcagtct 1021 ttgggatctg gggaggccgc ctggttttcc tccctccttc tgcacgtctg ctggggtctc 1081 ttcctctcca ggccttgccg tccccctggc ctctcttccc agctcacaca tgaagatgca 1141 cttgcaaagg gctctggtgg tcctggccct gctgaacttt gccacggtca gcctctctct 1201 gtccacttgc accaccttgg acttcggcca catcaagaag aagagggtgg aagccattag 1261 gggacagatc ttgagcaagc tcaggctcac cagcccccct gagccaacgg tgatgaccca 1321 cgtcccctat caggtcctgg ccctttacaa cagcacccgg gagctgctgg aggagatgca 1381 tggggagagg gaggaaggct gcacccagga aaacaccgag tcggaatact atgccaaaga 1441 aatccataaa ttcgacatga tccaggggct ggcggagcac aacgaactgg ctgtctgccc 1501 taaaggaatt acctccaagg ttttccgctt caatgtgtcc tcagtggaga aaaatagaac 1561 caacctattc cgagcagaat tccgggtctt gcgggtgccc aaccccagct ctaagcggaa 1621 tgagcagagg atcgagctct tccagatcct tcggccagat gagcacattg ccaaacagcg 1681 ctatatcggt ggcaagaatc tgcccacacg gggcactgcc gagtggctgt cctttgatgt 1741 cactgacact gtgcgtgagt ggctgttgag aagagagtcc aacttaggtc tagaaatcag 1801 cattcactgt ccatgtcaca cctttcagcc caatggagat atcctggaaa acattcacga 1861 ggtgatggaa atcaaattca aaggcgtgga caatgaggat gaccatggcc gtggagatct 1921 ggggcgcctc aagaagcaga aggatcacca caaccctcat ctaatcctca tgatgattcc 1981 cccacaccgg ctcgacaacc cgggccaggg gggtcagagg aagaagcggg ctttggacac 2041 caattactgc ttccgcaact tggaggagaa ctgctgtgtg cgccccctct acattgactt 2101 ccgacaggat ctgggctgga agtgggtcca tgaacctaag ggctactatg ccaacttctg 2161 ctcaggccct tgcccatacc tccgcagtgc agacacaacc cacagcacgg tgctgggact 2221 gtacaacact ctgaaccctg aagcatctgc ctcgccttgc tgcgtgcccc aggacctgga 2281 gcccctgacc atcctgtact atgttgggag gacccccaaa gtggagcagc tctccaacat 2341 ggtggtgaag tcttgtaaat gtagctgaga ccccacgtgc gacagagaga ggggagagag 2401 aaccaccact gcctgactgc ccgctcctcg ggaaacacac aagcaacaaa cctcactgag 2461 aggcctggag cccacaacct tcggctccgg gcaaatggct gagatggagg tttccttttg 2521 gaacatttct ttcttgctgg ctctgagaat cacggtggta aagaaagtgt gggtttggtt 2581 agaggaaggc tgaactcttc agaacacaca gactttctgt gacgcagaca gaggggatgg 2641 ggatagagga aagggatggt aagttgagat gttgtgtggc aatgggattt gggctaccct 2701 aaagggagaa ggaagggcag agaatggctg ggtcagggcc agactggaag acacttcaga 2761 tctgaggttg gatttgctca ttgctgtacc acatctgctc tagggaatct ggattatgtt 2821 atacaaggca agcatttttt tttttttttt aaagacaggt tacgaagaca aagtcccaga 2881 attgtatctc atactgtctg ggattaaggg caaatctatt acttttgcaa actgtcctct 2941 acatcaatta acatcgtggg tcactacagg gagaaaatcc aggtcatgca gttcctggcc 3001 catcaactgt attgggcctt ttggatatgc tgaacgcaga agaaagggtg gaaatcaacc 3061 ctctcctgtc tgccctctgg gtccctcctc tcacctctcc ctcgatcata tttccccttg 3121 gacacttggt tagacgcctt ccaggtcagg atgcacattt ctggattgtg gttccatgca 3181 gccttggggc attatgggtt cttcccccac ttcccctcca agaccctgtg ttcatttggt 3241 gttcctggaa gcaggtgcta caacatgtga ggcattcggg gaagctgcac atgtgccaca 3301 cagtgacttg gccccagacg catagactga ggtataaaga caagtatgaa tattactctc 3361 aaaatctttg tataaataaa tatttttggg gcatcctgga tgatttcatc ttctggaata 3421 ttgtttctag aacagtaaaa gccttattct aaggtgtatg tctgactcga taaatatcct 3481 tcaattaccc ttaaaaaaaa aaaaaaaaaa

By "Transforming Growth Factor β 3 (TGFP3) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No.

ABQ59024, version ABQ59024.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 4):

1 mkmhlqralv vlallnfatv slslstcttl dfghikkkrv eairgqilsk lrltsppept

61 vmthvpyqvl alynstrell eemhgereeg ctqentesey yakeihkfdm iqglaehnel

121 avcpkgitsk vfrfnvssve knrtnlfrae frvlrvpnps skrneqriel fqilrpdehi

181 akqryiggkn lptrgtaewl sfdvtdtvre wllrresnlg leisihcpch tfqpngdile

241 nihevmeikf kgvdneddhg rgdlgrlkkq kdhhnphlil mmipphrldn pgqggqrkkr

301 aldtnycfrn leenccvrpl yidfrqdlgw kwvhepkgyy anfcsgpcpy lrsadtthst

361 vlglyntlnp easaspccvp qdlepltily yvgrtpkveq lsnmvvksck cs

By "Growth/Differentiation Factor 15 (GDF15) nucleic acid molecule" is meant 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 taggagcgcc cccacctaaa gatcctcccc

61 ctaaatacac ccccagaccc cgcccagctg tggtcattgg agtgtttact ctgcaggcag 121 ggggaggagg gcgggactga gcaggcggag acggacaaag tccggggact ataaaggccg 181 gtccggcagc atctggtcag tcccagctca gagccgcaac ctgcacagcc atgcccgggc 241 aagaactcag gacggtgaat ggctctcaga tgctcctggt gttgctggtg ctctcgtggc 301 tgccgcatgg gggcgccctg tctctggccg aggcgagccg cgcaagtttc ccgggaccct 361 cagagttgca ctccgaagac tccagattcc gagagttgcg gaaacgctac gaggacctgc 421 taaccaggct gcgggccaac cagagctggg aagattcgaa caccgacctc gtcccggccc 481 ctgcagtccg gatactcacg ccagaagtgc ggctgggatc cggcggccac ctgcacctgc 541 gtatctctcg ggccgccctt cccgaggggc tccccgaggc ctcccgcctt caccgggctc 601 tgttccggct gtccccgacg gcgtcaaggt cgtgggacgt gacacgaccg ctgcggcgtc 661 agctcagcct tgcaagaccc caggcgcccg cgctgcacct gcgactgtcg ccgccgccgt 721 cgcagtcgga ccaactgctg gcagaatctt cgtccgcacg gccccagctg gagttgcact 781 tgcggccgca agccgccagg gggcgccgca gagcgcgtgc gcgcaacggg gaccactgtc 841 cgctcgggcc cgggcgttgc tgccgtctgc acacggtccg cgcgtcgctg gaagacctgg 901 gctgggccga ttgggtgctg tcgccacggg aggtgcaagt gaccatgtgc atcggcgcgt 961 gcccgagcca gttccgggcg gcaaacatgc acgcgcagat caagacgagc ctgcaccgcc 1021 tgaagcccga cacggtgcca gcgccctgct gcgtgcccgc cagctacaat cccatggtgc 1081 tcattcaaaa gaccgacacc ggggtgtcgc tccagaccta tgatgacttg ttagccaaag 1141 actgccactg catatgagca gtcctggtcc ttccactgtg cacctgcgcg gaggacgcga 1201 cctcagttgt cctgccctgt ggaatgggct caaggttcct gagacacccg attcctgccc 1261 aaacagctgt atttatataa gtctgttatt tattattaat ttattggggt gaccttcttg 1321 gggactcggg ggctggtctg atggaactgt gtatttattt aaaactctgg tgataaaaat 1381 aaagctgtct gaactgttca aaaaaaaaaa aaaaaaa

By "Growth/Differentiation Factor 15 (GDF15) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No.

EAW84694, version EAW84694.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 6):

1 mpgqelrtvn gsqmllvllv lswlphggal slaeasrasf pgpselhsed srfrelrkry 61 edlltrlran qswedsntdl vpapavrilt pevrlgsggh lhlrisraal peglpeasrl 121 hralfrlspt asrswdvtrp lrrqlslarp qapalhlrls pppsqsdqll aesssarpql 181 elhlrpqaar grrrararng dhcplgpgrc crlhtvrasl edlgwadwvl sprevqvtmc 241 igacpsqfra anmhaqikts lhrlkpdtvp apccvpasyn pmvliqktdt gvslqtyddl 301 lakdchci

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):

1 gtcggaggga gggagggagg gagagaaaga aagagagaaa aagaaggaaa gggagaggga 61 gacggctgga gcccgaggac gagcgcggag ccgcggaccg agcggggggc gggagacagg 121 aaggagggag gcgagcagag ggaaggggaa gaggtcgggg agcgagggcg ggagcggtcg 181 cggtcgcgat cgagcaagca agcgggcgag aggacgccct cccctggcct ccagtgcgcc 241 gcttccctcg ccgccgcccc gccagcatgc ccggcgtggc ccgcctgccg ctgctgctcg 301 ggctgctgct gctcccgcgt cccggccggc cgctggactt ggccgactac acctatgacc 361 tggcggagga ggacgactcg gagcccctca actacaaaga cccctgcaag gcggctgcct 421 ttcttgggga cattgccctg gacgaagagg acctgagggc cttccaggta cagcaggctg 481 tggatctcag acggcacaca gctcgtaagt cctccatcaa agctgcagtt ccaggaaaca 541 cttctacccc cagctgccag agcaccaacg ggcagcctca gaggggagcc tgtgggagat 601 ggagaggtag atcccgtagc cggcgggcgg cgacgtcccg accagagcgt gtgtggcccg 661 atggggtcat cccctttgtc attgggggaa acttcactgg tagccagagg gcagtcttcc 721 ggcaggccat gaggcactgg gagaagcaca cctgtgtcac cttcctggag cgcactgacg 781 aggacagcta tattgtgttc acctatcgac cttgcgggtg ctgctcctac gtgggtcgcc 841 gcggcggggg cccccaggcc atctccatcg gcaagaactg tgacaagttc ggcattgtgg 901 tccacgagct gggccacgtc gtcggcttct ggcacgaaca cactcggcca gaccgggacc 961 gccacgtttc catcgttcgt gagaacatcc agccagggca ggagtataac ttcctgaaga 1021 tggagcctca ggaggtggag tccctggggg agacctatga cttcgacagc atcatgcatt 1081 acgctcggaa cacattctcc aggggcatct tcctggatac cattgtcccc aagtatgagg 1141 tgaacggggt gaaacctccc attggccaaa ggacacggct cagcaagggg gacattgccc 1201 aagcccgcaa gctttacaag tgcccagcct gtggagagac cctgcaagac agcacaggca 1261 acttctcctc ccctgaatac cccaatggct actctgctca catgcactgc gtgtggcgca 1321 tctctgtcac acccggggag aagatcatcc tgaacttcac gtccctggac ctgtaccgca 1381 gccgcctgtg ctggtacgac tatgtggagg tccgagatgg cttctggagg aaggcgcccc 1441 tccgaggccg cttctgcggg tccaaactcc ctgagcctat cgtctccact gacagccgcc 1501 tctgggttga attccgcagc agcagcaatt gggttggaaa gggcttcttt gcagtctacg 1561 aagccatctg cgggggtgat gtgaaaaagg actatggcca cattcaatcg cccaactacc 1621 cagacgatta ccggcccagc aaagtctgca tctggcggat ccaggtgtct gagggcttcc 1681 acgtgggcct cacattccag tcctttgaga ttgagcgcca cgacagctgt gcctacgact 1741 atctggaggt gcgcgacggg cacagtgaga gcagcaccct catcgggcgc tactgtggct 1801 atgagaagcc tgatgacatc aagagcacgt ccagccgcct ctggctcaag ttcgtctctg 1861 acgggtccat taacaaagcg ggctttgccg tcaacttttt caaagaggtg gacgagtgct 1921 ctcggcccaa ccgcgggggc tgtgagcagc ggtgcctcaa caccctgggc agctacaagt 1981 gcagctgtga ccccgggtac gagctggccc cagacaagcg ccgctgtgag gctgcttgtg 2041 gcggattcct caccaagctc aacggctcca tcaccagccc gggctggccc aaggagtacc 2101 cccccaacaa gaactgcatc tggcagctgg tggcccccac ccagtaccgc atctccctgc 2161 agtttgactt ctttgagaca gagggcaatg atgtgtgcaa gtacgacttc gtggaggtgc 2221 gcagtggact cacagctgac tccaagctgc atggcaagtt ctgtggttct gagaagcccg 2281 aggtcatcac ctcccagtac aacaacatgc gcgtggagtt caagtccgac aacaccgtgt 2341 ccaaaaaggg cttcaaggcc cacttcttct cagaaaagag gccagctctg cagccccctc 2401 ggggacgccc ccaccagctc aaattccgag tgcagaaaag aaaccggacc ccccagtgag 2461 gcctgccagg cctcccggac cccttgttac tcaggaacct caccttggac ggaatgggat 2521 gggggcttcg gtgcccacca accccccacc tccactctgc cattccggcc cacctccctc 2581 tggccggaca gaactggtgc tctcttctcc ccactgtgcc cgtccgcgga ccggggaccc 2641 ttccccgtgc cctaccccct cccattttga tggtgtctgt gacatttcct gttgtgaagt 2701 aaaagaggga cccctgcgtc ctgctccttt ctcttgcaga aaaaaaa

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):

1 mpgvarlpll lgllllprpg rpldladyty dlaeeddsep lnykdpckaa aflgdialde 61 edlrafqvqq avdlrrhtar kssikaavpg ntstpscqst ngqpqrgacg rwrgrsrsrr 121 aatsrpervw pdgvipfvig gnftgsqrav frqamrhwek htcvtflert dedsyivfty 181 rpcgccsyvg rrgggpqais igkncdkfgi vhelghvvg fwhehtrpdr drhvsivren 241 iqpgvlhssl lllscgsrng asfpcsless ihqalcwtgl flrpspfprl plaaprtlra By "Shwachman-Bodian -Diamond Syndrome (SBDS) nucleic acid molecule" is meant a polynucleotide encoding a SBDS polypeptide. An exemplary SBDS nucleic acid molecule is provided at NCBI Accession No. NM_016038, version NM_016038.3, incorporated herein by reference, and reproduced below (SEQ ID NO: 9):

1 tttgggcgtg gaaagatggc gtaaaaagcc acaatacgca ggcgtcatcg ctcacttttc

61 ccctcccggc ttctgctcca cctgacgcct gcgcagtaag taagcctgcc agacacactg 121 tgacggctgc ctgaagctag tgagtcgcgg cgccgcgcac tggtggttgg gtcagtgccg 181 cgcgccgatc ggtcgttacc gcgaggcgct ggtggccttc aggctggacg gcgcgggtca 241 gccctggttc gccggcttct gggtctttga acagccgcga tgtcgatctt cacccccacc 301 aaccagatcc gcctaaccaa tgtggccgtg gtacggatga agcgtgccgg gaagcgcttc 361 gaaatcgcct gctacaaaaa caaggtcgtc ggctggcgga gcggcgtgga aaaagacctc 421 gatgaagttc tgcagaccca ctcagtgttt gtaaatgttt ctaaaggtca ggttgccaaa 481 aaggaagatc tcatcagtgc gtttggaaca gatgaccaaa ctgaaatctg taagcagatt 541 ttgactaaag gagaagttca agtatcagat aaagaaagac acacacaact ggagcagatg 601 tttagggaca ttgcaactat tgtggcagac aaatgtgtga atcctgaaac aaagagacca 661 tacaccgtga tccttattga gagagccatg aaggacatcc actattcggt gaaaaccaac 721 aagagtacaa aacagcaggc tttggaagtg ataaagcagt taaaagagaa aatgaagata 781 gaacgtgctc acatgaggct tcggttcatc cttccagtca atgaaggcaa gaagctgaaa 841 gaaaagctca agccactgat caaggtcata gaaagtgaag attatggcca acagttagaa 901 atcgtatgtc tgattgaccc gggctgcttc cgagaaattg atgagctaat aaaaaaggaa 961 actaaaggca aaggttcttt ggaagtactc aatctgaaag atgtagaaga aggagatgag 1021 aaatttgaat gacacccatc aatctcttca cctctaaaac actaaagtgt ttccgtttcc 1081 gacggcactg tttcatgtct gtggtctgcc aaatacttgc ttaaactatt tgacattttc 1141 tatctttgtg ttaacagtgg acacagcaag gctttcctac ataagtataa taatgtggga 1201 atgatttggt tttaattata aactggggtc taaatcctaa agcaaaattg aaactccaag 1261 atgcaaagtc cagagtggca ttttgctact ctgtctcatg ccttgatagc tttccaaaat 1321 gaaagttact tgaggcagct cttgtgggtg aaaagttatt tgtacagtag agtaagatta 1381 ttaggggtat gtctatacaa caaaaggggg ggtctttcct aaaaaagaaa acatatgatg 1441 cttcatttct acttaatgga acttgtgttc tgagggtcat tatggtatcg taatgtaaag 1501 cttggatgat gttcctgatt atctgagaaa cagatataga aaaattgtgc cggacttacc 1561 tttcattgaa catgctgcca taacttagat tattcttggt taaaaaataa aagtcactta 1621 tttctaattc ttaaagttta taatatatat taatatagct aaaattgtat gtaatcaata 1681 aaaccactct tatgtttatt aaactatggc ttgtgtttct agacaaaaaa aaaaaaaaaa 1741 aaaaaaaaa

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):

1 msiftptnqi rltnva vrm kragkrfeia cyknk vgwr sgvekdldev lqthsvfvnv 61 skgqvakked lisafgtddq teickqiltk gevqvsdker htqleqmfrd iativadkcv 121 npetkrpytv ilieramkdi hysvktnkst kqqalevikq lkekmkiera hmrlrfilpv 181 negkklkekl kplikviese dygqqleivc lidpgcfrei delikketkg kgslevlnlk 241 dveegdekfe

By "granulocyte-colony stimulating factor (G-CSF) nucleic acid molecule" is meant a polynucleotide encoding a G-CSF polypeptide. 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):

1 ctgccgcttc caggcgtcta tcagcggctc agcctttgtt cagctgttct gttcaaacac

61 tctggggcca ttcaggcctg ggtggggcag cgggaggaag ggagtttgag gggggcaagg 121 cgacgtcaaa ggaggatcag agattccaca atttcacaaa actttcgcaa acagcttttt 181 gttccaaccc ccctgcattg tcttggacac caaatttgca taaatcctgg gaagttatta 241 ctaagcctta gtcgtggccc caggtaattt cctcccaggc ctccatgggg ttatgtataa 301 agggccccct agagctgggc cccaaaacag cccggagcct gcagcccagc cccacccaga 361 cccatggctg gacctgccac ccagagcccc atgaagctga tgggtgagtg tcttggccca 421 ggatgggaga gccgcctgcc ctggcatggg agggaggctg gtgtgacaga ggggctgggg 481 atccccgttc tgggaatggg gattaaaggc acccagtgtc cccgagaggg cctcaggtgg 541 tagggaacag catgtctcct gagcccgctc tgtccccagc cctgcagctg ctgctgtggc 601 acagtgcact ctggacagtg caggaagcca cccccctggg ccctgccagc tccctgcccc 661 agagcttcct gctcaagtgc ttagagcaag tgaggaagat ccagggcgat ggcgcagcgc 721 tccaggagaa gctggtgagt gaggtgggtg agagggctgt ggagggaagc ccggtgggga 781 gagctaaggg ggatggaact gcagggccaa catcctctgg aagggacatg ggagaatatt 841 aggagcagtg gagctgggga aggctgggaa gggacttggg gaggaggacc ttggtgggga 901 cagtgctcgg gagggctggc tgggatggga gtggaggcat cacattcagg agaaagggca 961 agggcccctg tgagatcaga gagtgggggt gcagggcaga gaggaactga acagcctggc 1021 aggacatgga gggaggggaa agaccagaga gtcggggagg acccgggaag gagcggcgac 1081 ccggccacgg cgagtctcac tcagcatcct tccatcccca gtgtgccacc tacaagctgt 1141 gccaccccga ggagctggtg ctgctcggac actctctggg catcccctgg gctcccctga 1201 gcagctgccc cagccaggcc ctgcagctgg tgagtgtcag gaaaggataa ggctaatgag 1261 gagggggaag gagaggagga acacccatgg gctcccccat gtctccaggt tccaagctgg 1321 gggcctgacg tatctcaggc agcaccccct aactcttccg ctctgtctca caggcaggct 1381 gcttgagcca actccatagc ggccttttcc tctaccaggg gctcctgcag gccctggaag 1441 ggatctcccc cgagttgggt cccaccttgg acacactgca gctggacgtc gccgactttg 1501 ccaccaccat ctggcagcag gtgagccttg ttgggcaggg tggccaaggt cgtgctggca 1561 ttctgggcac cacagccggg cctgtgtatg ggccctgtcc atgctgtcag cccccagcat 1621 ttcctcattt gtaataacgc ccactcagaa gggcccaacc actgatcaca gctttccccc 1681 acagatggaa gaactgggaa tggcccctgc cctgcagccc acccagggtg ccatgccggc 1741 cttcgcctct gctttccagc gccgggcagg aggggtcctg gttgcctccc atctgcagag 1801 cttcctggag gtgtcgtacc gcgttctacg ccaccttgcc cagccctgag ccaagccctc 1861 cccatcccat gtatttatct ctatttaata tttatgtcta tttaagcctc atatttaaag 1921 acagggaaga gcagaacgga gccccaggcc tctgtgtcct tccctgcatt tctgagtttc 1981 attctcctgc ctgtagcagt gagaaaaagc tcctgtcctc ccatcccctg gactgggagg 2041 tagataggta aataccaagt atttattact atgactgctc cccagccctg gctctgcaat 2101 gggcactggg atgagccgct gtgagcccct ggtcctgagg gtccccacct gggacccttg 2161 agagtatcag gtctcccacg tgggagacaa gaaatccctg tttaatattt aaacagcagt 2221 gttccccatc tgggtccttg cacccctcac tctggcctca gccgactgca cagcggcccc 2281 tgcatcccct tggctgtgag gcccctggac aagcagaggt ggccagagct gggaggcatg 2341 gccctggggt cccacgaatt tgctggggaa tctcgttttt cttcttaaga cttttgggac 2401 atggtttgac tcccgaacat caccgacgtg tctcctgttt ttctgggtgg cctcgggaca 2461 cctgccctgc ccccacgagg gtcaggactg tgactctttt tagggccagg caggtgcctg 2521 gacatttgcc ttgctggatg gggactgggg atgtgggagg gagcagacag gaggaatcat 2581 gtcaggcctg tgtgtgaaag gaagctccac tgtcaccctc cacctcttca ccccccactc 2641 accagtgtcc cctccactgt cacattgtaa ctgaacttca ggataataaa gtgtttgcct 2701 ccagtcacgt ccttcctcct tcttgagtcc agctggtgcc tggccagggg ctggggaggt 2761 ggctgaaggg tgggagaggc cagagggagg tcggggagga ggtctgggga ggaggtccag 2821 ggaggaggag gaaagttctc aagttcgtct gacattcatt ccgttagcac atatttatct 2881 gagcacctac tctgtgcaga cgctgggcta agtgctgggg acacagcagg gaacaaggca 2941 gacatggaat ctgcactcga

By "granulocyte-colony stimulating factor (G-CSF) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No.

CAA27290, version CAA27290.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 12):

1 magpatqspm klmalqlllw hsalwtvqea tplgpasslp qs fllkcleq vrkiqgdgaa 61 lqeklcatyk lchpeelvll ghslgipwap lsscpsqalq lagclsqlhs glflyqgllq 121 alegispelg ptldtlqldv adfattiwqq meelgmapal qptqgampaf asafqrragg 181 vlvashlqsf levsyrvlrh laqp

Microarrays

Differential gene expression can also be identified, or confirmed using a microarray technique. In these methods, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. Just as in the RT- PCR method, 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.

In the microarray technique, 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.

In some cases, 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.

In some configurations, 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. In various configurations, the miniaturized scale of the hybridization can afford a convenient and rapid evaluation of the expression pattern for large numbers of genes. In various configurations, 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. In various configurations, 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)). In various configurations, 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-seq

RNA sequencing (RNA-seq), also called whole transcriptome shotgun sequencing (WTSS), uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment in time.

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.

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).

Transforming Growth Factor β Inhibitors

Described herein is the treatment of SDS with a TGFp inhibitor. As described herein, AVID200 is a TGF-β inhibitor for the treatment of anemia associated with myelodysplastic syndromes (Thwaites et al., Blood 2017, 130: 2532, incorporated herein by reference). Also, SD-208 is a selective TGF-PRl inhibitor, the compound of which is provided below.

SD-

Various TGFp inhibitors have either been approved for use by the United States Food and Drug Administration (USFDA) for specified indications or are currently in a clinical trial. Exemplary 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 (Esbriet; S-7701; AMR-69; FDA-approved),

LY333531 (Ruboxistaurin), and LY2109761 the structure of each of which TGFP inhibitor is provided below. LY2157299 Monohvdrate

TEW-7197

Bortezomib

P144

Pirfenidone

LY333531

LY2109761

Exemplary clinical uses of the TGFP receptor inhibitor compounds are provided below. 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

Pharmaceutical Therapeutics

For therapeutic uses, the compositions or agents described herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable 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. Formulation of Pharmaceutical Compositions

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.,

subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R.

Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical

Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

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. In certain embodiments it is envisioned that the dosage may vary from between about 1 μg 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. In other cases, 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. In other aspects, it is envisaged that doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body. In other embodiments, the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Of course, 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.

Pharmaceutical 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. The latter types of compositions are generally known as 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 the therapeutic agent to a particular cell type. For some applications, 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. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, 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.

Parenteral Compositions

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. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

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. Apart from the active agent that reduces or ameliorates SDS, 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.

Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable active SDS therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among 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). In cases where one of the compounds is only sparingly or slightly soluble in water, 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

Controlled release parenteral compositions may be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. Alternatively, the active drug may be incorporated in biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.

Materials for use in the preparation of microspheres and/or microcapsules are, e.g., 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).

Combination Therapy

In certain embodiments of the invention, 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.

Alternatively, the present inventive therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where 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. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 h of each other and, more preferably, within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (e.g., 2, 3, 4, 5, 6 or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 weeks) lapse between the respective administrations.

It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the inventive TGFP inhibition therapy.

Kits or Pharmaceutical Systems

The present compositions may be assembled into 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.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental

Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. EXAMPLES

Example 1 : Materials and Methods

The following materials and methods were utilized in the examples described herein. Sample processing

For scRNA-seq of all SDS samples, and normal donors Nl and N2: 7-20 ml of fresh BM were diluted to 35 ml in MACS buffer (phosphate buffered saline (PBS)/2mM

Ethylenediaminetetraacetic acid (EDTA)/0.5% Bovine Serum Albumin (BSA)), layered onto 15 ml Ficoll-paque (GE Healthcare, Uppsala, Sweden), and spun for 30 min at 1400 rpm and 20°C with no brakes. Mononuclear cells were collected from the interface, washed once, pelleted for 5 min at 1200 rpm and 20°C, and resuspended at 40 μΐ per 107,397 cells in MACS buffer + 1 μΐ/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). For samples N3 and N4, protocol conditions were modified to ascertain immunophenotypes from single cells in accordance with the newest available methods. For these samples, 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 μΐ/ml RNaseOUT. Cells were stained as described below. Single CD34⁺ cells were sorted into 5 μΐ 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 μΐ 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.

Antibodies and staining

Cells were stained at a density of lxlO⁶ per 100 μΐ in PBS + 1 μΐ/ml RNaseOUT because staining buffers contain proteins that can inhibit SMART er-seq (Clontech) cDNA synthesis reactions. The staining panel was adapted from an analysis of human cord blood progenitors by Laurenti et al. (Laurenti, E., Doulatov, S., Zandi, S., Plumb, I, Chen, J., et al., 2013 Nature Immunology, 14:756-763) in accordance with the parameters of the flow cytometer. 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

563965, 1 :20), allophycocyanin-anti-CD45RA (BD 550855, 1 :5), phycoerythrin-anti-CD135 (BD 558996, 1 :5), and allophycocyanin-cyanine 7-anti-CDlO (Biolegend 312212, 1 :20).

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.

Data processing and availability

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.

Gene selection based on bulk expression data

The Gini index (Jiang et al., 2016 Genome Biol., 17: 144) was used to identify cell type- specific genes from HSC, MPP, CLP, CMP, MEP, and GMP 100 cell libraries. The maximum TPM value of each gene was calculated, and genes with maximum value lower than the 20- quantile of all maximum values were filtered out because those genes could have high Gini index due to their low expression. The top 500 high Gini index genes were identified for each of the biological (n=2) and technical (n=2) replicates for each cell type. The cell type specific gene signatures were chosen as the intersection of high Gini genes across all replicates for each cell type.

tSNE analysis

Following Tirosh et al. (Tirosh et al., 2016 Science, 352: 189-196), 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)). The expression of the 79 genes identified by bulk data across the 583 single cells was used for Principal Component Analysis (PCA) in the Seurat Package in R (Satija et al., 2015 Nat. Biotechnol., 33 :495-502). Using a jackstraw approach implemented in the Seurat package with num. replicate = 200 and each time randomly permuting three genes, the top four principal components (PCs) were identified as significant (p-value < 1x10-4). To aid visualization, these top four PCs, were subjected to t-distributed Stochastic Neighbor Embedding (t-SNE) analysis (Van der Maaten, L., Hinton, G., 2008 Journal of Machine Learning Research, 2579-2605) in

Seurat with 2000 iterations.

Clustering analysis

The tSNE coordinates were used for partitioning around medoids (PAM), a

more robust version of k-means clustering implemented in the "cluster" package in R with default parameters (stat.ethz.ch/R-manual/R-devel/library/cluster/html/pam.html). To determine the optimal k, the average Silhouette value (Rousseeuw, P.J., 1986 Journal of Computational and Applied Mathematics, 20:53-65) was assessed for each clustering result (from k=2 to k=10) and selected k =5, which gave the largest mean Silhouette value.

Differential gene expression and pathway analysis

Differential gene expression analysis was performed on SDS versus normal cells in each cluster (and in all clusters combined) using the Model-based Analysis of Single Cell

Transcriptomics (MAST) package in R (Finak et al., 2015 Genome Biology, 16:278). P-values were adjusted for mutiple testing using the "p. adjust" function in R with "fdr" method

(Benjamini, Y.H., Yosef, 1995 Journal of the Royal Statistical Society, 57:289-300). Genes with an FDR adjusted p-value < 0.05 and |log2(fold 2 change)] >1 in at least one cluster were given most focus. Enriched pathways and functions were determined in Ingenuity Pathway Analysis (Qiagen) using the 11094 detected genes as the reference gene set. Split violin plots were generated using the "vioplot" package and "vioplot2" function in R.

Immunofluorescent staining and imaging

Primary 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). CD34⁺ cells were sorted using CD34 Microbeads (Millitenyi) according to manufacturer's protocol, and allowed to recover in culture medium for 14-16 h, plus an additional 2 h in the presence of 0.6 μg/ml AVID200 for relevant samples. 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-Alexa488 antibody (Invitrogen, A21206) in blocking solution for 1 h at RT; and washed 3X with 0.1% NP40 in IX PBS at RT for 10 min. Stained coverslips were mounted on glass slides with VectaShield with DAPI (H-1200, Vector

Laboratories, Burlingame, CA) diluted 1 : 1 in VectaShield without DAPI (H-1000). Slides were imaged on a LeicaSP5 confocal microscope with constant laser power (30% for DAPI, 70% for Alexa488) and identical resolution, offset, and gain settings for all slides. Z stack images were captured with 40-80 μπι step range, and the plane with the best nuclear representation was analyzed using Fiji software. Background was calculated using four randomly selected empty regions for each image. Mean signal intensity for p-SMAD2 (Alexa Fluor-488) was calculated within each nucleus, and background signal was subtracted.

Colony formation assays

Primary 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 μΜ SD208 (Tocris, Bristol, UK), and incubated for lhr at 37°C/5% C02. The 200 μΙ_, 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® proteomic analysis

SOMAscan® (SomaLogic, Βομ εΓ, CO) was performed on 50 μΐ 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.

Statistics

In 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. In 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. In FIG. 2B, the frequency of cells was compared between SDS and normal cells within each cluster. In FIG. 3C, log2 expression was compared between SDS and normal cells within each cluster. In FIG. 4D and FIG. 7B, relative colony number was compared between each drug dose and the 0 μΜ treatment. In 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.

SDS patients exhibit BM hypocellularity and peripheral cytopenias involving multiple lineages (Myers et al., 2014 The Journal of Pediatrics, 164: 866-870), consistent with defects in the CD34⁺ HSPC pool. It was hypothesized that the dynamic subpopulations that comprise the CD34⁺ pool may exhibit cell type-specific responses to SBDS mutations that influence clinical presentation. To simultaneously examine the consequences of SBDS mutations in all of these subpopulations, scRNA-seq was performed on CD34⁺ HSPC freshly isolated from normal donors (n=4) and SDS patients (n=4). The patients all exhibited BM hypocellularity or cytopenias at the time of sampling, with one patient being treated with Granulocyte-colony stimulating factor (G-CSF) for severe neutropenia (Table 2), and is discussed separately below. The CD34⁺ cells were selected from the mononuclear fraction without gating on additional markers, single cells were sequenced using SMART-seq technology for full length cDNA amplification (Clontech) (Ramskold et al., 2012 Nat. Biotechnol., 30:777-782; Picelli et al., 2013 Nat. Methods, 10: 1096-1098), and classified HSPC a posteriori based on transcriptional signatures of lineage commitment. This approach is well suited to capture cells along the CD34⁺ differentiation spectrum, which is still poorly understood in human BM (Notta et al., 2011 Science, 333 :218-221; Velten et al., 2017 Nat. Cell Biol., 19:271-281). Table 2: Summary of primary samples

The challenge for examining a rare patient population is that biological variables and batch effects can obscure disease signatures. To classify single cells with respect to

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. 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.

Table 1: Loading scores for significant principle components

- -

2 PCI PC2 PC3 PC4

|AVP 0.1320461 0.1.214926 -0.0272783 ! 0.0775371.!

4 RP11.377 22.2 0.0218935 0.0846749 -0.0094932! 0.0975026

5 |UNC01122 0.1026106 0.0816914 0.0027606! -0.0092333!

HtF 0.1524062 0.1109241 -0.011457! -0.168341!

7 CRHBP 0.2074693 0.1528988 -0.0233602 -0.0925432!

8 |RP11.598F7.3 0.0885881 0.0714256 -0.0153245! -0.0753122!

Q MEG 3 0.127617 0.1076118 -0.0356421! -0.1002352!

10 |A X)6998.2 0.1745456 0.1398457 -0.0182772! -0.065537!

11 jMECOM 0.1393703 0.1032365 -0.0478287! -0.0976923!

12 |AP001171.1 0,0731065 0.0322266 -0.0335455! 0.0180203 j

13 HOPX 0.1678224 0.126822 0.0200682! 0.0787952 !

14 MYTH 0.1615232 0.1105524 -0.0791853! -0.0895044

15 |EHD2 0.1314587 0.0905262 -0.0839661! 0.0267594

16 IL !^' 3 0.176181 0.0867134 -0.1370385! -0.11.34212! 7 CFH 0.1121748 0.0900791 -0.0489065! -0.1246504!

IS C16orf45 0.0937809 0.0625325 -0.0410034! -0.0894206!

19 |NKAIN2 0.0784577 0.0473798 -0.0623238! 0.0168014!

20 EGFEMiP 0.1115309 0.0914848 0.0103015! 0.0559955!

21 NAP 113 0.0782878 0.0531961 -0.0256782 ! 0.1088834!

77 EPGN 0.089259 0.0403301 -0.1000141 -0.1166885!

23 HROB04 0.1218013 0.0994334 -0.0069691! 0.0634939!

24 PROMI 0.1255486 0.0924065 0.0273368! 0.1812505!

NPR3 0.1778127 0.0990723 -0.0653727! 0.0462387

26 I KRT18 0.0859306 0.0730337 -0.0110101! 0.1605854!

27 STSSSA6 0.064566 0.0137221 -0.1095499! -0.026908

28 |^"Γ ΕΜ163 0.0810112 0.05752 :1 -0.0153564! -0.0312674!

29 R 0.0697643 0.0563821 0.0015357! 0.0699851!

30 E C 0.0961996 0.0659307 -0.0214091! -0.1178515!

31 P! 0.1455133 0.1156372 -0.0323406! 0.0421938!

32 ADGRG6 0.1631705 0.1434236 -0.0102104! -0.0178215!

33 |T EM98 0.0786712 0.0556391 -0.0357874! 0.0189145!

34 CRYGD 0.0676056 0.0422936 -0.0303043! 0.1620625!

35 |SPINK2 0.155921 0.0875274 0.0760921! 0.1532664!

36 !KRTS 0.0586355 0.0626256 -0.0110602! 0.0636611! NPDCi ! 0,0658072 0.0489092! 0.0170717! 0.1609581

!CLU ! 0.1288107 0.1090425! -0.078487! -0.1463032

ADA 2 I 0.1094508 0.1077622! -0.0110446! 0.1319325

|SULT1C4 I 0.0990531 0.0777179! -0.0072136! 0.0280261

ICALCRL 0.074104 0.0618792! 0.0066228! 0.0136645

!SYTL4 ! 0.1163233 0.0674076! -0.059845! -0.085879

GPAT3 I 0.0738851 0.1312256! -0.015853! 0.0315911

|CA1 I -0.0225528 -0,1092636! -030764! -0.0074177

! LFl ! -0.0212772 -0.0887386! -0.3231517! 0.0076864

GAIAl I -0.0198455 -0. 159343! -0.3196723! 0.0084901

CNRIPI I -0.0243812 -0.1016714! 0.3019711! 0.0061568

1 -0.0218239 -0.11578191 -0.3309531.! -0.0203408

|TFR2 ! -0.0074437 -0.0818365! -0.300442! 0.0168034

SLC25A15 I -0.019086 -0.05560291 -0.2059138 0.1150512

PVTl I -0.0131797 -0.0983553! -0.2872603! 0.0077355

D^AJA4 1 -0.0222893 -0.0338979! -0.147128! 0.1045361

|lCAM4 ! 0.0252937 -0.0202161! -0.2450637! 0.0158553

A JiP I -0.0003365 0.03898491 0.0010371! -0.0196366

FOVIR I -0.008076 -0.0256841! 0.0986885! -0.0086599

IANXA2P2 ! -0.129510 0.09083681 -0.0756982.! 0.1570692

!RAGl ! -0.0346368 -0.1045087! 0.1467186! -0.1588075

IF BO I -0.1674088 0.2139343! -0.0604106! -0.1198796

S100A8 I -0.1499692 0.1489021! -0.0254892! -0.1960018

|ANXA2 ! -0.1432736 0.08628581 -0.0510368! 0.1483287

! -0.1470133 0.1657405! -0.0384531! -0.0293699

S100A9 I -0.1671945 0.1683733! -0.0229211! -0.1885932

|NCF2 I -0.1492075 0.1724889! -0.0340067! 0.0126485

! -0.1067403 0.13494911 0.0105563! -0.111757

|RA831 ! -0.1363258 0.1524519! -0.0214783! -0.0124639

CTSS I -0.0868616 0.2150945! -0.0732163! 0.0167792

|PADf4 1 0.0075679 0.1007576! -0.00741.1.3! 0.0284111

! E ! -0.0381127 -0.113571! 0.1576306! -0.1680722

!TN!FRSFIS ! -0.1565318 0.1811902! -0.0433036! -0.1207563 70 MUDA I -0.1651804 ! 0.1678559 -0.0201976] -0.0789835

71 CTSH I -0.1501955 0.1600694 -0.0974547 ! 0.0509265

72 F L2 ! -0.1650565 ! 0.2161442 -0.0560556! -0.0959085

73 COTLl ! -0.147504! 0.1554705 -0,0861197 ! 0.0859106

74 CYBB ! -0.1823609 ! 0.2000469 -0.0385957 ! -0.0487986

75 HC ! -0.15335951 0.1690362 -0.0258271 ! 0.0306388

76 CECRl ! -0.1131758! 0.1788205 -0.003678 ! 0.0329332

77 IL3RA ! -0.0040183 ! 0.0598241 0.02115981 0.2685614

78 PLD4 ! -0.0203109 ! 0.120824 0,0219689 ! 0.3512634

79 IRF8 ! -0.1368248 ! 0.0989995 0.0398447 ! 0.2841403

SO GCDC50 ! -0.06177351 0.0289073 0.0116206! 0.2236485

81 TOP2A ! -0.03632891 -0.0783121 -0.0206165 ! 0.1482924

To associate regions of the map with specific lineages, mRNA expression

patterns of four lineage-predictive signature genes were examined: EVI1, IRF8, GATA1, and MME. 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. Gated HSCs, MPPs, MLPs, CMPs, GMPs or MEPs accounted for 68% of indexed cells; an additional 9% were CD34⁺ CD90^" CD38⁺ CD10⁺ CD45RA⁺ common lymphoid progenitors (CLPs). Cells from the different gates clustered in distinct regions of the map, consistent with mRNA expression patterns (FIG. ID). Thus, supervised transcriptional mapping distinguished the major branches of hematopoiesis among randomly sampled CD34⁺ cells. Example 3 : The cellular architecture of early hematopoiesis is altered in SDS

This single cell map of normal hematopoietic lineage commitment was used as a baseline from which to examine alterations in the cellular architecture of SDS hematopoiesis. 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 (χ2 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. Untreated SDS patients had a stark reduction in GMPs and a modest increase in HSC/MPP (FIG. 2B). The reduction in GMP was evident even in the absence of symptomatic neutropenia (FIG. 6), suggesting that it contributes to the neutropenia predisposition in SDS patients. G-CSF treatment in one patient rescued loss of GMP and depleted HSC/MPP from the BM (FIG. 2B), consistent with the drug's known mechanism (Thomas et al., 2002 Curr. Opin. Hematol., 9: 183-189).

Therefore, cells from this treated patient were excluded from comparative gene expression analyses.

Example 4: TGFp signaling is selectively activated in SDS stem and multipotent progenitors

Gene expression was compared between normal and SDS cells within each cluster except for GMP, which was excluded due to the low number of GMP in untreated SDS patients. Overall, 1680 genes were differentially expressed in at least one cluster (FDR<0.05, |log2(fold change)] >1). Strikingly, 81.5% of all differentially expressed genes were unique to

either HSC/MPP or CMP (FIG. 3 A). An additional 9.8% were commonly affected in HSC/MPP and CMP, but not in MLP/CLP or MEP. Thus, HSC/MPP and CMP are the primarily affected cell types in SDS, but the affected genes are distinct between cell types. These data indicate that despite the general biochemical functions of the SBDS protein in ribosomal subunit joining and mitotic spindle stabilization (Menne et al., 2007 Nature Genetics, 39:486-495; Ganapathi et al., 2007 Blood, 110: 1458-1465; Finch et al., 2011 Genes & Development, 25:917-929; Burwick et al., 2012 Blood, 120:5143-5152; Austin et al., 2008 J Clin. Invest, 118: 1511-1518), SBDS mutations lead to cell type-dependent consequences.

The Inflammatory Response was enriched among differentially-expressed genes in both the HSC/MPP and CMP clusters (maximum p-value 4.98xl0^"5 and 1.18xl0^"3, respectively). However, the genes contributing to the enrichment differed between the clusters (FIG. 3B). TGFb was the top regulator predicted for the HSC/MPP inflammatory response (p=4.03xl0^"15, z- score=0.891). It was also a significant upstream regulator among all differentially-expressed genes in HSC/MPP (p=1.27xl0^"2, z-score=0.417). Dysregulation of these TGFP targets was most significant in HSC/MPP, with lesser or no effect in other HSPC populations (FIG. 3C). 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). Thus, it was hypothesized that activation of TGFP in SDS HSC/MPP may contribute to BM failure in SDS.

Example 5: TGFP pathway activation through TGFpRl suppresses hematopoiesis in SDS BM progenitors

To confirm activation of TGFP signaling in SDS BM, primary CD34⁺ cells were stained for phospho-SMAD2 (p-SMAD2), a transcriptional modulator that translocates to the nucleus in response to TGFp. A subset of CD34⁺ cells from SDS BM had elevated levels of nuclear p- SMAD2 that were outside the normal range (FIG. 4A and FIG. 4B). Treating SDS cells with AVID200, a decoy receptor trap designed to specifically neutralize TGFpi and TGFP3, reduced the P-SMAD2 signal. The same trend was observed to varying degrees in two additional sample pairs (FIG. 4C). These data are consistent with single cell RNA-seq analysis demonstrating selective activation of the TGFp pathway in the HSC/MPP subset of SDS CD34⁺ cells.

BM cells from SDS patients exhibit impaired hematopoietic colony formation in vitro (Dror, Y. & Freedman, M.H., 1999 Blood, 94:3048-3054) (FIG. 7A). To determine whether attenuation of TGFP signaling improves SDS hematopoiesis, 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

Biotechnol, 29: 543-549, incorporated herein by reference). Both compounds improved hematopoietic colony formation in SDS patient samples, but not in normal donor controls (FIG. 4D, FIG. 7B). Taken together, the data supports a model in which activation of TGFpRl kinase activity by TGFpi and/or TGFP3 leads to increased concentration of p-SMAD2 in the nucleus and transcription of inflammatory response genes in SDS HSC/MPP (FIG. 4E).

To determine whether SDS patients express elevated levels of TGFp ligands, blood plasma proteins were screened from six SDS patients and six normal controls (Table 2) using SOMAscan, which is a highly-sensitive, aptamer-based proteomic platform (Gold et al., 2012 N Biotechnol., 29:543-549). 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).

Additional experiments determine the levels of TGFP3 in the bone marrow compartment, and identify the cell types that produce it.

Example 6: Targeting TGFP inhibition for Shwachman Diamond Syndrome Therapy

Described herein is the development of pharmacologic agents for more effective and less toxic treatments for bone marrow failure with a specific focus on Shwachman Diamond

Syndrome (SDS). 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). As described herein, the TGFp3 pathway is a therapeutic target for SDS (FIG. 4F; FIG. 7A; and FIG. 7B. Single cell RNA sequencing (RNA-seq) of SDS patients' CD34⁺ bone marrow cells to identified increased expression of TGFp pathway genes in HSC. Also, 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.

First, the efficacy of 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.

Described herein is: (1) whether TGFP signaling in hematopoietic progenitors promotes bone marrow failure in SDS patients, and (2) whether 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.

Prior to the invention described herein, there were no SDS animal models of bone marrow failure. Described herein are two human systems for ex vivo analysis of SDS

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.

Leveraging these unique resources, several TGFP inhibitors (SD208 and Galunisertib) are dose-escalated on normal-donor CD34⁺ cells knocking down SBDS. To identify the progenitor cells and genes affected by each inhibitor, 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. These experiments improve specificity and minimize side effects of TGFP inhibitors for SDS therapy.

Impact

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.

While development of rationally-designed and targeted treatments would improve outcomes for SDS patients, historically, and prior to the invention described herein, agents identified for patients with rare bone marrow failure disorders have also had broader medical applications for the general population. The identification of TGFP inhibitors that improve hematopoiesis in SDS are tested more broadly in additional bone marrow failure disorders.

As described herein, 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

Unraveling of a complex disease with single cell analysis

Dissecting the pathogenic mechanisms that contribute to complex disease is a major biomedical challenge. This challenge can be simplified by viewing disease as an amalgam of heterogeneous processes in different cell types that promote or antagonize normal organ function. Moreover, rare cells such as somatic stem cells play outsized roles in pathogenesis. Stated simply, complex disease is rooted in single cell biology. Technological advances in DNA, RNA, and protein profiling now enable unprecedented access to the single cells that serve as building blocks of complex disease (Tang et al., 2009 Nature Methods, 6: 377-382; Ramskold et al., 2012 Nat Biotechnol, 30: 777-782; Picelli et al., 2013 Nature Methods, 10: 1096-1098;

Bendall et al., 2011 Science, 332, 687-696). Hematologic diseases, such as bone marrow (BM) failure, are 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.

Defining cellular mechanisms of SDS that can be targeted by rational therapies

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

Hematology, 18: 30-35; Finch et al., 2011 Genes & Development, 25: 917-929). It is

characterized by BM failure with predisposition to myelodysplasia and acute myeloid leukemia (AML), exocrine pancreatic dysfunction, and other organ anomalies. AML and other

complications of BM failure are major causes of mortality. Prior to the invention described herein, hematopoietic stem cell (HSC) transplant was the only curative treatment for hematologic dysfunction in SDS, but outcomes were limited by the inability to predict which patients will develop complications that outweigh the significant risks of transplant. Prior to the invention described herein, rational therapies for BM failure and clonal disease were urgently needed.

Despite the simple genetic basis of SDS, 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

thrombocytopenia, megakaryocyte dysplasia, anemia, and erythroid dysplasia occurring in smaller percentages of patients (Huang, J.N. & Shimamura, 2010 Current Opinion in

Hematology, 18: 30-35; Myers et al., 2014 The Journal of Pediatrics, 164: 866-870). The development of rational therapies requires a deeper understanding of why some cell types are selectively sensitive to the same genetic mutation in different individuals or at different times during the course of disease. Single cell analysis is the most direct way to determine how cells within a complex milieu respond to a genetic mutation, either directly or indirectly due to the breakdown of the normal microenvironment.

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). Thus, insights gained from single cell analysis of SDS advance the general

understanding of clonal progression in the context of BM failure. The results presented herein provide a paradigm to link defects in individual cells to complex disease phenotypes.

Single cell RNA-sequencing (scRNA-seq)

As described herein, BM hypocellularity and the involvement of multiple hematopoietic lineages in SDS suggested a defect in CD34⁺ hematopoietic stem and progenitor cells (HSPC). HSPC heterogeneity influences the production of mature blood cells, and enables rapid and dynamic responses to systemic perturbations (e.g. bleeding or infection). Known surface markers do not isolate pure populations from heterogeneous HSPC (Velten et al., 2017 Nat Cell Biol, 19: 271-281; Crisan, M. & Dzierzak, E., 2016 Development, 143 : 4571-4581). Thus, single cell RNA-sequencing (scRNA-seq) was performed on unsorted HSPC from fresh normal and SDS BMs. Five clusters of cells that coincide with stages of lineage commitment were identified(FIG. 2A). Heterogeneity within each cluster likely reflects differences in cellular function (e.g.

quiescent versus proliferating HSC). Comparative gene expression analysis between normal and SDS cells within each cluster revealed heterogeneous activation of TGFp target genes among individual SDS HSCs (FIG. 3C).

In contrast, TGFp target genes were not activated in lineage-committed

progenitors(FIG. 3C) . TGFP acts on many different cells, but the downstream effectors and phenotypic outcomes vary depending on cell type and functional state. To test whether TGFp activation in rare HSCs contributes to SDS BM failure, the pathway in normal and SDS BM cells was inhibited. Remarkably, 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. The data establishes TGFp signaling in rare HSCs as a therapeutic target in SDS, and further suggests that the role of TGFp in SDS HSCs is distinct from its role in normal hematopoiesis (Jacobsen et al., 1995 Blood, 86: 2957-2966; Sing et al., 1988 Blood, 72: 1504-1511; Park et al., 2014 J Exp Med, 211 : 71-87), and potentially from its role in other BM disorders for which it is being investigated for treatment (Zhou et al., 2011 Cancer Res, 71 : 955-963; Zingariello et al., 2013 Blood, 121 : 3345-3363; Suragani et al., 2014 Nat Med, 20: 408-414; Zhang et al., 2016 Cell Stem Cell, 18: 668-681; Zhou et al., 2008 Blood, 112: 3434-3443). Overall, these findings translate insights from single cell biology into a therapy.

As described herein, 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. Prior to the invention described herein, 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.

Accordingly, described herein is the definition of context-specific TGFP targets in functional subsets of SDS HSCs. The data described herein supports a clinical trial using TGFP inhibitors to treat BM failure. However, 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. To help develop a safer, targeted strategy for SDS therapy, this protocol requires:

• Obtaining bone marrow aspirates from eight SDS patients, and four normal donors;

• Profiling mRNA and protein expression in individual HSCs by scRNA-seq and mass cytometry (CyTOF); and

• Performing integrated analysis of mRNA and protein expression to map TGFP signaling to HSC subset(s).

The 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.

Also described herein is the rescue of SDS hematopoiesis by targeting TGFp using an Organ-on-a-Chip technology. Animal models of SDS do not faithfully recapitulate BM failure. Existing human cellular models of SDS (such as primary CD34⁺ culture and iPSC) dissociate HSPCs from the cellular milieu of BM which is critical for TGFP production and regulation. To develop a human cellular model of SDS that mimics the BM microenvironment, this protocol requires:

• Adapting BM-on-a-Chip technology to analyze the hematopoietic potential of SBDS- depleted HSCs;

• Defining the mechanistic relationship between TGFP and SBDS in HSCs and other resident BM cells; and

• Rescuing SDS phenotypes by inhibiting downstream effectors of TGFP signaling in HSCs.

These results generate the first ex vivo model of the human SDS BM microenvironment using Organ-on-a-Chip technology, and validate molecular targets for SDS therapies that precisely inhibit the pathogenic effect of TGFP signaling in HSCs.

As described herein, single cell transcriptomics pipeline for fresh HSPC has been designed, which 1) preserves natural biology by minimizing ex vivo manipulations; and 2)

bioinformatically classifies heterogeneous cells based on custom transcriptional signatures.

Using this pipeline, TGFp activation was defined as a molecular defect in SDS that specifically affects rare HSCs. In the experiment described herein, 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.

Define context-specific TGFp targets in functional subsets of SDS HSCs.

Fresh BM from eight SDS patients and four normal donors were provided. If fresh samples are limiting, frozen, clinically-annotated BM mononuclear cells from SDS patients may also be provided. For 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. For 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). Unfortunately, scRNAseq and CyTOF cannot be performed on the same single cell, but the datasets are integrated based on surface marker expression patterns that are commonly measured on all cells. Thus, TGFp signaling activity in single cells is deduced based on the combined status of protein mediators and transcriptional targets.

To determine whether key molecular features of SDS pathogenesis map to specific HSC subsets, sophisticated algorithms for visualizing high dimensional single cell data are applied. For example, viS E23 is used to spatially arrange cells in two dimensions based on

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.

Accordingly, the results presented herein comprehensively map human HSC heterogeneity, providing new insight into a cell population with critical roles in hematologic disease and malignancy.

Rescue of SDS hematopoiesis by targeting TGFb using a novel Organ-on-a-Chip technology.

To define the mechanisms of TGFp activation in SDS HSCs, SBDS-depleted primary human HSCs were cultured. However, it is ill-advised to assess activated TGFp signaling in purified HSCs ex vivo because the numerous cell types that produce and regulate TGFp in BM are absent. It is proposed to develop a new cellular model of SDS that incorporates key cell types from the BM microenvironment using Organ-on-a-Chip technology. Briefly, 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. These

experiments determine the cell types that produce and activate TGFp in SDS BM, and validate cell type-specific responses to TGFp. Lastly, SDS phenotypes are rescued by inhibiting signal transduction complexes (with aptamers or small molecules) or manipulating transcriptional targets (with short hairpin RNA (shRNAs) or cDNAs) that act downstream of TGFp in HSC subsets. Candidates that are selectively affected in the SDS target cell population are prioritized based on data obtained above, previous single cell analysis of HSPCs, and publicly available datasets. These

experiments identify putative targets for rational SDS therapies that precisely inhibit the pathogenic effect of TGFp signaling in HSCs.

The 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.

Prior to the invention described herein, there were currently no rational therapies to treat BM failure and deadly clonal progression to AML that occurs in up to one-third of SDS patients. The 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. In summary, the results presented herein lie at the intersection of basic and translational cancer research, and transforms SDS patient care.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and

modifications are within the scope of the following claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference.

Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is claimed is:

1. A method of treating or preventing bone marrow failure in a subject having or at risk of developing bone marrow failure comprising administering a transforming growth factor beta (TGFP) inhibitor to the subject, thereby treating or preventing bone marrow failure in the subject.

2. The method of claim 1, wherein the bone marrow failure comprises Shwachman-Diamond syndrome (SDS), Fanconi anemia (FA), dyskeratosis congenita (DC), congenital

amegakaryocytic thrombocytopenia (CAMT), Blackfan-Diamond anemia (BDA), or reticular dysgenesis (RD).

3. The method of claim 2, wherein the bone marrow failure comprises SDS.

4. The method of claim 3, wherein the TGFP inhibitor reduces or inhibits a symptom or sequelae associated with SDS, and wherein the symptom or sequelae associated with SDS is selected from the group consisting of neutropenia, anemia, thrombocytopenia, exocrine pancreatic dysfunction, growth retardation, chronic steatorrhea, metaphyseal dysplasia, myelodysplasia, megakaryocyte dysplasia, erythroid dysplasia, acute myeloid leukemia (AML), and generalized osteopenia.

5. The method of claim 3, wherein the TGFP inhibitor increases hematopoietic colony formation in the bone marrow of the subject by at least 5%.

6. The method of claim 5, wherein the TGFP inhibitor increases hematopoiesis in bone marrow hematopoietic stem or progenitor cells (HSPCs) by at least 5%.

7. The method of claim 1, wherein the TGFP inhibitor comprises AVID200, SD208,

LY2157299, TEW-7197, bortezomib, P144, pirfenidone, LY333531, or LY2109761.

8. The method of claim 7, wherein the TGFP inhibitor is administered at a dose of from about 0.1 nM to about 1 M.

9. The method of claim 7, wherein the TGFP inhibitor is administered at a frequency of from about once per day to about once per month.

10. The method of claim 7, wherein the TGFP inhibitor is administered for a duration of between one week and one year.

11. The method of claim 1, wherein TGFP receptor 1 expression or activity is inhibited by at least 5%.

12. The method of claim 1, wherein the TGFP inhibitor is administered orally or systemically.

13. The method of claim 1, further comprising performing an allogeneic bone marrow transplant or allogeneic hematopoietic stem cell transplant in the subject.

14. The method of claim 1, further comprising administering a granulocyte-colony stimulating factor (G-CSF) polypeptide to the subject.

15. The method of claim 1, further comprising administering a Shwachman-Bodian-Diamond Syndrome (SBDS) polypeptide to the subject.

16. A method of treating SDS in a subject having or at risk of developing SDS comprising contacting a CD34⁺ hematopoietic stem or progenitor cell (HSPC) of the subject with a TGFp inhibitor.

17. The method of claim 16, wherein the CD34+ HSPC is isolated from the subject prior to contacting the TGFP inhibitor, and further comprising administering the CD34⁺ HSPC to the subject.

18. The method of claim 17, wherein the CD34⁺ HSPC is isolated from the bone marrow of the subject.

19. The method of claim 16, wherein the CD34⁺ HSPC comprises a hematopoietic stem cell (HSC) or a multipotent progenitor cell (MPP).

20. A method of treating SDS in a subject having or at risk of developing SDS comprising contacting a CD34⁺ HSPC of the subject with an inhibitor or agonist of a polypeptide or nucleotide in the TGFP signaling pathway.

21. The method if claim 20, wherein the inhibitor of the nucleotide in the TGFP signaling pathway inhibits the expression or activity of TGFP3, growth/differentiation factor 15 (GDF15), or bone morphogenic protein 1 (BMP1).

22. A method of determining whether a subject has SDS comprising:

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.

23. The method of claim 22, wherein the gene comprises TGFP3, growth/differentiation factor 15 (GDF15), or bone morphogenic protein 1 (BMP1).

24. The method of claim 22, wherein the test sample is obtained from blood, serum, or plasma.