Treatment of sickle cell disease, vaso-occlusive crises-associated diseases, and / or thrombosis with a hemoglobinase enzyme

Abstract

Provided herein are compositions and methods comprising a purified hemoglobinase enzyme, or fragments thereof, and their use to treat Sickle Cell Disease (SCD) and / or one or more diseases associated with vaso-occlusive crises and / or thrombosis.

Description

Docket No. UM-43849.601TREATMENT OF SICKLE CELL DISEASE (SCD) AND / OR DISEASES ASSOCIATED WITH VASO-OCCLUSIVE CRISES AND / OR THROMBOSIS WITH A PURIFIED HEMOGLOBINASE ENZYME RELATED APPLICATION INFORMATION

[0001] The present application claims priority to United States Provisional Patent Application Serial Number 63 / 721,781, filed November 18, 2024, the disclosure of which is herein incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The text of the computer readable sequence listing filed herewith, titled “43849-601 SEQUENCE LISTING '. created November 18, 2025. having a file size of 25,902 bytes, is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0003] Provided herein are compositions and methods comprising a purified hemoglobinase enzyme, or fragments thereof, and their use to treat sickle cell disease (SCD) and / or one or more diseases associated with vaso-occlusive crises and / or thrombosis.BACKGROUND

[0004] Sickle cell disease (SCD) is one of the most common inherited blood disorders in the U.S., affecting around 100,000 people in the country' and millions worldwide. It occurs in about 1 in every 500 African American births and results from a mutation in the (3-globin gene, leading to the substitution of glutamic acid with valine in the [3-globin chain. This mutation causes hemoglobin to polymerize when deoxygenated, leading to sickled erythrocytes. These sickled cells obstruct blood flow, resulting in painful vaso-occlusive crises, chronic multiorgan damage, disability', and premature mortality'. Venous thromboembolism (VTE) and stroke are significant complications in SCD. with VTE affecting a quarter of adult patients and strokes occurring in about 24% of individuals by age 45.

[0005] Current therapies for SCD include hematopoietic cell transplantation, blood transfusions, hydroxyurea, and P-selectin antibodies. While hematopoietic cell transplantation is a therapeutic option, its use is restricted by the need for HLA-matched donors and increased risks for patients with pre-existing conditions, such as pulmonary’ or neurological complications. Hydroxyurea, though a standard treatment, works by raising hemoglobin levels to reduce sickling episodes however, this therapeutic carries long-term risks likeDocket No. UM-43849.601leukemogenesis and carcinogenesis. Similarly, blood transfusions are effective in managing symptoms and preventing some complications, such as anemia, vaso-occlusive crises, and stroke, however, blood transfusions can lead to issues like hemolysis, alloimmunization, infections, and iron overload. Given these limitations, there remains a critical need for safer, more effective, and widely accessible treatments for SCD, especially for patients who do not respond adequately to existing therapies.SUMMARY

[0006] Provided herein are compositions and methods comprising a purified hemoglobinase enzyme, or fragments thereof, and their use to treat sickle cell disease (SCD) and / or one or more diseases associated with vaso-occlusive crises and / or thrombosis.

[0007] In some embodiments, the present disclosure provides methods and compositions (e.g., pharmaceutical compositions) comprising a purified hemoglobinase enzy me, or a fragment thereof. In some embodiments, the purified hemoglobinase has an amino acid sequence that is at least about 50%, 55%, 60%. 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of the respective amino acid sequences of SEQ ID NOs: 1-20. In some embodiments, the purified hemoglobinase enzyme comprises at least one biologically active sub-fragment (e.g., comprising, for example, at least 20, at least 50, at least 100, at least 200. at least 300, at least 400 amino acids) of a hemoglobinase having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of the respective amino acid sequences of SEQ ID NOs: 1-20. In some embodiments, the hemoglobinase enzyme comprises engineered amino acid substitutions or modifications to increase stability, reduce immunogenicity, or enhance catalytic efficiency.

[0008] In some embodiments, the hemoglobinase enzyme is derived from Plasmodium falciparum Plasmepsin-2, Schistosoma japonicum Hemoglobinase (HAEM), or Schistosoma mansoni. In some embodiments of the above compositions and methods, the hemoglobinase enzyme is derived from Plasmodium falciparum Plasmepsin I.

[0009] In some embodiments, the hemoglobinase enzyme is an aspartic protease. In some embodiments, the hemoglobinase enzyme is a serine protease. In some embodiments, the hemoglobinase enzy me is a cysteine protease. In some embodiments, the hemoglobinase enzyme is a metalloprotease protease.Docket No. UM-43849.601

[0010] In some embodiments, the hemoglobinase is recombinantly expressed. In some embodiments, the recombinant hemoglobinase enzyme is expressed in yeast, E. coli, Baculovirus and / or mammalian cell.

[0011] In some embodiments, the recombinantly expressed hemoglobinase, or at least one subfragment thereof, comprises a protein tag at an N- and / or C-terminal.

[0012] In some embodiments, the hemoglobinase is derived from a hematophagous organism. In some embodiments, the hematophagous organism is selected from the group consisting of an insect, a triatomine, a mosquito, a sand fly, an arachnid, a tick, a mite, a hookworm, a mammal, a vampire bat, a lamprey, a plant, and / or a non-human mammal. In some embodiments, the hematophagous organism is a parasite.

[0013] In some embodiments, the hemoglobinase enzyme degrades extracellular hemoglobin in the bloodstream, reducing oxidative stress, nitric oxide scavenging, endothelial activation, and vascular inflammation.

[0014] In some embodiments, the pharmaceutical composition further comprises one or more agents that bind free hemoglobin. Such agents may function to sequester or neutralize extracellular hemoglobin released during hemolysis, thereby preventing its pro-oxidative, pro-inflammatory, and vasoactive effects. In some embodiments, the one or more agents that bind free hemoglobin comprise haptoglobin or hemopexin. Haptoglobin is a plasma glycoprotein that binds free hemoglobin with high affinity' to form a haptoglobin-hemoglobin complex. Hemopexin is a plasma protein that specifically binds free heme derived from hemoglobin degradation, facilitating its clearance through receptor-mediated endocytosis.

[0015] In some embodiments, inclusion of haptoglobin and / or hemopexin in the pharmaceutical composition supports the hemoglobinase enzyme by both degrading and scavenging free hemoglobin and its breakdown products. This combined approach can reduce oxidative stress, inflammation, nitric oxide depletion, and endothelial dysfunction associated with hemolysis and vaso-occlusive events. In some embodiments, haptoglobin and / or hemopexin are naturally derived, recombinant, or synthetically produced. In some embodiments, the hemoglobin-binding agents are formulated in free form, encapsulated form, or conjugated to a delivery vehicle such as a nanoparticle, liposome, hydrogel, or dendrimer.

[0016] In some embodiments, the hemoglobinase is provided in a pharmaceutical composition further comprising a pharmaceutically acceptable salt and / or a pharmaceutically acceptable excipient.Docket No. UM-43849.601

[0017] In some embodiments, the pharmaceutically acceptable excipient is selected from the group consisting of: a buffer, a stabilizer, a diluent, a binder, a vehicle, a filler, a preservative, a surfactant, and any combination thereof.

[0018] In some embodiments, the vehicle is selected from the group consisting of a, micelle, a dendrimer, a hydrogel, a emulsion, a nanoparticle, or a liposome.

[0019] In some embodiments, the nanoparticles or liposomes are made from materials selected from the group consisting of lipids, polymeric substances, and biodegradable materials.

[0020] In some embodiments, the present disclosure provides methods comprising: administering a pharmaceutically acceptable composition comprising a purified hemoglobinase, or fragment thereof, to a subj ect.

[0021] In some embodiments of the above method, the subject has one or more diseases or disorders associated with vaso-occlusive crises and / or thrombosis.

[0022] In some embodiments of the above method, the pharmaceutically acceptable composition reduces free blood plasma hemoglobin.

[0023] In some embodiments of the above method, the administering is selected from the group consisting of Intravenous (IV) administration, Intramuscular (IM) administration, and an Subcutaneous (SC) administration. In some embodiments of the above method, the administering is Intraperitoneal (IP) administration.

[0024] In some embodiments of the above method, the one or more diseases or disorders associated with vaso-occlusive crises and / or thrombosis in a subject is selected from the group consisting of sickle cell disease, deep vein thrombosis, stroke, pulmonary embolism, pulmonary hypertension, pain crisis, myocardial infarction, splenic sequestration, Factor V Leiden mutation, cancer-associated thrombosis, chronic venous insufficiency, atrial fibrillation, disseminated intravascular coagulation, nephrotic syndrome, thrombotic thrombocytopenic purpura, inflammatory bowel disease, varicose veins, peripheral artery disease, antiphospholipid syndrome, acute chest syndrome, splenic sequestration and the like.

[0025] In some embodiments of the above method, the subject is identified as at risk of new or recurrent thrombotic events.

[0026] In some embodiments of the above method, the pharmaceutically acceptable composition is administered in combination and / or sequentially with at least one antithrombotic agent. In some embodiments of the above method, the antithrombotic agent is low-dose aspirin, a direct factor Xa inhibitor, a thrombin inhibitor, a PY12 inhibitor, warfarin, heparin, or a combination thereof. In some embodiments, the pharmaceutical compositionDocket No. UM-43849.601further comprises antioxidants, anti-inflammatory agents, or antithrombotic agents to complement the activity of the hemoglobinase enzyme.

[0027] In some embodiments of the above method, the subject is identified as at risk of new or recurrent vaso-occlusive crises events. In some embodiments, the subject is identified as at risk for new or recurrent vaso-occlusive events or thrombotic complications based on genetic, biochemical, or clinical markers. In some embodiments, the pharmaceutically acceptable composition comprising a purified hemoglobinase. or fragment thereof, is administered prophylactically to a subject. In some embodiments, prophylactic administration is intended to prevent, delay, or reduce the severity or frequency of one or more diseases, disorders, or clinical symptoms associated with vaso-occlusive crises and / or thrombosis. In some embodiments of the above method, the method comprises treating the subject identified as at risk of new or recurrent vaso-occlusive crises events with at least one support therapy. In some embodiments of the above method, the support therapy is an anti -infl ammatory agent, a corticosteroid, fluid replenishment, an intravenous (IV) fluid, a nonsteroidal anti-inflammatory drug (NS AIDs), an opioid, oxygen therapy, a blood transfusion, a disease-modifying therapy, a hydroxyurea, crizanlizumab, voxelotor, or any combination thereof.

[0028] In some embodiments, the present disclosure relates to use of a purified hemoglobinase or a fragment thereof (e.g., in a pharmaceutical composition). In some embodiments the present disclosure relates to the use of a purified hemoglobinase or a fragment thereof (e.g., in a pharmaceutical composition) for the treatment, prevention, or reduction of diseases or disorders associated with vaso-occlusive crises and / or thrombosis in a subject. In some embodiments, the disease or disorder is selected from the group consisting of sickle cell disease, deep vein thrombosis, stroke, pulmonary embolism, pulmonary hypertension, pain crisis, myocardial infarction, splenic sequestration, Factor V Leiden mutation, cancer-associated thrombosis, chronic venous insufficiency, atrial fibrillation, disseminated intravascular coagulation, nephrotic syndrome, thrombotic thrombocytopenic purpura, inflammatory bowel disease, varicose veins, peripheral artety disease, antiphospholipid syndrome, acute chest syndrome, splenic sequestration and the like.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Having thus described the presently disclosed subj ect matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:Docket No. UM-43849.601

[0030] Figure 1 demonstrates the digestion of sickle hemoglobin with Plasmodium falciparum Plasmepsin I (PMI). Both (A) monomer and (B) dimer of hemoglobin S reduced after digestion.

[0031] Figure 2 demonstrates measurements of free hemoglobin. (A) Shows a graph indicating free hemoglobin in sickle cell disease (SCD) plasma and serum. (B) Shows a graph indicating free hemoglobin in SCD plasma after being treated with saline or Plasmodium falciparum Plasmepsin I (PMI).

[0032] Figure 3 demonstrates the effects of PMI treatment on thrombi size in a femoral vein thrombosis model. (A) Shows a schematic diagram of femoral vein thrombosis surgery. (B) Shows a representative images of thrombi size in wild type (WT; left), SCD (center) and SCD + PMI (right) mice. (C) Shows a graph indicating femoral vein thrombi size in WT saline (left), SCD saline (center), SCD PMI (right). * p<0.05, **** p<0.0001 Data were analyzed with one way ANOVA.

[0033] Figure 4 demonstrates the effects of plasma hemoglobin in SCD mice before and after femoral vein thrombosis (FVT) treatment (SCD Pre; left), SCD mice after FVT (SCD FVT; center) and SCD mice after FVT with PMI treatment (SCD FVT PMI; right).

[0034] Figure 5 demonstrates a histological analysis of femoral vein thrombosis in SCD mice. Graphs of IHC staining analysis of femoral vein thrombi for (A) hemoglobin, (B) fibrin / fibrinogen, (C) CD68, and (D) MPO in WT saline (left), SCD saline (center) and SCD PMI (right) mice. * p<0.05, ** p<0.01 Data were analyzed with one way ANOVA.

[0035] Figure 6 demonstrates that PMI treatment reduced infarct area in a stroke model. (A) Shows representative images of brain sections stained with 4% TTC to assess stroke infarct size in SCD mice in WT saline (top left), WT PMI (top right), SCD saline (bottom left) and SCD PMI (bottom right) mice. (B) Stroke size in mice treated with saline or PMI. ** pO.OI, ***p<0.0001 Data were analyzed with on way ANOVA.

[0036] Figure 7 demonstrates femoral vein thrombus from a wild-type or sickle mouse. (A) A schematic diagram of femoral vein thrombosis surgery. (B) Wild type thrombus. Arrow: polyhedrocytes. Scale: 2 pm (C) Sickle thrombus. Arrow: fiber-like structure connecting two sickle RBCs. Arrowhead: elongated sickle RBC. Scale: 2 pm (D) Sickle thrombus. Arrow: a damaged sickle RBC with ruptured membrane. Scale: 2 pm (E) PMI (10 pg / ml) treatment reduced free hemoglobin in SCD mouse plasma. *** p<0.001 Data were analyzed with t-test.(F) Representative pictures of thrombus in a femoral vein thrombosis model. (G) Thrombus length in wild type or SCD mice treated with saline or PMI. n=10 for WT mice treated with saline, n=6 for SCD mice treated with saline. n=7 for SCD mice treated with PMI * p<0.05, **** p<0.0001 Data were analyzed with one way ANOVA.Docket No. UM-43849.601

[0037] Figure 8 shows a morphological study of femoral vein thrombi using H&E staining. (A, scale: 100 pm for the left, 25 pm for the right) and hemoglobin (B. scale: 100 pm for the left, 25 pm for the right). * p<0.05, ** p<0.01 Data were analyzed with one way ANOVA.

[0038] Figure 9 shows sickle hemoglobin involved in coagulation. (A) Free hemoglobin measured in plasma isolated from sickle whole blood sitting for 0 or 2 hours. (B) Free hemoglobin in SCD plasma and serum. (C) Plasma hemoglobin in SCD mice before and after femoral vein thrombosis and PMI treatment. (D) In vitro clotting assay was performed on a stir plate (Left) in a glass tube 1.5 cm above the surface (Middle, the arrow points to the reference line on an adjacent tube and the arrowhead points to the mirror under the tube to show the bar rotation at the bottom) with bar rotation recorded (Right, the arrow points to the mirror image of the stir bar at the bottom of the tube). (E) Addition of sickle hemoglobin shortened clotting time compared with wild type hemoglobin. * p<0.05, ** p<0.01 Data were analyzed with t-test.DEFINITIONS

[0039] Unless otherw ise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0040] The terms “comprise(s),” “include(s),” ‘‘having,” ‘'has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0041] The term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / -15%. + / -10%,Docket No. UM-43849.601+1-5%, + / -4%, + / -3%, +1-2%, and + / -!%. The recitation of numerical ranges by endpoints includes all numbers, e.g.. whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0042] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0043] As used herein, “protein” is used synonymously with “peptide,” “polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein, peptide, or peptide fragment is substantially free of cellular material or other contaminating proteins from the cell, tissue, or cell-free source from which the amino acid sequence is obtained, or substantially free from chemical precursors or other chemicals when chemically synthesized.

[0044] The term “administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g.. pharmaceutical compositions of the present disclosure) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through the eyes (e.g., intraocularly, intravitreally, periocularly, ophthalmic, etc.), intrathecal (within or into the space around the membranes enveloping the spinal cord and brain), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0045] The term “co-administration” refers to the administration of at least two agent(s) (e.g., a purified hemoglobinase) or therapies to a subject. In some embodiments, the coadministration 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).

[0046] The terms “sample,” “test sample,” “specimen,” “sample from a subject.” “biological sample,” and “patient sample” as used interchangeably herein may be a sample of blood, suchDocket No. UM-43849.601as whole blood (including for example, capillary blood, venous blood, dried blood spot, etc.), tissue, urine, saliva, nasal mucus, serum, plasma, amniotic fluid, lower respiratory specimens such as, but not limited to, sputum, endotracheal aspirate or bronchoalveolar lavage, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

[0047] The terms “subject” and “patient” as used herein typically refers to a human, but can also refer to test subjects (e.g., vertebrate animal models such as mice, rats, Guinea pigs, rabbits, pigs, dogs, etc.) or non-human animals that may benefit from treatment by a hemoglobinase (e.g., deer, sheep, fish, etc.)

[0048] The terms “thrombosis” or “thrombotic event” as used interchangeably herein refers to when a blood clot forms in at least one vein, at least one artery7, and / or in at least one chamber of the heart of a subject. Diseases caused by thrombosis include stroke, heart attack, peripheral vascular disease, superficial venous thrombosis, deep vein thrombosis (DVT) and pulmonary embolism.

[0049] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated. Supplementary7active ingredients, such as antibiotics, antifungals, antimicrobials, can also be incorporated into the compositions. In addition, various adjuvants such as are commonly used in the art may be included. These and other such compounds are described in the literature, e.g., in the Merck Index, Merck & Company, Rahway, N.J. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (2006); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 11th Ed., The McGraw-Hill Companies.

[0050] By “therapeutically effective amount” or “pharmaceutically effective amount” is typically one which is sufficient to achieve the desired effect and may vary7according to the nature and severity of the disease condition, the nature of the subject, and the potency of the composition. It will be appreciated that different concentrations may be employed forDocket No. UM-43849.601prophylaxis than for treatment of an active disease. This amount can further depend upon the patient's height, weight, sex, age, and medical history.

[0051] A therapeutic effect relieves, to some extent, one or more of the symptoms of the disease, and includes curing a disease. “Curing” means that the symptoms of active disease are eliminated. However, certain long-term or permanent effects of the disease may exist even after a cure is obtained (such as tissue damage). The terms “treat,” “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and / or injury, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a pharmaceutical composition to a subject that is not at the time of administration afflicted with the disease. “Preventing” also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease. “Treatment” and “therapeutically,” refer to the act of treating, as “treating” is defined above.

[0052] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.DETAILED DESCRIPTION

[0053] Provided herein are compositions and methods comprising a purified hemoglobinase enzyme, or fragments thereof, and their use to treat sickle cell disease (SCD) and / or one or more diseases associated with vaso-occlusive crises and / or thrombosis.

[0054] Sickle cell disease (SCD) is characterized by hemolytic anemia and widespread vasoocclusive events12. Deoxygenated sickle hemoglobin leads to formation of insoluble fibers causing erythrocytes to become rigid and assume a “sickled” shape13. Subsequent trapping ofDocket No. UM-43849.601erythrocytes in microvasculature leads to tissue hypoxia and premature lysis of erythrocytes. Release of free hemoglobin from lysed erythrocytes may contribute to vascular comorbidities in SCD14. In the setting of low oxygen content following vaso-occlusion, local high concentrations of free hemoglobin may be particularly toxic, leading to propagation of the vascular occlusion14. The low oxygen content of the venous system may predispose to erythrocyte sickling which may then accelerate further following reduction of blood flow. The oxygen content at the site of a venous thrombus in a mouse model is reduced from 78 to 8 mmHg in thrombi analyzed 24 hours after occlusion13. The incidence of deep venous thrombosis (DVT), recurrent DVT, and pulmonary emboli are higher in SCD patients compared to the general population16, although the specific contribution of SCD to venous thrombogenesis remains unclear.

[0055] Potential risk factors for venous thromboembolism (VTE) in SCD include enhanced platelet function17, coagulation cascade activation18, and impaired fibrinolysis19. Increased circulating free hemoglobin due to intravascular hemolysis may also contribute to vasoconstriction and platelet activation20-21. While antiplatelet and anticoagulant therapies have been evaluated for the treatment of sickle cell disease (SCD)22, these interventions have not provided clear or reproducible clinical benefit, highlighting the need for alternative approaches.

[0056] Under hypoxic conditions, polymerization of sickle hemoglobin alters the morphology and structural integrity of red blood cells (RBCs), resulting in intravascular hemolysis and extemalization of phosphatidylserine (PS), a highly procoagulant phospholipid, on the RBC membrane23. The resulting sickled RBCs exhibit decreased deformability, which can reduce fibrin network permeability and increase the resistance of clots to fibrinolysis24. Entrapment of sickle RBCs within thrombi has been reported to be mediated by the platelet-free cellular fraction of sickle blood23. In addition, cellular components other than PS have been shown to promote thrombin generation in sickle cell disease (SCD)25.

[0057] Experiments were conducted during the development of the present technology to investigate the role of SCD in venous thrombosis and to evaluate potential therapeutic interventions such as the role of treatment targeting free hemoglobin in SCD using Plasmepsin, a hemoglobinase.

[0058] The present technology overcomes the limitations and failures of prior methodologies through the use of, for example, compositions and methods comprising a purified hemoglobinase enzyme, or fragments thereof, and their use to treat sickle cell disease (SCD) and / or one or more diseases associated with vaso-occlusive crises and / or thrombosis. Experiments conducted during the development of the present technology demonstrated thatDocket No. UM-43849.601hemoglobinase can be utilized to treat vascular complications related to SCD. The hemoglobinase evaluated in these studies was recombinant Plasmodium falciparum plasmepsin I (PMI). Hemoglobinases derived from other biological sources, including ticks and hookworms, were also indicated to exhibit similar activity.

[0059] The present technology' found that, in a murine stroke model, intraperitoneal administration of the hemoglobinase (PMI) resulted in a reduction of brain infarct size in SCD mice by approximately 50% compared with SCD mice receiving vehicle control, although infarct size remained greater than in wild-type control mice. In a murine femoral deep vein thrombosis model, intraperitoneal administration of the hemoglobinase (PMI) reduced clot size in SCD mice by approximately 30% compared with SCD mice treated with vehicle control. The present invention demonstrates that enzymatic degradation of free or sickle hemoglobin by hemoglobinase represents a therapeutic strategy' for mitigating thrombotic and vasoocclusive complications associated with SCD.COMPOSITIONS

[0060] Embodiments of the disclosure relate to compositions (e.g., pharmaceutical compositions) comprising a purified hemoglobinase or fragments thereof (e.g.. fragments that are enzymatic active) and their use to treat sickle cell disease (SCD) and / or one or more diseases associated with vaso-occlusive crises and / or thrombosis.

[0061] Sickle cell disease (SCD) is a hereditary' hemoglobin disorder characterized by the production of abnormal hemoglobin, known as sickle hemoglobin or hemoglobin S. This abnormal hemoglobin results from a single amino acid substitution in the [3-globin chain of normal adult hemoglobin (hemoglobin A). Under low oxygen tension or other stress conditions, sickle hemoglobin polymerizes, causing red blood cells (RBCs) to assume a rigid, sickle-like shape. These misshapen cells have reduced deformability and an increased tendency to adhere to the vascular endothelium and to one another, resulting in impaired blood flow and increased hemolysis.

[0062] SCD primarily affects individuals with genetic inheritance of tw o abnormal P-globin genes (homozygous HbS), although compound heterozy gous states involving other abnormal hemoglobins (e.g., HbSC, HbSp-thalassemia) can also cause disease manifestations. In some embodiments, clinical manifestations include chronic anemia, recunent pain episodes, organ damage, and increased risk of stroke and infection.

[0063] A major complication of SCD is vaso-occlusive crisis (VOC), a condition in which sickled RBCs obstruct small blood vessels, restricting blood flow and oxygen delivery toDocket No. UM-43849.601tissues. This vascular blockage causes severe pain and may lead to ischemic tissue injury. Repeated VOC events contribute to cumulative organ damage over time. In some embodiments, in addition to vaso-occlusion, patients with SCD exhibit an increased risk of venous and arterial thrombosis, including deep vein thrombosis (DVT), pulmonary embolism, and stroke. These thrombotic complications arise in part from hemolysis, endothelial activation, exposure of phosphatidylserine on RBC membranes, and the release of free hemoglobin into plasma, which promotes oxidative stress and nitric oxide scavenging.

[0064] Hemoglobinases are proteolytic enzymes capable of degrading hemoglobin into smaller peptides and amino acids. Hemoglobinases can be obtained from or derived from any of a wide variety of organisms. In some embodiments, the hemoglobinase is derived from a hematophagous organism. In some embodiments, the hematophagous organism is an insect, a triatomine, a mosquito, a sand fly, an arachnid, a tick, a mite, a hookworm, a mammal, a vampire bat, a lamprey, a plant, and / or a non-human mammal. In some embodiments, the hematophagous organism is a parasite. In some embodiments, the parasite is a Haemaphysalis longicomis. In some embodiments, the parasite is a Rhipicephalus (Boophilus) microplus. In some embodiments, the parasite is a Ixodes ricinus. In some embodiments, the triatomine is a Rhodnius prolixus. In some embodiments, the hematophagous organism is a dipteran. In some embodiments, the triatomine is a Glossina morsitans. In some embodiments, the hemoglobinase enzyme is derived from Plasmodium falciparum Plasmepsin-2, Schistosoma japonicum Hemoglobinase (HAEM), or Schistosoma mansoni. In some embodiments, the hemoglobinase enzyme is derived from Plasmodium falciparum Plasmepsin I. In some embodiments, the hemoglobinase is recombinantly expressed.

[0065] In some embodiments, the hemoglobinase enzyme is a protease capable of cleaving hemoglobin or hemoglobin-derived peptides. The hemoglobinase enzyme may belong to one or more classes of proteolytic enzymes, including aspartic proteases, serine proteases, cysteine proteases, or metalloproteases, depending on the nature of the catalytic residue and reaction mechanism. Aspartic proteases utilize aspartic acid residues in the active site and are typically active under acidic conditions. In some embodiments, the hemoglobinase enzyme is an aspartic protease. In some embodiments, the aspartic protease is Cathepsin D. Serine proteases employ a serine residue for peptide bond hydrolysis and may function under neutral to slightly basic conditions. In some embodiments, the hemoglobinase enzyme is a serine protease. Cysteine proteases employ a cysteine residue as the catalytic nucleophile and may contribute to hemoglobin degradation under physiological or lysosomal conditions. In some embodiments, the hemoglobinase enzyme is a cysteine protease. In some embodiments, the cysteine proteaseDocket No. UM-43849.601is a Cathepsin B. In some embodiments, the cysteine protease is a Cathepsin C. In some embodiments, the cysteine protease is a Cathepsin L. In some embodiments, the cysteine protease is a Legumain. In some embodiments, the hemoglobinase enzyme is a metalloprotease protease. In some embodiments, hemoglobinase enzyme is a protease. In some embodiments, the protease is BmCl 1. In some embodiments, the protease is RmLCE. In some embodiments, the protease is VIDCE. In some embodiments, the protease is IrCC. In some embodiments, the protease is IrCDl. In some embodiments, the protease is BYC.

[0066] In some embodiments, the composition comprises a hemoglobinase of one of SEQ ID Nos: 1-20 or variants or fragments thereof. For example, in some embodiments, the composition comprises a purified hemoglobinase enzy me having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%. 75%. 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of the respective amino acid sequences of SEQ ID NOs: 1-20, or a fragment thereof. In some embodiments, the composition comprises a variant of a natural hemoglobinase. Such engineered hemoglobinases may comprise one or more (e.g., 1, 2, 3, 4. 5, 6, 7, 8, 9. 10, 15, 20, 25, or more, etc.) amino acid substitutions as compared to starting sequences (e.g.. SEQ ID NOs: 1-20). An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic”. An aromatic amino acid includes an aromatic ring. Examples of aromatic amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly grouped as aliphatic. Examples of aliphatic amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr). cysteine (C or Cys). proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).

[0067] The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other and therefore resembleDocket No. UM-43849.601each other most in their impact on the overall protein structure (Schulz and Schirmer). Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free — OH can be maintained, and glutamine for asparagine such that a free — NH2 can be maintained. “Semiconservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same sub-group. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. "Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.

[0068] In some embodiments, hemoglobinase enzy mes digests free hemoglobin that accumulates in the plasma during hemolysis. By degrading extracellular hemoglobin, these enzymes reduce oxidative stress, diminish endothelial activation, and mitigate downstream thrombotic and vaso-occlusive complications.

[0069] In some embodiments, compositions comprising hemoglobinase enzymes or active fragments thereof are utilized to reduce pathological effects associated with sickle hemoglobin and free hemoglobin in sickle cell disease (SCD) and related vascular disorders. In some embodiments, vascular diseases include, but are not limited to, sickle cell disease, thalassemia, hemolytic anemia, ischemia-reperfusion injury, and other conditions associated with hemolysis or vascular dysfunction.

[0070] In some embodiments, the composition may further include one or more agents that bind free hemoglobin, which can enhance clearance or neutralization of hemoglobin and limit its deleterious effects. Such agents may include natural (endogenous) proteins, such as haptoglobin and hemopexin, which bind free hemoglobin and heme, respectively, facilitating their removal from circulation. In some embodiments, the compositions may also include recombinantly produced or modified forms of haptoglobin or hemopexin, or synthetic molecules and antibodies capable of selectively binding free hemoglobin, thereby reducing oxidative damage, inflammation, and vascular complications.

[0071] In some embodiments, the co-administration of hemoglobinase enzymes with hemoglobin-binding agents provides a synergistic therapeutic effect, where the enzyme degrades free hemoglobin while the binding agents neutralize and remove residual hemoglobin, further protecting the vasculature and reducing the risk of thrombotic or vaso-occlusive events.Docket No. UM-43849.601

[0072] In some embodiments, the composition is formulated to stabilize the one or more hemoglobinase enzymes, e.g., to preserve the enz matic activity of the enzymes.

[0073] In some embodiments, the composition is a pharmaceutical composition comprising a hemoglobinase and a pharmaceutically acceptable salt or excipient (e.g., a buffer, a stabilizer, a diluent, a binder, a vehicle, a filler, a preservative, a surfactant, and any combination thereof).

[0074] Stabilization techniques and / or vehicles can limit or prevent auto-degradation of the one or more enzymes in a composition and help maintain enzymatic activity, increase shelflife, and aid in the tolerance of the activity of the compositions to changes in temperature, humidity, and storage conditions.

[0075] In some embodiments, the pharmaceutical composition comprises a carrier that protects the hemoglobinase from inactivation or degradation in vivo and / or reduces or prevents undesired interactions with the subject’s immune system.

[0076] In some embodiments, the composition includes a targeting component that causes binding or association with target tissue or molecules so as to increase local concentration of the hemoglobinase at or near its intended target. For example, in some embodiments, a clottargeting or thrombus-targeting agent (e.g., a fibrin-specific targeting agent) is included in the composition. Fibrin-specific agents include alteplase (tPA), reteplase (recombinant plasminogen activator [r-PA]), and tenecteplase. Antibodies and / or antibody fragments may also be used as targeting components (e.g., anti-fibrin antibodies).

[0077] In some embodiments, the one or more enzymes in the composition are encapsulated in or attached to a vehicle such as a micelle, a dendrimer, a hydrogel, a emulsion, a nanoparticle or a liposome. In some embodiments, the nanoparticles or liposomes are made from materials selected from the group consisting of lipids, polymeric substances, and biodegradable materials.

[0078] In other embodiments, variations in excipients, pH, enzyme inhibitors, etc. are employed to aid in stabilizing the enzymes. Appropriate stabilization techniques and / or vehicles will depend on the intended application for the composition, the route of administration, the form of the composition, the intended site of delivery / activity, and other factors, and can be determined by those having ordinary skill in the art.METHODS OF ADMINISTERING THE PHARMACEUTICALLY ACCEPTABLE COMPOSITION TO A SUBJECT

[0079] Embodiments of the present disclosure provide a method comprising: administering the pharmaceutical composition, as described herein, to a subject.Docket No. UM-43849.601

[0080] In some embodiments, the subject has one or more diseases or disorders associated with vaso-occlusive crises and / or thrombosis. Such diseases are ty pically characterized by abnormal blood flow, vessel obstruction, or clot formation leading to tissue ischemia, inflammation, and organ dysfunction. In some embodiments, the disorder is sickle cell disease (SCD), a hereditary hemoglobinopathy in which sickled red blood cells promote hemolysis, vascular occlusion, and recurrent vaso-occlusive crises. In some embodiments, the disorder is thalassemia, paroxysmal nocturnal hemoglobinuria (PNH), or another hemolytic anemia, in which intravascular hemolysis releases free hemoglobin that contributes to endothelial dysfunction and thrombosis.

[0081] In some embodiments, the subject has a thrombotic disorder including, but not limited to, deep vein thrombosis (DVT), pulmonary embolism (PE), stroke, myocardial infarction, or microvascular thrombosis associated with inflammation or infection. In some embodiments, the disorder is a vaso-occlusive or ischemic condition such as peripheral artery disease, retinopathy, or renal vaso-occlusion.

[0082] Diagnosis of such diseases and disorders may include one or more laboratory7, imaging, or clinical assessments. In some embodiments, diagnosis is established based on hematological testing (e.g., complete blood count, reticulocyte count, hemoglobin electrophoresis, or genetic testing for hemoglobin variants). In some embodiments, biochemical markers such as plasma-free hemoglobin, lactate dehydrogenase (LDH), bilirubin, or haptoglobin levels are used to confirm hemolysis. In other embodiments, coagulation tests (e.g., D-dimer, fibrinogen, or platelet count) or vascular imaging (e.g., Doppler ultrasound, MRI angiography, or CT angiography) are employed to detect thrombotic or vaso-occlusive events.

[0083] In some embodiments, the subject may also present with clinical symptoms such as pain crises, fatigue, shortness of breath, swelling of extremities, neurological deficits, or organspecific ischemic injury, which guide diagnosis and treatment decisions.

[0084] The 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 or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), 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, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g.. intrathecal or intraventricular, administration. Pharmaceutical compositions and formulations for topical administration may include personal lubricants.Docket No. UM-43849.601transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. 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, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0085] 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.

[0086] 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.

[0087] 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 nucleic acid(s) of the formulation.Docket No. UM-43849.601

[0088] In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, specific protein stabilizers (e.g., polysorbate 20 or 80), anti-adsorption agents (e.g., polyethylene glycol), pH buffering agents, antioxidants (e.g., methionine), and solubility enhancers, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, amino acids (e.g., glycine, arginine), and sugars or polyols (e.g., sucrose, mannitol).

[0089] Injectables are designed for local and systemic administration. In one aspect, a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1 % w / w up to about 90% w / w or more, in certain embodiments more than 1% w / w of the hemoglobinase enzymes to the treated regions / tissue(s).

[0090] In some embodiments, a therapeutically effective amount of pharmaceutically acceptable composition to a subject is from 0.01 to 1000 mg / kg. In other embodiments, a therapeutically effective amount of pharmaceutically acceptable composition to a subject is from 0.01 to 100 mg / kg. In some embodiments, a therapeutically effective amount of pharmaceutically acceptable composition to a subject is from 1 to 20 mg / kg (e.g., 1, 2, 3. 4, 5, 6, 7, 8, 9, 10. 11. 12. 13. 14. 15, 16, 17, 18, 19, 20 mg / kg).METHODS OF TREATMENT

[0091] Pharmaceutical compositions described previously can be used to treat, prevent, or reduce the severity of one or more diseases or disorders associated with vaso-occlusive crises and / or thrombosis in a subject. In some embodiments, the disease or disorder is selected from the group consisting of sickle cell disease, deep vein thrombosis, stroke, pulmonary' embolism, pulmonary hypertension, pain crisis, myocardial infarction, splenic sequestration, Factor V Leiden mutation, cancer-associated thrombosis, chronic venous insufficiency, atrial fibrillation, disseminated intravascular coagulation, nephrotic syndrome, thrombotic thrombocytopenic purpura, inflammatory bowel disease, varicose veins, peripheral artery disease, antiphospholipid syndrome, acute chest syndrome, and splenic sequestration.

[0092] In some embodiments, the subject is identified as at risk of new or recurrent thrombotic events. Identification of at-risk subjects can be based on clinical history, laboratory testing, genetic predisposition, or biomarkers indicative of hemolysis, endothelial dysfunction, or hypercoagulability7. In some embodiments, the subject may present with elevated levels of plasma-free hemoglobin, D-dimer, fibrinogen, C-reactive protein, or other indicators of oxidative or thrombotic stress. In some embodiments, risk is determined based on genetic testing for mutations associated with coagulation abnormalities (e.g.. Factor V Leiden,Docket No. UM-43849.601prothrombin G20210A, or MTHFR variants), or by clinical features such as recurrent vasoocclusive pain, previous thrombotic episodes, or organ ischemia.

[0093] In some embodiments, the compositions may be administered prophylactically to prevent disease onset or recurrence, or therapeutically to alleviate symptoms, reduce the extent of vascular occlusion, or mitigate complications resulting from hemolysis and thrombosis.

[0094] In other embodiments, the pharmaceutically acceptable composition is administered in combination and / or sequentially with at least one antithrombotic agent. In some embodiments, the antithrombotic agents is low-dose aspirin, a direct factor Xa inhibitor, a thrombin inhibitor, a PY12 inhibitor, warfarin, heparin, or a combination thereof. In some embodiments, the antithrombotic agent is a low-dose aspirin. In some embodiments, the antithrombotic agent is a direct factor Xa inhibitors. In some embodiments, the antithrombotic agent is a thrombin inhibitors. In some embodiments, the antithrombotic agent is a PY12 inhibitors. In some embodiments, the antithrombotic agent is a warfarin. In some embodiments, the antithrombotic agent is a heparin.

[0095] In another embodiment, the subject is identified as at risk of new or recurrent vasoocclusive cases events. Such subjects may include individuals with sickle cell disease or other hemolytic or vascular disorders who exhibit clinical, genetic, or biochemical markers associated with vaso-occlusive complications. In some embodiments, the method comprises treating or preventing vaso-occlusive crises in the subject with at least one supportive or adjunctive therapy alone, in combination with, or in addition to, administration of a hemoglobinase enzyme composition. In some embodiments, the support therapy is an antiinflammatory agent, a corticosteroid, fluid replenishment, an intravenous (IV) fluid, a nonsteroidal anti-inflammatory drug (NSAIDs), an opioid, oxygen therapy, a blood transfusion, a disease-modifying therapy, a hydroxyurea, crizanlizumab, voxelotor, or any combination thereof. In some embodiments, anti-inflammatory agents or corticosteroids are used to reduce vascular inflammation and endothelial activation associated with vaso-occlusive events. In some embodiments, fluid replenishment or IV fluid therapy is used to maintain blood volume and reduce erythrocyte sickling by improving circulation and hydration. In some embodiments, NSAIDs or opioids are used for pain management during acute crises. In certain embodiments, oxygen therapy may be provided to correct hypoxia and minimize further sickling of red blood cells. In some embodiments, blood transfusions may be administered to restore oxygen-carrying capacity and reduce circulating sickled cells. In some embodiments, the support therapy is a disease-modifying therapy. In certain embodiments, the supportive therapy may further comprise administration of hydroxyurea, crizanlizumab, or voxelotor, orDocket No. UM-43849.601any combination thereof. In some embodiments, disease-modifying therapies such as hydroxyurea, crizanlizumab, and voxelotor are administered to reduce the frequency and severity of vaso-occlusive crises, improve hemoglobin function, and decrease hemolysis. Hydroxyurea may act by increasing fetal hemoglobin levels, crizanlizumab by inhibiting P-selectin-mediated cell adhesion, and voxelotor by enhancing hemoglobin oxygen affinity and reducing polymerization of sickle hemoglobin.

[0096] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable and may be made using suitable equivalents without departing from the scope of the present disclosure or the embodiments and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some embodiments and embodiments of the present disclosure and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.EXAMPLES

[0097] The present disclosure has multiple embodiments, illustrated by the non-limiting examples as described herein.

[0098] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.

[0099] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.Example 1Materials and Methods

[0100] Animals. Male C57BL / 6J (wild-type,stock #000664), and homozygous humanized sickle cell disease (SCD) mice (SCD, stock #013071, Townes model) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). All animal use protocols complied with the Principle of Laboratory and Animal Care established by the National SocietyDocket No. UM-43849.601for Medical Research and were approved by the University of Michigan Institutional Committee on Use and Care of Animals.

[0101] Bone marrow transplantation. To generate SCD and control wild type mice on a homogeneous genetic background, bone marrow transplantation (BMT) was used. 8 week-old male WT mice were used as recipients that received bone marrow from 8 week-old SCD or wild type male donors. Bone marrow was harvested from the donor mice by flushing their femurs and tibias with RPMI medium (Gibco / Invitrogen, Carlsbad, CA. USA) containing 10% fetal bovine serum (Gibco / Invitrogen, Carlsbad, CA, USA). Cells were then centrifuged at 300g and resuspended in phosphate-buffered saline before injection. Each recipient mouse was irradiated (2x650 rad [0.02x6.5 Gy]) and injected with 4xl06bone marrow cells via the tail vein in a 200 pL bone marrow suspension in phosphate- buffered saline. Acid water (6 mM HC1, pH=2.5) was provided to animals beginning 4 days before BMT and continued for 4 eeks following BMT. 10 weeks following BMT, the phenotype was confirmed with more than 95% hemoglobin S measured with hemoglobin electrophoresis as previously described11.

[0102] Femoral vein thrombosis model. SCD and WT mice were anesthetized with isoflurane. A 1 centimeter (cm) skin incision w as made on the ventral aspect of the left leg. The left femoral vein was exposed, and the adjacent femoral artery separated by blunt dissection. The femoral vein w as then ligated 5 millimeter (mm) proximal to the superficial inferior epigastric vein. The superficial inferior epigastric vein and visible side branches were ligated with 6.0 suture, and the skin incision was closed with surgical staples. Mice were euthanized for three days following the procedure at which time the femoral vein segment containing visible thrombus w as removed and measured for length.

[0103] Histological analysis of femoral vein thrombus. Samples were washed with 0.1 M sodium cacodylate buffer 15 minutes x 3, treated with 2% OsCL for 2 hours and washed with water three times for 15 minutes, then dehydrated with 30%, 50%, 70%, 90%, 95% and 100% ethanol. The samples were then dried using a critical point dryer (Leica CPD 300) and coated with 10 nm gold before scanning electron microscopy (SEM) imaging (Zeiss EVO 15 SEM).

[0104] Femoral vein thrombi from WT or SCD mice were fixed in formalin, sectioned and stained with hematoxylin / eosin and antibodies to hemoglobin (Abeam, Waltham, MA), fibrin / fibrinogen, C68, and MPO followed by detection of the biotin-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TEXAS BD Biosciences, San Jose, CA) wtith AEC substrate kit (Vector Laboratories, Newark, CA). Negative controls consisted of tissues handled identically to experimental samples except that the primary antibody was omitted. The detection system was streptavidin-HRP, and endogenous peroxidase was quenched withDocket No. UM-43849.601hydrogen peroxide. All images were analyzed by automated detection of positive stained areas using Nikon MetaMorph software.

[0105] Hemoglobin purification. Whole blood from WT or SCD mice was drawn from the retro-orbital venous plexus into ethylenediamine tetra-acetic acid (EDTA)-lined polyethylene tubes (BD. Ref: 365974). After washing 3 times with saline and centrifugation at 1000 rpm for 10 minutes, cells were lysed with three freezing thaw cycles. The cell lysates were dialyzed against saline with a Pur-A-Lyzer Midi 1000 Dialysis Kit (Sigma) for hemoglobin purification. The protein concentration was measured with a Pierce BCA Protein Assay Kit (Thermo Scientific).

[0106] In vitro clotting assay. For clotting analysis, all samples were tested with whole blood withdrawn from the same WT mouse by cardiac puncture. Blood was drawn into an EDTA-lined polyethylene tube (BD, Ref: 365974) and then transferred to a centrifuge tube kept on a shaker. In a glass cuvette (Chrono-Log P / N 312) with a stir bar (Chrono-Log P / N 311) held 1.5 cm above a stir plate (Coming, Model PC-520) 100 pl whole blood was spiked with 10 pl purified wild type or sickle hemoglobin at a concentration of 40 mg / ml and then stirred for 2 minutes before 2 pl 0.5 M CaCE was added. The rotation of the stir bar on the bottom was videotaped. The time from CaCh addition to stir bar stoppage was recorded as clotting time.

[0107] Plasmepsin treatment. Recombinant Plasmodium falciparum Plasmepsin I (10 ug / mouse / day); CUSABIO Technology LLC, Houston, TX) or Saline was injected daily (intraperitoneal) into SCD and WT mice following femoral vein ligation. For the stroke model, SCD and WT mice were treated with saline or 10 pg daily Plasmepsin I IP for two weeks and then three days following induction of stroke. The first dose was administered immediately after the surgery.

[0108] Stroke model. Stroke was induced by photochemical -mediated injury to the middle cerebral artery (MCA) in SCD or WT mice anesthetized with isoflourane. Rose Bengal (50 mg / kg) was injected via the tail vein while a 1-5-mW green light laser (540 nm) was applied to the MCA for 30 minutes to induce thrombotic occlusion. The temporal muscle and skin were then sutured back in place. Three days later the brains were harvested, sectioned, and stained with 2% 2, 3. 5-triphenyltetrazolium chloride (TTC) for infarct area measurement.

[0109] Measurement of cell-free hemoglobin. Blood samples were withdrawn from the retro-orbital venous plexus into ethylenediamine tetra-acetic acid (EDTA)-lined polythene tubes (BD) to collect plasma or silica and gel containing tubes (BD) to collect serum. In vitro and in vivo serum and plasma hemoglobin levels were measured with a Human Hemoglobin ELISA Kit (Fisher Scientific) following manufacturer’s instruction.Docket No. UM-43849.601

[0110] Digestion of sickle hemoglobin with Recombinant Plasmodium falciparum Plasmepsin I (PMI). Sickle red blood cells were collected from SCD mice from whole blood by centrifugation. Sickle hemoglobin was prepared by lysing sickle red blood cells with three freeze / thaw cycles followed by centrifugation (10000 rpm for 30 min) to discard cell debris. The protein concentration was measured with BCA protein assay kit (Thermo Scientific) and adjusted to 1 mg / ml with saline. For hemoglobin digestion. 10 pg hemoglobin was incubated with saline or PMI (16.7 or 167 ug / ml in acidic buffer at pH 4.0) at 37 °C for 1 hour. After digestion, hemoglobin was mixed with reducing 4X Laemmli sample buffer (Bio-Rad) and incubated at 95°C for 10 minutes before loading on a 8-16% PAGE gel (SurePAGE, Bis-Tris, GenScript) and run with Tris-MOPS-SDS running buffer (GenScript). Bands of hemoglobin were detected with Recombinant Anti-Hemoglobin subunit alpha antibody (Abeam) and Donkey anti-Rabbit secondary antibody (Li-COR). PMI at both concentrations digested sickle hemoglobin.Example 2Free hemoglobin consumption during coagulation.

[0111] Whole blood was collected by retro-orbital bleeding from Townes humanized sickle cell mice (Strain #:013071, Jackson laboratory) and divided into two groups to collect serum or plasma, respectively. Free hemoglobin in serum or plasma was measured with Human Hemoglobin ELISA Kit (Fisher) following manufacturer’s instruction. Free hemoglobin concentration was reduced significantly in serum compared with plasma indicating sickle free hemoglobin was integrated into thrombus during coagulation (FIG. 2A). To confirm the ability of PMI to digest sickle hemoglobin in physiological conditions, PMI was spiked into sickle plasma (10 ug / ml) and incubated at 37°C for 1 hour. Free hemoglobin concentration was reduced significantly after PMI digestion (FIG. 2B).Example 3Effect of PMI on femoral vein thrombosis.

[0112] Mice were anesthetized with isoflurane. A small opening (around 1cm in length) of skin was made on left leg and the left femoral vein was exposed by blunt dissection. The accompanying femoral artery’ was carefully separated, and the femoral vein was 5 mm proximal to the superficial inferior epigastric vein. The superficial inferior epigastric vein and other side branches were also ligated. The skin incision was closed with surgical staples. The mice were treated with saline or 10 pg daily recombinant plasmodium falciparum plasmepsin I (PMI)Docket No. UM-43849.601intraperitoneal (IP.) injections for 3 days before euthanization. The first dose was administered immediately after surgery. Thrombi were then removed and measured for length.Example 4Free plasma hemoglobin in sickle mice after femoral vein thrombosis and PMI treatment.

[0113] Free hemoglobin was measured in sickle mice plasma after femoral vein thrombosis and PMI treatment. Consistent with results of in vitro experiments, free hemoglobin was reduced significantly after thrombosis and even further after PMI treatment.Example 5Histological analysis of femoral vein thrombus.

[0114] WT thrombi that were partially occupied by red blood cells (RBCs), RBCs occupied most of the SCD thrombus (FIG. 8A). Hemoglobin immunostaining was also increased in SCD thrombus, indicating more hemoglobin integrated into thrombi in SCD mice. Following PMI treatment, hemoglobin immunostaining was reduced in SCD thrombi (FIG. 8B).

[0115] The apparent higher thrombus hemoglobin content in SCD thrombi along with larger thrombus burden that is reduced with PMI indicates a prothrombotic contribution of sickle hemoglobin towards venous thrombosis in SCD.

[0116] Femoral vein thrombi from WT or sickle mice were fixed, sectioned, and stained for hemoglobin, fibrin / fibrinogen, C68, and MPO. Compared with WT thrombi, hemoglobin content increased in sickle thrombi, indicating more hemoglobin integrated into thrombi in sickle mice. After PMI treatment, hemoglobin content of thrombi was reduced (FIG. 5A). Fibrin / fibrinogen content of thrombi was also reduced in sickle mice after PMI treatment (FIG.5B). No difference were observed in markers of macrophage (Cd68) or neutrophil (MPO) markers.Example 6Effect of PMI on stroke size in sickle cell mice.

[0117] Sickle or WT bone marrow transplant (BMT) mice were treated with saline or 10 pg daily Recombinant Plasmodium falciparum Plasmepsin I (PMI) intraperitoneal (I P ). injection for two weeks. Then, stroke was induced by photochemical-mediated injury to the middle cerebral artery (MCA)11. Rose Bengal (50 mg / kg) was injected via the tail vein while a T5-mW green light laser (540 nm) was applied to the MCA for 30 minutes to induce thrombotic occlusion. The temporal muscle and skin were then sutured back in place. The mice wereDocket No. UM-43849.601treated for 3 more days before the brains were harvested, sectioned, and stained with 2% 2, 3, 5-triphenyltetrazolium chloride (TTC) for infarct area measurement.Example 7Effect of SCD on femoral vein thrombosis.

[0118] The infrarenal inferior vena cava (IVC) is the most widely used vein for murine VTE thrombosis models. Thrombosis is typically induced by ligation. However, there is an observed uniform mortality in SCD mice after IVC ligation23. Therefore, a femoral vein ligation model was established (FIG. 7A). Three days following femoral vein and branch ligation, a RBC-rich thrombus w as observed in the majority of WT mice. A thrombus was observed in all SCD mice with no mortality.

[0119] To characterize the presence of WT versus SCD erythrocytes in femoral venous thrombi, scanning electron microscopy (SEM) was performed. In WT mice, polyhedrocytes were observed compressed by fibrin and platelets26(FIG. 7B), while in SCD mice, rigid elongated RBCs were intertwined together to form unique structures (FIG. 7C and FIG. 7D). Additionally, protruding fiber like structures connecting sickle RBCs were common (FIG. 7C). Some RBCs in SCD thrombi appeared damaged with membrane disruption (FIG. 7D). Prior studies have demonstrated that increased deposition of acellular material within SCD venous thrombi21. It has also been reported that clots from SCD patients are more resistant to fibrinolysis, and this effect is mediated by the cellular fraction23.

[0120] In SCD, intracellular hemoglobin polymerization leads to rigid sickle shaped RBCs while extracellular hemoglobin polymerization likely occurs in the hypoxic environment of a thrombus15. These observations indicate sickle hemoglobin may affect thrombosis in SCD. To explore the role of hemoglobin in SCD thrombosis, recombinant Plasmodium falciparum Plasmepsin I (PMI), a hemoglobinase, was used to digest hemoglobin and the efficacy of PMI was confirmed in SCD mouse plasma with reduction of free hemoglobin (FIG. 7E). PMI activity7at the site of thrombosis may be enhanced due to local acidosis caused by blood stasis and subsequent tissue hypoxia27’28.

[0121] To determine in vivo effects of hemoglobinase on thrombosis, PMI was administered daily for 3 days following femoral vein ligation, after which venous thrombi were harvested. The thrombus was larger in SCD mice compared to WT mice (FIG. 7F and FIG. 7G). This finding supports a causal role for SCD towards increased venous thrombosis. Thrombus size was reduced in SCD mice after PMI treatment indicating the contribution of sickle hemoglobin to thrombosis in SCD (FIG. 7F and FIG. 7G).Docket No. UM-43849.601Example 8Effect of sickle hemoglobin on coagulation.

[0122] Damaged cell membranes observed on some RBCs in SCD thrombi (FIG. 7D) indicate that the exposure of phosphatidylserine may contribute to enhanced thrombogenicity in SCD23,25’29. Rupture of cell membranes could also cause leaking of other cellular components including hemoglobin. To assess plasma changes over time, samples were isolated from whole blood immediately or 2 hours after collection. Free plasma hemoglobin increased with the 2 hour incubation indicating release of hemoglobin from static SCD blood (FIG. 9A). Elevated free plasma hemoglobin in SCD may promote coagulation. SCD whole blood was divided into two groups to collect plasma or serum. Free hemoglobin concentration was reduced in serum compared with plasma, indicating consumption of free sickle hemoglobin during coagulation (FIG. 9B). In SCD mice, free plasma hemoglobin was also reduced after VTE (FIG. 9C). Previous work has shown that a cellular contribution other than RBC phosphatidylserine exposure enhances coagulation in SCD23,25, the present technology assessed the capacity of sickle hemoglobin to promote coagulation in SCD.

[0123] To determine if sickle hemoglobin affects coagulation, normal or sickle hemoglobin samples were spiked into normal blood, and an in vitro coagulation assay was performed. Coagulation was triggered with addition of CaCh to EDTA anticoagulated mouse whole blood (FIG. 9D). Sickle hemoglobin shortened the clotting time compared with normal hemoglobin (FIG. 9E). Enhanced coagulation has been previously observed in homozygous SCD patients whole blood using thromboelastography (TEG)30. Experiments conducted during the development of the present technology found that hemoglobin provides a therapeutic target for thrombosis in SCD.

[0124] Hemoglobin binds to haptoglobin and is cleared by monocytes / macrophages. As a second line of defense, hemopexin binds with heme31. SCD is characterized by low levels of haptoglobin and hemopexin which are consumed by high levels of free hemoglobin31. Reducing free hemoglobin in SCD with haptoglobin / hemopexin administration has been associated with reduced pulmonary hypertension32, decreased tissue iron deposition32,33, and suppressed vaso-occlusion and inflammation34. In human SCD pulmonary' hypertension, macrophages with accumulated oxidative markers after hemoglobin endocytosis were found located at vascular regions of fibrosis and remodeling-'5. In a rat model mimicking SCD pulmonary hypertension, depleting circulating macrophages significantly attenuated the response to combined hemoglobin and hypobaric hypoxia related to inflammation, oxidativeDocket No. UM-43849.601stress, and pulmonary' vascular remodeling, indicating a contribution of macrophages to SCD pulmonary hypertension after hemoglobin ingestion35. It is contemplated that digestion of free hemoglobin in SCD with hemoglobinase is particularly beneficial as macrophage activation may be mitigated. The presently disclosed subject matter indicates a prothrombotic effect of sickle hemoglobin that is relevant to venous thrombosis. Experiments conducted during the development of the present technology indicate that targeting sickle hemoglobin in SCD provides a therapeutic approach for thrombotic complications.SEQUENCE LISTING SEQ ID NO 1LENGTH: 334TYPE: Recombinant Plasmodium falciparum Plasmepsin I (PMI) AGDSVTLNDVANVMYYGEAQIGDNKQKFAFIFDTGSANLWVPSAQCNTIGCKTKNL YDSNKSKTYEKDGTKVEMNYVSGTVSGFFSKDIVTIANLSFPYKFIEVTDTNGFEPAY TLGQFDGIVGLGWKDLSIGSVDPVVVELKNQNKIEQAVFTFYLPFDDKHKGYLTIGG IEDRFYEGQLTYEKLNHDLYWQVDLDLHFGNLTVEKATAIVDSGTSSITAPTEFLNKF FEGLDVVKIPFLPLYITTCNNPKLPTLEFRSATNVYTLEPEYYLQQIFDFGISLCMVSIIP VDLNKNTFILGDPFMRKYFTVFDYDNHTVGFALAKKKL SEQ ID NO 2LENGTH: 452TYPE: Plasmepsin I (Aspartic hemoglobinase I) (Q7KQM4)MALSIKEDFS S AFAKNES AVNS STFNNNMKTWKIQKRFQILYVFFFLLITGALFYYLI DNVLFPKNKKINEIMNTSKHVIIGFSIENSHDRIMKTVKQHRLKNYIKESLKFFKTGLT QKPHLGNAGDSVTLNDVANVMYYGEAQIGDNKQKFAFIFDTGSANLWVPSAQCNTI GCKTKNLYDSNKSKTYEKDGTKVEMNYVSGTVSGFFSKDIVTIANLSFPYKFIEVTD TNGFEPAYTLGQFDGIVGLGWKDLSIGSVDPVVVELKNQNKIEQAVFTFYLPFDDKH KGYLTIGGIEDRFYEGQLTYEKLNHDLYWQ VDLDLHFGNLTVEKATAIVDS GTS SIT APTEFLNKFFEGLDVVKIPFLPLYITTCNNPKLPTLEFRSATNVYTLEPEYYLQQIFDF GISLCMVS1IPVDLNKNTF1LGDPFMRKYFTVFDYDNHTVGFALAKKKLSEQ ID NO 3LENGTH: 23TYPE: Aspartic hemoglobinase II (malaria parasite P. falciparum) XXNDNIELVDFQNIMFYGDAEVG SEQ ID NO 4Docket No. UM-43849.601LENGTH: 329TYPE: Recombinant Plasmodium falciparum Plasmepsin-2 SSNDNIELVDFQNIMFYGDAEVGDNQQPFTFILDTGSANLWVPSVKCTTAGCLTKHL YDSSKSRTYEKDGTKVEMNYVSGTVSGFFSKDLVTVGNLSLPYKFIEVIDTNGFEPT YTASTFDGILGLGWKDLSIGSVDPIVVELKNQNKIENALFTFYLPVHDKHTGFLTIGGI EERFYEGPLTYEKLNHDLYWQITLDAHVGNIMLEKANCIVDSGTSAITVPTDFLNKM LQNLDVIKVPFLPFYVTLCNNSKLPTFEFTSENGKYTLEPEYYLQHIEDVGPGLCMLN IIGLDFPVPTFILGDPFMRKYFTVFDYDNHSVGIALAKKNL SEQ ID NO 5LENGTH: 453TYPE: Plasmepsin II (PLM II) (Aspartic hemoglobinase II) (P46925) MDITVREHDFKHGFIKSNSTFDGLNIDNSKNKKKIQKGFQILYVLLFCSVMCGLFYYV YENVWLQRDNEMNEILKNSEHLTIGFKVENAHDRILKTIKTHKLKNYIKESVNFLNS GLTKTNYLGSSNDNIELVDFQNIMFYGDAEVGDNQQPFTFILDTGSANLWVPSVKCT TAGCLTKHLYDSSKSRTYEKDGTKVEMNYVSGTVSGFFSKDLVTVGNLSLPYKFIEV IDTNGFEPTYTASTFDGILGLGWKDLS1GSVDPIVVELKNQNKIENALFTFYLPVHDK HTGFLTIGGIEERFYEGPLTYEKLNHDLYWQITLDAHVGNIMLEKANCIVDSGTSAIT VPTDFLNKMLQNLDVIKVPFLPFYVTLCNNSKLPTFEFTSENGKYTLEPEYYLQHIED VGPGLCMLNIIGLDFPVPTFILGDPFMRKYFTVFDYDNHSVGIALAKKNL SEQ ID NO. 6LENGTH: 256TYPE: Recombinant Schistosoma japoni cum Hemoglobinase (HAEM) HKWAVLVAGSNGFENYRHQADVCHAYHVLLSKGVKPEHIITFMYDDIAHNKENPFP GKIFNDYRHKDYYKGVVIDYKGKKVNPKTFLQVLKGDKRAGGKVLKSGKNDDVFI YFTDHGAPGILAFPDDDLHAKPFINTLKYLRQHRRYSKLVIYVEACESGSMFAGLLPT DINIYATTAARPDESSYATFCDDPRISSCLADLYSYDWIVDSEKHQLTQRTLDQQYKE VKFETNLSHVQRYGDKKMGKLYLSEFQGS SEQ ID NO. 7LENGTH: 423TYPE: Schistosoma japonicum Hemoglobinase (Antigen Sj32) MFYSIFFIHILRIVLVDCNEYSEENVDDRHKWAVLVAGSNGFENYRHQADVCHAYH VLLSKGVKPEHIITFMYDDIAHNKENPFPGKIFNDYRHKDYYKGVVIDYKGKKVNPK TFLQVLKGDKRAGGKVLKSGKNDDVFIYFTDHGAPGILAFPDDDLHAKPFINLKYLR QHRRYSKLVIYVEACESGSMFAGLLPTDINIYATTAARPDESSYATFCDDPRISSCLADocket No. UM-43849.601DLYSYDWIVDSEKHQLTQRTLDQQYKEVKFETNLSHVQRYGDKKMGKLYLSEFQG SRKKASTEHDEPPMKPKDSIPSRDIPLHTLHRRIMMANNMNDKTLLMKILGLKLKRR DLIKDTMEVIDQFMFNVKQPNSNATIDETMDCIEVVYKEFQSKCFKIQQAPEITGYLS TLYNYCQKGYSAENINGVIRKVCG SEQ ID NO. 8LENGTHS 15TYPE: Schistosoma japonicumHemoglobinase (A0A4Z2CLM0) MFYSIFFIHILCIVLVDCNEYSEENVDDRHKWAVLVAGSNGFENYRHQADVCHAYH VLLSKGVKPEHIITFMYDDIAHNKENPFPGKIFNDYRHKDYYKGVVIDYKGKVSDKR AGGKVLKSGKNDDVFIYFTDHGAPGILSFPDDDLHAKPFINTLKYLRQHRRYSKLVI YVEACESGSMFAGLLPTDINIYATTAARPDESSYATFCDDPRISSCLADLYSYDWIVD SEKHQLTQRTLDQQYKEVKFETNLSHVQRYGDKKMGKLYLSEFQGSRKKASTEHD EPPMKPKDSIPSRDIPLHTLHRRIMMANNMNDKNLLMKILGLKLKRRDLIKDTMELIE QFMFNVKQPNSNATIDETMDCIEVVYKEFQSKCFKIQTGTRTLQVLINTLQVLSKRLS AENINEVIRKVAVKLK SEQ ID NO. 9LENGTH: 352TYPE: Schistosoma japonicumHemoglobinase (A0A4Z2CLY2) MYDDIAHNKENPFPGKIFNDYRHKDYYKGVVIDYKGKKVNPKTFLQVLKGDKRAG GKVLKSGKNDDVFIYFTDHGAPGILAFPDDDLLAKPFINTLKYLRQHRRYSKLVIYVE ACESGSMFAGLLPTDINIYATTAARPDESSYATFCDDPRISSCLADLYSYDWIVDSEK HQLTQRTLDQQYKEVKFETNLSHVQRYGDKKMGKLYLSEFQGSRKKASTEHDEPP MKPKDSIPSRDIPLHTLHRRIMMANNMNDKNLLMKIFGLKLKRRDLIKDTMELIEQF MFNVKQPNSNATIDETMDCIEVVYKEFQSKCFKIQQAPEITGYLSTLYNYCQKGYSA ENINEVIMKVCG SEQ ID NO. 10LENGTH: 179TYPE: Schistosoma japonicumHemoglobinase (A0A4Z2D3N7) RIQYVRLVPFETRRLDILASGCCCSQRDSNSVCFVSKDSESQPIKFTFTCFRKPKDSIPS RDIPLHTLHRRIMMANNMNDKNLLMKILGLKLKRRDLIKDTMELIEQFMFNVKQPN SNATIDETMDCIEVVYKEFQSKCFKTQQAPEITGYLSTLYKYCQKGYSAENINEVIMK VWRLS SEQ ID NO. 11LENGTH: 260Docket No. UM-43849.601TYPE: Recombinant Schistosoma mansoni Hemoglobinase VSDNNKWAVLVAGSNGYPNYRHQADVCHAYHVLRSKGIKPEHIITMMYDDIAYNL MNPFPGKLFNDYNHKDWYEGVVIDYRGKNVNSKTFLKVLKGDKSAGGKVLKSGK NDDVFIYFTDHGAPGLIAFPDDELYAKEFMSTLKYLHSHKRYSKLVIYIEANESGSMF QQILPSNLSIYATTAANSTECSYSTFCGDPTITTCLADLYSYNWIVDSQTHHLTQRTLD QQYKEVKRETDLSHVQRYGDTRMGKLYVSEFQGS SEQ ID NO. 12LENGTH: 429TYPE: Schistosoma mansoni Hemoglobinase (Antigen SM32) MMLFSLFLISILHILLVKCQLDTNYEVSDETVSDNNKWAVLVAGSNGYPNYRHQAD VCHAYHVLRSKGIKPEHIITMMYDDIAYNLMNPFPGKLFNDYNHKDWYEGVVIDYR GKNVNSKTFLKVLKGDKSAGGKVLKSGKNDDVFIYFTDHGAPGLIAFPDDELYAKE FMSTLKYLHSHKRYSKLVIYIEANESGSMFQQILPSNLSIYATTAANSTECSYSTFCGD PTITTCLADLYSYNWIVDSQTHHLTQRTLDQQYKEVKRETDLSHVQRYGDTRMGKL YVSEFQGSRDKSSSENDEPPMKPRHSIASRDIPLHTLHRQIMMTNNAEDKSFLMQILG LKLKRRDLIEDTMKLIVKVMNNEE1PNTKAT1DQTLDCTESVYEQFKSKCFTLQQAPE VGGHFSTLYNYCADGYTAETINEAIIKICG SEQ ID NO. 13LENGTH: 1,377TYPE: Hemoglobin-binding protease hbp autotransporter MNRIYSLRYSAVARGFIAVSEFARKCVHKSVRRLCFPVLLLIPVLFSAGSLAGTVNNE LGYQLFRDFAENKGMFRPGATNIAIYNKQGEFVGTLDKAAMPDFSAVDSEIGVATLI NPQYIASVKHNGGYTNVSFGDGENRYNIVDRNNAPSLDFHAPRLDKLVTEVAPTAV TAQGAVAGYLDKERYPVFYRLGSGTQYIKDSNGQLTKMGGAYSWLTGGTVGSLSS YQNGEMISTSSGLVFDYKLNGAMPIYGEAGDSGSPLFAFDTVQNKWVLVGVLTAGN GAGGRGNNWAVIPLDFIGQKFNEDNDAPVTFRTSEGGALEWSFNSSTGAGALTQGT TTYAMHGQQGNDLNAGKNLIFQGQNGQINLKDSVSQGAGSLTFRDNYTVTTSNGST WTGAGIVVDNGVSVNWQVNGVKGDNLHKIGEGTLTVQGTGINEGGLKVGDGKVV LNQQADNKGQVQAFSSVNIASGRPTVVLTDERQVNPDTVSWGYRGGTLDVNGNSL TFHQLKAADYGAVLANNVDKRATITLDYALRADKVALNGWSESGKGTAGNLYKY NNPYTNTTDYFILKQSTYGYFPTDQSSNATWEFVGHSQGDAQKLVADRFNTAGYLF HGQLKGNLNVDNRLPEGVTGALVMDGAADISGTFTQENGRLTLQGHPVIHAYNTQS VADKLAASGDHSVLTQPTSFSQEDWENRSFTFDNLKNTDFGLGRNATLNTTIQADNS SVTLGDSRVFIDKNDGQGTAFTLEEGTSVATKDADKSVFNGTVNLDNQSVLNINDIFDocket No. UM-43849.601NGGIQANNSTVNISSDSAVLGNSTLTSTALNLNKGANALASQSFVSDGPVNISDATLS LNSRPDEVSHTLLPVYDYAGSWNLKGDDARLNVGPYSMLSGNINVQDKGTVTLGG EGELSPLDLTLQNQMLYSLFNGYRNIWSGSLNAPDATVSMTDTQWSMNGNSTAGN MKLNRTIVGFNGGTSPFTTLTTDNLDAVQSAFVMRTDLNKADKLVINKSATGHDNSI WVNFLKKPSNKDTLDIPLVSAPEATADNLFRASTRVVGFSDVTPILSVRKEDGKKEW VLDGYQVARNDGQGKAAATFMHISYNNFITEVNNLNKRMGDLRDINGEAGTWVRL LNGSGSADGGFTDHYTLLQMGADRKHELGSMDCFTGVMATYTDTDASADLYSGKT KSWGGGFYASGLFRSGAYFDVIAKYIHNENKYDLNFAGAGKQNFRSHSLYAGAEAV GYRYHLTDTTFVEPQAELVWGRLQGQTFNWNDSGMDVSMRRNSVNPLVGRTGVV SGKTFSGKDWSLTARAGLHYEFDLTDSADVHLKDAAGEHQINGRKDSRMLYGVGL NARFGDNTR LGLEVERSAF GKYNTDDAIN ANIRYSF SEQ IDNO. 14LENGTH: 79TYPE: Clonorchis sinensis Hemoglobinase (G7YY68) MEACYSGSMFHDVLPSNMGVFVTTSAKEDEQSWSAFCHDKRINICLANEYSYAWIT DSQYKDLKKRTLDQQYEEVDKRT SEQ IDNO. 15LENGTH: 397TYPE: Clonorchis sinensis Hemoglobinase (A0A8T1MMC6) MMYRGTLLMTFLLYVNYAAWLGAVCVGSRLLHNDPAKNWVVLVAGSNGWENYR HQADIFHAYQIVKQHNVPAENIITFAYDDIAFNTLNPFKGQVFNDYAHKDVYEGVQI DYKKEDVTPNFLRALKGDKELELAGKKVLNSGPEDYVFIYFSDHGADGIIAFPEDELS ATDLNKTLSYMHTHGMYKKLVLYVEACESGSMFEGILPSNIGSKFVHDLNQYSLDQ QFDSVKQSTVQSHVSKFGEMDMGSLPVGEFQGHSKQSIRLDSSTMSQVLDSRPSRW AHLTTMSRRLMQAETVEEHELAARKLYRALQLGQIVKQTFDDIVMDVTTFHQPTIH VLSKSEELQCYEAVFQQFKKRCFTIRQVPEVAQHATRLRKLCKEGYATGIIIEAIHNL CS SEQ IDNO. 16LENGTH: 432TYPE: Clonorchis sinensis Hemoglobinase (A0A8T1MKT2) MRRSCLLIAFFYVNYAAWLGSVCVGSRLLHSDPTKNWVVLVAGSNGWGNYRHQA DVFHAYQILRHNNISAEQIITFAYDDIANNSENPFMGKVFNDYYHIDVYEGVIIDYRG EDVTPQNFLRVLRGDKELEAAGKKVLKSGPEDHVFIYFSDHGGDGIISFPEDELSATD LNKTLGYMYKNGKYKKLVLYVEACESGSMFEGILPSNIGIYVTTAANNQEASWATFDocket No. UM-43849.601CHDEVIDTCLADEYSYNWLTDSEEHDLTHRTLDQQFKSVKRRTKRSHVSRFGEMDV GRLPVGDFQGHSEQSMLLDSATMTQVLHSRPSRWAHLTTISRRLVHAESVEEHELA ARKLYRTLQLGHIVKQTFDDIVMDVTTFHQPTIHELSKSEELQCYEAVFKQFRKRCFT IRQVPEVAQYAGYLRKLCKKGYETKILIQSVHKVCS SEQ IDNO. 17LENGTH: 309TYPE: Schistosoma haematobium Hemoglobinase (A0A095A0I7) MLRFSLLLIHCLSIVLVQSRFNSTIEYFDENLSDKNKWAILVAGSNGFYNYRHQADVC HAYHVLRSKGIKPEHIITMMYDDIAHNKMNPFRGKIFNDYSHRDWYKGVVIDYKGK KVNSETFLKVLKGDQSAGGKVLKSGKNDDVFIYFTDHGAPGLIAFPDDELYAKRFM ATLKYLHRHKRYSRLVIYIEACESGSMFQGLLPSNLNIYATTAASPTESSYATFCDDP KIVACLADLYSYDWIVDSQTHQLTRRTLDQQYREVKRETNLSHVQRYGDTNMGKL HVGEFQGSRNKDSPENDEPPMKVNHT SEQ IDNO. 18LENGTH: 125TYPE: Schistosoma haematobium Hemoglobinase (A0A095A410) PKDFVASRDIPLHTLRRQIMMTNNGEDKNLLIEILGLKLKRRDFIEDTMKLVVKVMN NENKPIAKATIDQTLDCTESVYEQFKSKCFTLQQCERVIYPNNCLSDLSRENLKYIEA RLQVYSDFAL SEQ IDNO. 19LENGTH:280TYPE: Schistosoma haematobium Hemoglobinase (A0A419PHQ8) MNFRPRHKKLQVSLEIMRGYSLLTAFLFCINHVAWLEAAGVHNLSAIFNENPSKNW VVLVAGSNTWKNYRHQADVYHAYQVVRANKVPAENIITLAYDDIAKNPKNPFKGK VFHDYEHEDVYKGVVIDYRGKDVTAKNFLKVLRGDKTLEANRKKVLKSGPDDYVF IFYSGHGLDGLLTFPVGDYKKLVMYVEACYAGSMFRDVLPSNMGVYVTTSSNPVEQ SWSVFCLDKFIDVCLADEYSYAWITDSQYVSFSRTVAAKPHEGDLWEFSNHDLPAAP K SEQ ID NO. 20LENGTH: 333TYPE: Schistosoma haematobium Hemoglobinase (A0A4E0RLP5) MQFILLILSLLINFTLGLEDNGRTHWAVLVAGSNGWSNYRHQADVCHAYHVLRKNG MPPENIITMIYDDIANNTNNPFPGKLFNDYQHKDVYAGVKIDYRGENVTADIFLRVL EGDRKLKESGLKVLESGPEDNVFIYYNDHGAPNLLTFPKELLYAKQLNKTLANMYRDocket No. UM-43849.601EKRFKKMVIYIEACNSGSMFRWILPHNINVWAVTAANPTESSWATFCEDPAITTCLS DEFSYQWMNDTEKYRGQLFNRSMLDQYLNVKPAVKGSHVMEYGDIKISLLPVGEF QGNSTHVGSSGFNGDSVNQVMCTSIWIAIKTYTISTKSSASVFNKYRKLQKN REFERENCES

[0125] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

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Concurrent homozygous sickle-cell disease and severe haemophilia A: Thromboelastography profiles. Haemophilia. 2019;25(2):el24-el26.31. Santiago RP, Guarda CC, Figueiredo CVB, et al. Serum haptoglobin and hemopexin levels are depleted in pediatric sickle cell disease patients. Blood Cells Mol Dis. 2018;72:34-36.Docket No. UM-43849.60132. Irwin DC, Baek JH, Hassell K, et al. Hemoglobin-induced lung vascular oxidation, inflammation, and remodeling contribute to the progression of hypoxic pulmonary hypertension and is attenuated in rats with repeated-dose haptoglobin administration. Free Radic Biol Med. 2015;82:50-62.33. Shi PA, Choi E, Chintagari NR, et al. Sustained treatment of sickle cell mice with haptoglobin increases HO-1 and H-ferritin expression and decreases iron deposition in the kidney without improvement in kidney function. Br J Haematol. 2016: 175(4): 714-723.34. Belcher JD, Chen C, Nguyen J, et al. Haptoglobin and hemopexin inhibit vaso-occlusion and inflammation in murine sickle cell disease: Role of heme oxygenase- 1 induction. PLoS One. 2018;13(4):e0196455.Redinus K, Baek JH, Yalamanoglu A, et al. An Hb-mediated circulating macrophage contributing to pulmonary vascular remodeling in sickle cell disease. JCI Insight. 2019:4(15).

Claims

Docket No. UM-43849.601CLAIMSWhat is claimed is:

1. A pharmaceutical composition comprising a purified hemoglobinase enzyme or an active fragment thereof.

2. The composition of claim 1, wherein the hemoglobinase comprises an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. 99%. or 100% identical to one or more of the respective amino acid sequences of SEQ ID NOs: 1-20.

3. The pharmaceutical composition of claim 1, wherein the hemoglobinase is recombinantly expressed.

4. The pharmaceutical composition of claim 1, wherein the hemoglobinase enzyme is derived from Plasmodium falciparum Plasmepsin-2, Schistosoma japonicum Hemoglobinase (HAEM), or Schistosoma mansoni.

5. The pharmaceutical composition of claim 1, wherein the hemoglobinase enzyme is derived from Plasmodium falciparum Plasmepsin I.

6. The pharmaceutical composition of claim 1, wherein the hemoglobinase enzyme is an aspartic protease.

7. The pharmaceutical composition of claim 1, wherein the hemoglobinase enzyme is a serine protease.

8. The pharmaceutical composition of claim 1, wherein the hemoglobinase enzyme is a cysteine protease.

9. The pharmaceutical composition of claim 3, wherein the recombinant hemoglobinase enzyme is in expressed in yeast, E. coli, Baculovirus or mammalian cell.

10. The pharmaceutical composition of claim 1, further comprising one or more agents that bind free hemoglobin.

11. The pharmaceutical composition of claim 10. wherein the one or more agents is haptoglobin or hemopexin.Docket No. UM-43849.60112. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable salt or an excipient.

13. The pharmaceutical composition of claim 12, wherein the excipient is selected from the group consisting of a buffer, a stabilizer, a diluent, a binder, a vehicle, a filler, a preservative, a surfactant, and any combination thereof.

14. The pharmaceutical composition of claim 13, wherein the vehicle is selected from the group consisting of, a micelle, a dendrimer, a hydrogel, a emulsion, a nanoparticle, or a liposome.

15. A method comprising: administering the pharmaceutical composition of any one of claims 1-14 to a subject.

16. The method of claim 15, wherein said subject has one or more diseases or disorders associated with vaso-occlusive crises and / or thrombosis.

17. The method of claim 15, wherein the administering reduces free blood plasma hemoglobin.

18. The method of claim 15, wherein of the one or more diseases or disorders associated with vaso-occlusive crises and / or thrombosis in a subject is selected from the group consisting of sickle cell disease, deep vein thrombosis, stroke, pulmonary embolism, pulmonary hypertension, pain crisis, myocardial infarction, splenic sequestration, Factor V Leiden mutation, cancer-associated thrombosis, chronic venous insufficiency, atrial fibrillation, disseminated intravascular coagulation, nephrotic syndrome, thrombotic thrombocytopenic purpura, inflammatory bowel disease, varicose veins, peripheral artery disease, antiphospholipid syndrome, acute chest syndrome and splenic sequestration.

19. The method of claim 18, wherein the subject is identified as at risk of new or recurrent thrombotic events.

20. The method of claim 15, wherein the pharmaceutically acceptable composition is administered in combination and / or sequentially with at least one antithrombotic agent.

21. The method of claim 20, wherein the antithrombotic agent is low-dose aspirin, a direct factor Xa inhibitor, a thrombin inhibitor, aPY12 inhibitor, warfarin, heparin, or a combination thereof.Docket No. UM-43849.60122. The method of claim 15, wherein the subject is identified as at risk of new or recurrent vaso-occlusive crises events.

23. The method of claim 15, wherein the method comprises treating the subj ect with at least one support therapy.

24. The method of claim 23, wherein the support therapy is an anti-inflammatory agent, a corticosteroid, fluid replenishment, an intravenous (IV) fluid, a nonsteroidal anti-inflammatory drug (NSAIDs), an opioid, oxygen therapy, a blood transfusion, a disease-modifying therapy, a hydroxyurea, crizanlizumab, voxelotor, or any combination thereof.

25. Use of a pharmaceutical composition of any one of claims 1-14.

26. Use of a pharmaceutical composition of any one of claims 1-14, for the treatment, prevention, or reduction of diseases or disorders associated with vaso-occlusive crises and / or thrombosis in a subject, wherein the disease or disorder is selected from the group consisting of sickle cell disease, deep vein thrombosis, stroke, pulmonary embolism, pulmonary hypertension, pain crisis, myocardial infarction, splenic sequestration, Factor V Leiden mutation, cancer-associated thrombosis, chronic venous insufficiency, atrial fibrillation, disseminated intravascular coagulation, nephrotic syndrome, thrombotic thrombocytopenic purpura, inflammatory bowel disease, varicose veins, peripheral artery disease, antiphospholipid syndrome, acute chest syndrome and splenic sequestration.

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