Compositions and methods for treating anemia

Abstract

Provided herein are compositions and methods for treating anemia. In particular, provided herein are compositions and methods for blocking SCD1 to treat anemia and / or prevent relapses of anemia.

Description

UM-44851.601COMPOSITIONS AND METHODS FOR TREATING ANEMIASTATEMENT OF RELATED APPLICATIONSThis application claims priority to and the benefit of provisional patent application 63 / 778,934, filed March 27, 2025, which is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with government support under DK127013 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELDProvided herein are compositions and methods for treating anemia. In particular, provided herein are compositions and methods for blocking SCD 1 to treat anemia and / or prevent relapses of anemia.BACKGROUNDDiamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome that manifests as a variably penetrant macrocytic anemia, which can spontaneously enter remission for unclear reasons.DBA anemia is characterized by normocytic or macrocytic anemia (low red blood cell counts) with decreased erythroid progenitor cells in the bone marrow. DBA usually develops during the neonatal period. About 47% of affected individuals also have a variety of congenital abnormalities, including craniofacial malformations, thumb or upper limb abnormalities, cardiac defects, urogenital malformations, and cleft palate. Low birth weight and generalized growth delay are sometimes observed. DBA patients further have a modest risk of developing leukemia and other malignancies.Treatments include corticosteroids, leucine, blood transfusions, allogeneic bone marrow transplant. However, these treatments all have significant side effects or risks.Improved treatments for DBA are needed.SUMMARYUM-44851.601Provided herein are compositions and methods for treating anemia. In particular, provided herein are compositions and methods for blocking SCD1 to treat anemia and / or prevent relapses of anemia.The present disclosure meets an existing need for improved treatments for anemia such as DBA. The treatments both reduce signs or symptoms of anemia and prevent relapse in subjects in remission from anemia.For example, in some embodiments, provided herein is a method of treating or preventing a relapse of anemia, comprising: administering to a subject diagnosed with anemia an agent that inhibits one or more activities of stearoyl-CoA desaturase (SCD1).The present disclosure is not limited to particular agents that inhibit SCD 1. In some embodiments, the agent is a small molecule (e.g. Aramchol, MK-8245, MTI-301, A939572, CAYI0566, MF-438, MF-152, CVT-11127, CVT-12012, T-3764518, BZ36, SSI4, SAR707, XEN103, XEN723, SW208108, SW203668 or a combination thereof).In certain aspects, a genetic therapy is used to decrease the expression of SCD1 (e.g., in vivo or ex vivo genetic therapy).The present disclosure is not limited to particular types of anemia. In some embodiments, the anemia is Diamond Blackfan anemia (DBA). In some embodiments, the subject has a variant RPL5 gene or allele. In some embodiments, the subject is in remission or is not in remission from anemia.The present disclosure is not limited to particular subjects. In some embodiments, the subject is a juvenile (e.g., under the age of 18, under the age of 12, under the age of 6 months, under the age of two months, or a newborn).In some embodiments, the administration increases erythropoiesis and / or red blood cell levels in the subject.In certain embodiments, the method further comprises administering an additional therapy or agent to the subject. Examples include but are not limited to, corticosteroids, leucine, blood transfusions, allogeneic bone marrow transplant, iron supplements, vitamin supplements or a combination thereof.Also provided is the use of an agent that inhibits one or more activities of SCD1 to treat anemia and / or prevent the relapse of anemia in a subject.Additional embodiments are described herein.BRIEF DESCRIPTION OF THE DRAWINGSUM-44851.601FIG. 1 shows that Rpl5+ / _mice show delayed erythroid recovery after Poly I:C treatment. (A) Adult female mice were injected with Poly I:C at lOmg / kg or PBS by IP. (B) Summary of the weekly plots of RBC, Hgb and MCV. Rpl5+ / - mice showed significantly lower RBC (C) and Hgb (D) at Day 14 after Poly EC injection compared to WT mice, with a delay in recovery to normal. (E) Mice injected with Poly EC showed significantly increased MCV level at Day 14 and Day 28.FIG. 2 shows that Rpl5Skax23‘Jus / +mice (Rpl5+ / - mice) show delayed erythroid recovery after phenylhydrazine treatment. (A) Adult (1.5-3.5 month) mice were injected with phenylhydrazine (Phz) (60pg / g IP) or PBS on days 0, 2, 21, and 23, and weekly blood count obtained until recovery (n=8 for each group:, 50 / 50 M:F ratio). (B) Rpl5+ / - Phz-treated mice had a more significant macrocytic anemia with lower RBC after both Phz treatments compared with WT. WT and Rpl5+ / - both showed recovery by days 21 and 49, however Rpl5+ / _mice experienced slower recovery of RBC counts after Phz treatment and death occurcd in 3 mutant mice (C) Graph of the RBC counts at each day of analysis in panel (B). (D) Analysis of erythropoiesis in the spleen using CD71 and Teri 19 on day 4 with quantification in (E) (n=6 for each group:, 50 / 50 M:F ratio). (F) Analysis of CFU-E and PreCFU-E in the spleen on day 4 with quantification in (G).FIG. 3 shows that treatment with SCD1 inhibitor improves erythropoiesis. (A) Adult mice (female, n=6 per group, age 2-4 months) were pretreated with SCD1 inhibitor (CAY10566) or DMSO for two weeks (day -14-0) and then treated with phenylhydrazine on days 0 and 2. (B) Analysis of blood counts at day 7 showed an improvement in RBC number in mutant mice after SCD1 treatment. Analysis of HSPC subsets by flow cytometry at day 4 (C, D) showed a significant increase in CFU-E and a decrease in pre-CFU-E in WT mice. (E, F) Analysis of erythroid progenitors at day 4 by CD71 and Teri 19 showed a decrease in earlier progenitors (II) and an increase in late progenitors (IV) in WT mice. (G, H) Further quantification of terminal erythropoiesis (Teri 19+ gate) using CD44 vs FSC shows significant improvement in erythropoiesis in WT animals after SCDl-i treatment.FIG. 4 shows treatment with SCD1 inhibitor. (A) Weights (B) blood counts were measured at baseline (day -14) and then weekly. (C) Quantification of bone marrow and spleen cellularity.FIG. 5 shows that mutant E12.5 FL with lower cellularity shows significant downregulation of lipid metabolism gene Scdl. (A) Bulk RNA-seq using total RNA from sorted population II (CD71+ Teri 19-) cells from E12.5 FL (WT, n=3; Rpl5+ / - n=6). (B) The heatmap visualizes the variance stabilizing transformation (VST) log-scaled expressions forUM-44851.601all the significant genes unioned from the four types of comparisons (M-high vs. WT, M-low vs. WT, M-all vs. WT, and M-low vs. M-high). (C) Volcano and (D) summary plot showing the comparisons between mutants and wild-types, highlighting the positions of the five significant genes in the M-low vs. M-high comparison. (E) Summary of common genes involved in the significant GO terms across all comparisons of the two RNA-seq experiments. Triangles indicate no significance (FDR q-value > 0.05). (F) Validation of significant genes in population II by RT-qPCR.FIG. 6 shows that steroids inhibit SCD expression in human HSPS cells. (A) CD34+ cells treated with dexamethasone show reduced growth and (B) differentiations on day 7. (C) Quantification of QI and Q2 cells treated with or without steroids. (D) Quantification RT-PCR shows increased SCD at day 11 in no treatment group. (E) Relative SCD expression with or without Dex treatment. (F) Quantification of monounsaturated / saturated fatty acids.DEFINITIONSTo facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.As used herein, the term “subject diagnosed with anemia” refers to a subject who has been tested and found to have anemia. A “subject diagnosed with anemia” includes a subject that is in remission from anemia. As used herein, the term “initial diagnosis” refers to a test result of initial disease that reveals the presence or absence of disease.As used herein, the term “non-human animals” refers to all non-human animals including, but not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, raminants, lagomorphs, porcines, caprines, equines, canines, felines, etc.As used herein, the term “in vitro” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.The terms “test compound” and “candidate compound” refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease,UM-44851.601illness, sickness, or disorder of bodily function (e.g., anemia). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present disclosure.As used herein, the term “sample” is used in its broadest sense. A "biological sample", as used herein, includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such substances include, but are not limited to, blood, (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, cells, organs, tissues, bone marrow, lymph nodes and spleen. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.As used herein, the term “effective amount” refers to the amount of an agent (e.g., an agent described herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not limited to or intended to be limited to a particular formulation or administration route.As used herein, the term “co-administration” refers to the administration of at least two agent(s) (e.g., an agent described herein and an additional agent) or therapies to a subject. In some embodiments, the co-administration of two or more agents / therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents / therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents / therapies are co-administered, the respective agents / therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents / therapies lowers the requisite dosage of a known potentially harmful (e.g., toxic) agent(s).As used herein, the term “pharmaceutical composition” refers to the combination of an active agent or agents with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, or ex vivo.As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants. (See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] ).UM-44851.601DETAILED DESCRIPTION OF THE DISCLOSUREProvided herein are compositions and methods for treating anemia. In particular, provided herein are compositions and methods for blocking SCD1 to treat anemia and / or prevent relapses of anemia.Experiments described herein identified SCD1 as a target for treating anemia such as DBA. In particular, SCD1 inhibitors were found to improve erythropoiesis in an animalmodel of anemia. Accordingly, SCD1 inhibitors find use in the treatment of a variety ofXanemias and related conditions. oooI. Therapies IAny suitable therapy may be utilized to decrease SCD1 activity and / or expression.\ / XExamples include but are not limited to SC g / \ , X#■D1 inhibitors (e.g., pharmaceutical agents) orC -genetic therapies.Inhibitors / / \ / _ \ / ( ) / The present disclosure is not limited to particular agents that inhibit SCD1. In some / _ / \ / embodiments, the agent is a small molecule or mimetic (e.g., steroid mimetic). ExemplaryQ / \ ........ / -■ \ ' / \inhibitors are shown in Table 1 and in Sun et al., Biomedicine & Pharmacotherapy 170(2024) 115586, herein incorporated by reference in its entirety.Table 1Name Structure ReferenceMK-8245 Oballa, et al., J. Med. Chem. 54I P (14) (2011) 5082-5096JL .A A J " OHK "O'Aramchol Tardiff, et al., Mol. Neurobiol. 59(4) (2022) 2171-2189UM-44851.601 A939572 0 H i - || von Roemeling, et al., Clin.Hz-'x .A. A. A. Cancer Res. 19 (9) (2013) 2368- 1 1 X ) * ' I ' 2380xACAY10566 j r Williams, el al., J. Med. Chem. 63\(17) (2020) 9773-9786 o a“ \ / \ / \MF-438 Mohammadzadeh, et al., IUBMB AT FA \ _\~~ ff Life 71 (3) (2019) 340-346 X AT \ / [ <1 A Y I k -• > 1-k •y ,'hk 'N Adx f i1 N-N >-- MF- 152 Li, et al., Bioorg. Med Chem.0 Jj Lett. 19 (17) (2009) 5214-5217 H2N" | \ / \Ai k .4* - w \ — / N - \ f'>_ / CFg\„ / CVT-11127 'Ck Koltun, et al., Bioorg. Med._ 1 1 ' Chem. Lett. 19 (7) (2009) 2048-a. V'’ A 'TICv2052V N’ N ?k' 'scr 1 JHIOUM-44851.601 CVT-12012 Koltun, et al., Bioorg. Med.F f | H Chem. Lett. 19 (15) (2009) 4070- 4074F L Jx0 Y NHO.. JL A Jk.N' Y ''H II0T-3764518 F f Imamura, et al., Bioorg. Med N ‘X z N N x / z\ z > r Chem. 25 (14) (2017) 3768-3779F BZ36 0 Br Fritz, et al., Mol. Cancer Ther. 9A X. (6) (2010) 1740-1754.1 ,J OA X J ' ,10SSI4 H Li et al., Mol. Pharmaceutics ......A<...Y> 2023, 20, 8, 4129-4137- ” » l 1 X 1SSM^SAR707 / > 'X Z' F r Voss, et al., Eur. J. Pharm. 707 / / z — » XX, / G F \ X Z -NZ\ 11 N— / \ / X-F<( N NV. y--t (1-3) (2013) 140-146 z / y\ / SW208108 Theodoropoulos, et al., Nat.I .-... Chem. Biol. 12 (4) (2016) 218-0. I X f li 1 .V 22IxH ’rX'o' 5,NH. f Y'cr'UM-44851.601SW203668 Theodoropoulos, et al., Nat.Chem. Biol. 12 (4) (2016) 218- 225XEN103 F Zhang, et al., J. Med. Chem. 56K I b / / =\ (2) (2013) 568-5831 J / \n A. f V!x bT ” M I ~li ) HN O XEN723 Sun, et al., Bioorg. Med. Chem.f NC" f Y y-< K K X. J Lett. 24 (2) (2014) 520-525 | i i XMTI-301 2- { [4-(2-chlorophenoxy)piperidine- 1 - BHUPATHI et al., Aspet 2025;carbonyl] amino }-N-methylpyri dine-4- April 3-6; Abstract ID: 168237 carboxamideThe present disclosure further provides pharmaceutical compositions (e.g.,comprising the compounds described above). The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluentsUM-44851.601and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.The pharmaceutical formulations of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / or aromatic substances and the like which do not deleteriously interact with the compounds of the formulation.Genetic therapiesIn some embodiments, the expression of SCD1 is reduced or eliminated using genetic therapies (e.g., therapies that decrease expression of SCD1 by removing or modifying genes directly expressing SCD1 or modifying genes that indirectly modify SCD1 expression).Genetic therapies can be performed in vivo or ex vivo. In some embodiments, the present disclosure provides ex vivo modification of a subject’s CD34 cells or in vivo targeting of hematopoietic stem cells to ultimately reduce SCD1 expression in a subject (e.g., in boneUM-44851.601marrow). The present disclosure is not limited to particular genetic therapies. Additional genetic therapies are specifically contemplated.Genetic therapies to reduce expression of SDC1 can utilize any number of gene editing or disruption methods. Examples include but are not limited to, CRISPR / Cas9 (or other CRISPR editing systems), recombination, or expression of an antisense, shRNA, miRNA, or RNAi nucleic acid.Both in vivo and ex vivo genetic therapies utilize viral vectors or other delivery methods for modifying cells (e.g., to decrease expression of SCD1).A number of viruses have been used for human gene therapy, including viruses such as lentivirus, adenoviruses, herpes simplex, vaccinia, and adeno-associated virus.Adenovirus viral vectors (Ad) temporarily modify a cell's genetic expression with genetic material that is not integrated into the host cell's DNA (Bulcha JT, Wang Y, Ma H, Tai PW, Gao G (February 2021). Signal Transduction and Targeted Therapy. 6 (1): 53). As of 2017, such vectors were used in 20% of trials for gene therapy (Ginn SL, Amaya AK, Alexander IE, Edelstein M, Abedi MR (May 2018). The Journal of Gene Medicine. 20 (5): e3015).Lentiviral vectors based on lentivirus, a retrovirus, can modify a cell's nuclear genome to permanently express a gene, although vectors can be modified to prevent integration. Retroviruses were used in 18% of trials before 2018 (Ginn et al. supra).Adeno-associated virus (AAV) is a virus that is incapable of transmission between cells unless the cell is infected by another virus, a helper virus. Adenovirus and the herpes viruses act as helper viruses for AAV. AAV persists within the cell outside of the cell's nuclear genome for an extended period of time through the formation of concatemers mostly organized as episomes (Sabatino DE, Bushman FD, Chandler RJ, Crystal RG, Davidson BL, Dolmetsch R, et al. (August 2022). Molecular Therapy. 30 (8): 2646-2663). Genetic material from AAV vectors is integrated into the host cell's nuclear genome at a low frequency and likely mediated by the DNA-modifying enzymes of the host cell (Sabatino DE, Bushman FD, Chandler RJ, Crystal RG, Davidson BL, Dolmetsch R, et al. (August 2022). Molecular Therapy. 30 (8): 2646-2663).Methods for non- viral gene therapy include the injection of naked DNA, electroporation, the gene gun, sonoporation, magnetofection, the use of oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles. These therapeutics can be administered directly or through scaffold enrichment (Krasilnikova, O.: Yakimova, A.; Ivanov, S.:Atiakshin, D.; Kostin, A. A.; Sosin, D.; Shegay, P.; Kaprin, A.D.; Klabukov, I. (2023).UM-44851.601International Journal of Molecular Sciences. 24 (22): 16250; Foldvari, M.; Chen, D.W.; Nafissi, N.: Calderon, D.; Narsineni, L.; Rafiee, A. (2016). Journal of Controlled Release. 240: 165-190).IL Treatment methodsThe compositions and methods described herein find use in the treatment of a variety of types of anemia. While the present disclosure is exemplified with DBA, the compositions and methods described herein find use in the treatment of any number of anemias. Examples include but are not limited to iron-deficiency anemia, vitamin deficiency anemias (e.g., megaloblastic anemia or pernicious anemia), Fanconi anemia, thalassemia, hemolytic anemia, microcytic anemia, anemia of chronic disease, autoimmune hemolytic anemia, macrocytic anemia, normocytic anemia, aplastic anemia, and sickle cell anemia.In some aspects, administration of SCD1 inhibitors or genetic therapies reduces one or more signs or symptoms of anemia (e.g., by increasing red blood cell levels and / or increasing erythropoiesis). In some embodiments, administration of SCD1 targeted treatment prevents relapse of anemia in subjects that are in remission from anemia.In some embodiments, treatments are administered to subjects with symptoms with anemia and treatment is stopped when symptoms have resolved. In other aspects, treatment is administered indefinitely (e.g., to prevent relapse).The present disclosure is not limited to particular subjects. In some embodiments, the subject is a juvenile (e.g., under the age of 18 years, 17 years, 16 years, 15 years, 14 years, 13 years, 12 years, 11 years, 10 years, 9 years, 8 years, 7 years, 6 years, 5 years, 4 years, 3 years, 2 years, 1 year, 6 months, or 2 months). In some embodiments, the subject is a newborn. In some embodiments, the subject is an adult (e.g., over the age of 18).Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual agents or compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from O.Olpg to 100g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosingUM-44851.601based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state (e.g., relapse of anemia), wherein the agents or compounds are administered in maintenance doses, ranging from 0.01 pg to 100g per kg of body weight, once or more daily, to once every 20 years.Some embodiments of the present disclosure provide methods for administering an effective amount of a compound or therapy of the disclosure and at least one additional therapeutic agent (including, but not limited to, corticosteroids (e.g., prednisone, prednisolone, and dexamethasone), iron supplements, vitamin supplements, and / or leucine) and / or therapeutic technique (e.g., blood transfusions, dietary modification (e.g., low carbohydrate diet) and / or bone marrow transplant).EXPERIMENTALThe following examples arc provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present disclosure and are not to be construed as limiting the scope thereof.Example 1MethodsRpl5ska^-3-Jus / +mjce(MGI: 3046775), also referred to as Rpl5+ / mice, were previously characterized (Yu et al. 2021). Timed matings (Rpl5+ / ~ x wildtype (WT)) were performed to obtain El 2.5 fetal liver (FL) cells and embryo sections were analyzed for the presence of VSD or craniofacial malformation as previously described (Yu et al. 2021). E12.5 FL cells were stained with anti-mouse CD7L Teri 19 antibodies, and CD71 positive / Terl 19 negative cells were sorted to obtain early erythroid progenitor cells (BD FACS ARIA III or Sony MA900 Cell Sorter). Total RNA was extracted using RNeasy® Micro Kit (Qiagen, Cat 74004) and sent for RNA-seq analysis. RNA-seq was performed twice: (1) using pooled embryo FL n=6 WT and 5 MT to generate 3 replicates per group (mRNA analysis) and (2) on 3 non-pooled WT FLs and 6 mutant FLs (total RNA analysis). During the second RNA-seq run, mutants were divided into two groups based on liver cellularity with the hypothesis that mutants with very low cellularity (M-low) were the ones with impending erythroid failure and death, while those with close to normal cellularity (M-high) had a higher chance of spontaneous recovery. Analysis was performed using DESeq2, GO enrichment analysisUM-44851.601described below. Blood was drawn from the lateral saphenous vein and collected in EDTA tubes. Blood counts were obtained using Drew Scientific Hemavet 950FS.SCD-1 inhibitor (CAY 10566) stock was dissolved in DMSO (lOpg / pL) and diluted (1:10) in PBS 30% 2-Hydroxypropyl-|3-cyclodextrin (Sigma Aldrich H107-5G). SCD-1 inhibitor treatment was administered daily via oral gavage in 5 mg / kg doses. Mice treated with SCD-1 inhibitor developed blepharoconjunctivitis and were treated with topical analgesic and antibiotic. Phenylhydrazine (Sigma Aldrich 114715-5g) was dissolved in PBS (10 mg / mE solution) and administered in 5 mg / kg doses. Poly EC HMW VacciGrade (InvivoGen vac-pic) was dissolved in endotoxin-free physiological water and administered in 10 mg / kg doses. Both phenylhydrazine and Poly EC were administered via intraperitoneal injection.ResultsRpl5x:ax-^Jlli / +mice have a heterozygous intronic Rpl5 mutation that leads to Rpl5 haploinsufficiency (Yu et al. 2021). Mutant mice have a small size, kinked tail, macrocytic anemia, and cardiac defect (VSD) at birth. The severe phenotype in this model aligns closely to human DBA patients with RPL5 mutations (Ulirsch et al. 2018). Histological characterization of four litters at E15.5 (WT, n=ll; Rpl5+ / ~, n=13) now revealed that about 20% of Rpl5+ / ' mice (3 out of 13) had a VSD and 30% of Rpl5+ / ~ mice (4 out of 13) showed a lack of palatal fusion. The presence of a VSD did not always correspond to a lack of palatal fusion.Prior work in ppi§skax33~Jas / +mice demonstrated postnatal macrocytic anemia, which worsens in some mice, leading to early mortality after birth, but surviving adult mice and aged mice had no evidence of anemia or bone marrow failure. It was contemplated that the lack of anemia in adult animals might be partly due to the lack of normal hematological challenges (i.e., absence of infections and / or environmental / dietary toxins) in normal mouse husbandry conditions, as seen in Fanconi mouse models, which do not develop bone marrow failure without stress (Garaycoechea and Patel 2014; Walter et al. 2015). Stress was induced with Poly EC, which is a synthetic double-stranded RNA that simulates a viral infection (Figure 1 A). Animals were dosed every other day for 14 days and blood counts were analyzed weekly. It was found that Poly EC induced significant anemia in both WT and Rpl5+ / ~ mice by day 7. However, WT mice started to recover after day 7 despite being administered additional doses of Poly EC, while Rpl5+ / mice slowly had slow recovery to baseline by day 28 (Figure 1B-E). Phenylhydrazine, which causes erythroid and progenitorUM-44851.601cell hemolysis, was next used to induce anemia (Figure 2A). After phenylhydrazine treatment, a bigger nadir in the RBC count and delayed recovery after the 1st and 2nd treatments with phenylhydrazine was observed, indicating that erythropoiesis in adult mutant mice was more severely affected by this hematological challenge (Figure 2B-C). In this experiment, 3 out of 4 male Rpl5+ / ' mice treated with phenylhydrazine died, whereas there were no deaths in other groups. One mouse died on day 21 and another on day 27. Fhe third was found ill-appearing on day 27. Blood was drawn from this mouse on days 27 and 28 and similar RBC (4.23M / pF) and RBC (4.16M / pF) levels were found on days 27 and 28 respectively. This mouse required euthanasia on day 35 after a 5g weight loss from day 21 to day 28, despite having an improvement in RBC (8.43M / pL).Stress erythropoiesis occurs predominantly in the mouse spleen, which generates erythroid output rapidly in response to anemic stress (Paulson et al. 2020). An analysis of peripheral blood counts was performed on days 3-5 (i.e. starting 1 day after 2nd phcnylhydrazinc dose) in order to determine the RBC nadir and ideal time point to assay erythroid progenitors and it was found that the nadir occurred on day 4. When spleen erythroblasts were examined on at day 4, the CD71+Terll9+cells (population 111) in spleen were reduced significantly in Rpl5+ / ' mice (Figure 2D) while this was less significant when assessed at day 7. Total cells, absolute cells / g BW, and % live cells (Figure 2E) showed a statistically significant decline in this progenitor population in all three analyses. There was an increase in the % live CD7F Teri 19‘ population (population I) in Rpl5+ / ~ mice but this was not statistically significant when absolute cell numbers were calculated. Bone marrow erythroid progenitors showed no change after phenylhydrazine treatment. The increase in erythropoiesis during stress recovery comes from erythroid progenitors. Therefore, the early and late erythroid progenitors in the spleen were quantified after phenylhydrazine treatment. In wildtype mice, the number of preMegE, preCFU-E, and CFU-E cells were elevated as early as four days after initial phenylhydrazine treatment (Figure 2F-G), demonstrating efficient regeneration response. While the preMegE and preCFU-E numbers were not significantly impaired in the Rpl5+ / ~ adult spleen in comparison to the WT spleen (Figure 2F-G), the CFU-E population was almost diminished (Figure 2F-G), which may be the cause of delayed production of CD71+Terl 19+stress erythroid progenitors and delayed mature RBC production in the circulation after Poly EC or phenylhy drzine.To identify genes down-regulated in erythroid progenitors, RNA-seq was performed using total RNA extracted from FL erythroid progenitors from single E12.5 embryos, comparing mutant (n=6) and wild-type (WT) mice (n=3). Mutant samples were separated intoUM-44851.6012 groups based on liver cellularity with the hypothesis that mutants with significantly lower cellularity in the erythroid progenitor gate (M-low) were likely to succumb to complete erythroid failure when compared to mutants with higher cell counts (M-high) (Figure 5A). To investigate the potential pathways involved in driving the cellularity differences between mutant groups, DE analysis among four types of comparisons between (1) all mutants and wildtype (M-all vs. WT); (2) mutants with higher cellularity and wildtype (M-high vs. WT); (3) mutants with lower cellularity and wildtype (M-low vs. WT); (4) M-low vs. M-high was performed. 390, 231, and 407 variable genes were identified in the three comparisons to the WT, respectively, where 210 were common (Figure 5B). No significantly dysregulated noncoding RNA was found in this data. Differentially expressed genes were enriched in the GO terms relating to erythrocyte development and oxidative stress. There were five genes (Scdl, ScarnalO, Rpphl, Itga2b, and Hbb-bhl) identified from the M-high vs. M-low comparison (Figure 5C, D). Scdl showed consistently significant down-regulation through all the comparisons here (Figure 5E). Dysregulated genes were validated by RTq-PCR and Scdl downregulation was demonstrated in E12.5 FL mutant erythroid progenitors (Figure 5F). Hbb-bhl was also significantly elevated in M-low but not M-high, which indicates that these two mutant groups have distinct erythroid features.Mice were pretreated with an available SCD1 inhibitor (SCD-i) or vehicle control (DMSO) for 14 days and then phenylhydrazine was administered to all mice on days 0 and 2 (Figure 3A). The mice showed no significant weight loss or change in weekly peripheral blood counts with SCDl-i (Figure 4A-B). There was a slight increase in bone marrow cellularity in WT mice treated with SCDl-i, but spleen cellularity was similar in all groups (Figure 4C). Rpl5+ / ~ mice treated with vehicle control showed a significant decrease in Ilgb and RBC compared with WT whereas SCDl-i treated mice had no or less significant differences in Hgb and RBC, respectively (Figure 3B). Analysis was stopped after day 7. In order to explore the effect of the drug on erythropoiesis, HSPC progenitors were examined by flow cytometry. WT mice treated with SCDl-i showed a significant increase in CFU-E counts in the bone marrow and decreased preCFU-E counts in both bone marrow and spleen, compared to WT mice treated with DMSO (Figure 3C-D). There was no corresponding change in Rpl5+ / ' mice. When later erythroid progenitors were analyzed, improvement in erythropoiesis in WT mice treated with SCDl-i with a significant decrease in early CD71+Terll9+(II) progenitors and an increase in late CD71 Terll9+(IV) progenitors in bone marrow was observed (Figure 3E-F). A corresponding change in spleen erythroid progenitors was not observed. Analysis of terminal erythropoiesis (CD44 vs. FSC) showed aUM-44851.601significant increase in mature bone marrow erythroid cells (IV + V) and a non-significant upward trend in Rpl5+ / mice (Figure 3G-H).Example 2This example demonstrates that steroid treatment leads to SCD downregulation and altered fatty acid ratio during erythropoiesis. To evaluate the role of SCD in human erythropoiesis, in vitro studies were performed using human hematopoietic stem (CD34+) cells using a cell culture system that utilizes steroids (hydrocortisone(HC) 1|1M) from days 0-7. When HC was replaced with dexamethasone IpM (amore potent steroid) in the culture media, there was decreased proliferation and differentiation, whereas complete removal of steroids led to increased expansion and premature differentiation (Fig. 6A-C). SCD mRNA levels were measured at days 7, 11 and 14 of CD34 cell differentiation using 5 different donors. There was a statistically significant decline in SCD levels at day 14 when compared with day 7 with normal culture conditions (HC luM) (Fig. 8D, E). In the cells grown without steroids, SCD levels were higher than the steroid treatment groups at day 11 of differentiation, indicating that steroids are involved in SCD downregulation during erythropoiesis. Free fatty acid ratio in cells was measured via LC-MS (Liquid Chromatography Mass Spectrometry). Human CD7 I CD235A CD34 cells were sorted by FACS (Fluorescence-Activated Cell Sorting) at day 7 of differentiation and 1 x 106cell pellets were rinsed with 150 mM ammonium acetate and snap-frozen in liquid nitrogen. Quantitative lipid analysis was performed and normalized to internal control and identified a decline in the 18:1 / 18:0 ratio ( monounsaturated / saturated fatty acid (MUFA / SFA)) ratio after steroid treatment (Fig. 6F). This is a consequence of Scdl / SCD downregulation, indicating that steroids also have the same downstream on-target effect on cellular fatty acid species.All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled relevant fields are intended to be within the scope of the following claims.

Claims

UM-44851.601CLAIMSWe claim:

1. A method of treating anemia and / or preventing a relapse of anemia, comprising: administering to a subject diagnosed with anemia an agent that inhibits one or more activities of stearoyl-CoA desaturase (SCD1).

2. The method of claim 1 , wherein said agent is a small molecule inhibitor of SCD1.

3. The method of claim 2, wherein said small molecule inhibitor of SCD1 is selected from Aramchol, MK-8245, A939572, CAY10566, MF-438, MI-301, MF-152, CVT-11127, CVT-12012, T-3764518, BZ36, SSI4, SAR707, XEN103, XEN723, SW208108, and SW203668.

4. The method of any one of the preceding claims, wherein said anemia is Diamond Blackfan anemia (DBA).

5. The method of any one of the preceding claims, wherein said subject is under the age of 18.

6. The method of claim 5, wherein said subject is under the age of 12.

7. The method of claims 5 or 6, wherein said subject is a newborn.

8. The method of any one of the preceding claims, wherein said subject has a variant Ribosomal Protein L5 (RPL5) gene or allele.

9. The method of any one of the preceding claims, wherein said subject is in remission from said anemia.

10. The method of any one of the preceding claims, wherein said subject is not in remission from said anemia.UM-44851.60111. The method of any one of the preceding claims, wherein said administration increases erythropoiesis in said subject.

12. The method of any one of the preceding claims, wherein said administration increases red blood cell levels in said subject.

13. The method of any one of the preceding claims, said method further comprises administering an additional therapy or agent to said subject.

14. The method of claim 13, wherein said additional therapy or agent is selected from corticosteroids, leucine, blood transfusions, and allogeneic bone marrow transplant.

15. The use of an agent that inhibits one or more activities of SCD1 to treat anemia and / or prevent the relapse of anemia in a subject.

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