Drug conditioning regimen for sickle cell disease

Abstract

Disclosed herein are methods of mobilizing hematopoietic stem and progenitor cells (HSPC) in a subject in need thereof, the methods include administering one or both of (1) an inhibitor of the polymerization of hemoglobin S and (2) a P-selectin or E-selectin inhibitor, prior to administering a hematopoietic stem cell mobilizing agent.

Description

Attorney Docket No.58434-0039WO1 DRUG CONDITIONING REGIMEN FOR SICKLE CELL DISEASE CLAIM OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 566,102, filed on March 15, 2024, U.S. Provisional Application Serial No.63 / 566,730, filed on March 18, 2024, and U.S. Provisional Application Serial No.63 / 704,960, filed on October 8, 2024. The entire contents of these applications are incorporated by reference in their entireties. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant Nos. P01 HL149626 and R35 HL161239 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0003] Sickle cell disease (SCD) is a painful inherited hemoglobinopathy which causes acute and chronic vaso-occlusion in various organs and tissues in the body, as well as early mortality. Gene therapy has emerged as a potentially curative treatment for SCD. Gene modification of an adequate number of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) for gene therapy requires hematopoietic stem and progenitor cells (HSPC) mobilization, with or without apheresis collection. Current ex vivo gene therapy protocols require the patient to undergo conditioning for HSPC mobilization and collection via several months of chronic red cell transfusions (red cell exchange). However, red cell transfusion poses transfusion-specific risks such as delayed hemolytic transfusion reactions and hyperhemolysis. Thus, improved methods of hematopoietic stem and progenitor cells (HSPC) conditioning are needed in SCD patients. SUMMARY

[0004] Current strategies for mobilizing hematopoietic stem and progenitor cells (HSPC) from the bone marrow to the peripheral blood typically involve red cell transfusions as a conditioning step which are associated with increased safety risks and unsatisfactory mobilization efficacy. Among other things, the present disclosure provides an insight that administering one or both of (1) an inhibitor of the polymerization of hemoglobin S and (2) aAttorney Docket No.58434-0039WO1 P-selectin or E-selectin inhibitor, prior to administering a hematopoietic stem cell mobilizing agent, may provide improved HSPC mobilization efficacy and reduced safety risks, for example, as compared to mobilization regimens that utilize red cell transfusion.

[0005] Accordingly, in some aspects, provided herein are methods of mobilizing hematopoietic stem and progenitor cells (HSPC) in a subject in need thereof, the method comprising administering one or both of (1) an inhibitor of the polymerization of hemoglobin S and (2) a P-selectin or E-selectin inhibitor, prior to administering a hematopoietic stem cell mobilizing agent.

[0006] In some embodiments, the methods include administering an inhibitor of the polymerization of hemoglobin S. In some embodiments, the inhibitor of the polymerization of hemoglobin S is a pyruvate kinase activator, voxelotor, ILX-002, GBT021601, or VZHE-039. In some embodiments, the inhibitor of the polymerization of hemoglobin S is a pyruvate kinase activator. In some embodiments, the pyruvate kinase activator is mitapivat, etavopivat, or tebapivat. In some embodiments, the pyruvate kinase activator is administered for at least about 3 weeks prior to administering the hematopoietic stem cell mobilizing agent. In some embodiments, the pyruvate kinase activator is administered at a dose of about 100-300 mg / day.

[0007] In some embodiments, the methods include administering a P-selectin inhibitor. In some embodiments, the P-selectin inhibitor is crizanlizumab or inclacumab. In some embodiments, the P-selectin inhibitor is administered about 12-48 hours prior to administering the hematopoietic stem cell mobilizing agent.

[0008] In some embodiments, the methods include administering an E-selectin inhibitor. In some embodiments, the E-selectin inhibitor is GMI-1687. In some embodiments, the E- selectin inhibitor is administered about 12-48 hours prior to administering the hematopoietic stem cell mobilizing agent.

[0009] In some embodiments, the hematopoietic stem cell mobilizing agent is a CXCR4 antagonist (such as, but not limited to, plerixafor or motixafortide). In some embodiments, the CXCR4 antagonist is plerixafor. In some embodiments, the hematopoietic stem cell mobilizing agent is a CXCR2 agonist (such as, but not limited to, N-terminal truncated GRO-β or MGTA- 145). In some embodiments, the hematopoietic stem cell mobilizing agent is a VLA-4 antagonist (such as, but not limited to, firategrast and BIO5192).

[0010] In some embodiments, the methods further include administering to the subject aspirin prior to administering the hematopoietic stem cell mobilizing agent. In someAttorney Docket No.58434-0039WO1 embodiments, aspirin is administered about 1-30 days prior to administering the hematopoietic stem cell mobilizing agent. In some embodiments, aspirin is administered at a dose of about 75-85 mg (e.g., about 81 mg / day). In some embodiments, the methods include administering an inhibitor of the polymerization of hemoglobin S and aspirin, prior to administering the hematopoietic stem cell mobilizing agent. In some embodiments, the methods include administering a P-selectin or E-selectin inhibitor and aspirin, prior to administering the hematopoietic stem cell mobilizing agent. In some embodiments, he methods include administering an inhibitor of the polymerization of hemoglobin S, a P-selectin or E-selectin inhibitor, and aspirin, prior to administering the hematopoietic stem cell mobilizing agent.

[0011] In some embodiments, mobilization of hematopoietic stem and progenitor cells (HSPC) from the bone marrow to the peripheral blood in the subject is increased compared to mobilization without the administration of the inhibitor of the polymerization of hemoglobin S and the P-selectin or E-selectin inhibitor. In some embodiments, the number of vaso-occlusive crises in the subject is reduced compared to mobilization without the administration of the inhibitor of the polymerization of hemoglobin S and the P-selectin or E-selectin inhibitor. In some embodiments, the subject has sickle cell disease. In some embodiments, the subject receives gene therapy for sickle cell disease after mobilization of hematopoietic stem and progenitor cells (HSPC). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGs.1A-1G depict the effects, compared to untreated control SS mice, of a single dose of anti-P-selectin (clone RB40.34, 5 mg / kg IV) or control isotype injected intravenously approximately 24 hours prior to mobilization with a single subcutaneous dose of plerixafor (10 mg / kg) on peripheral complete blood counts. Shown are white blood cells (WBC; FIG. 1A), neutrophils (ANC; FIG. 1B), lymphocytes (LYM; FIG. 1C), monocytes (MONO; FIG. 1D), red blood cells (RBC; FIG.1E), hemoglobin (Hb; FIG.1F), and platelets (PLT; FIG 1G). Each data point represents a single mouse.

[0013] FIGs.2A-2D depict the effects, compared to untreated control SS mice, of a single dose of murine anti-P-selectin (clone RB40.34, 5 mg / kg IV) or control isotype injected intravenously approximately 24 hours prior to mobilization with a single subcutaneous dose of plerixafor (10 mg / kg) on murine peripheral blood hematopoietic progenitor cells (HPCs), the LSK cells (Lin−Sca-1+c-kit+). FIGS. 2A and 2B depict the concentration of LSK cells inAttorney Docket No.58434-0039WO1 peripheral blood, with and without CD45+ gating. FIGS. 2C and 2D depict LSK cells in peripheral blood as a percentage of live cells and CD45+ cells, respectively. Each data point represents a single mouse.

[0014] FIGs.3A-3D depict the effects, compared to untreated control SS mice, of a single dose of murine anti-P-selectin (clone RB40.34, 5 mg / kg IV) or control isotype injected intravenously approximately 24 hours prior to mobilization with a single subcutaneous dose of plerixafor (10 mg / kg) on murine peripheral blood hematopoietic stem cells (HSCs), the LSKF cells (Lin−Sca-1+c-kit+Flt3-CD135-). FIGs.3A and 3B depict the concentration of LSKF cells in peripheral blood, with and without CD45+ gating. FIGS. 3C and 3D depict LSKF cells in peripheral blood as a percentage of live cells and CD45+ cells, respectively. Each data point represents a single mouse.

[0015] FIGs.4A-4C depict the effects, compared to untreated control SS mice, of a single dose of murine anti-P-selectin (clone RB40.34, 5 mg / kg IV) or control isotype injected intravenously approximately 24 hours prior to mobilization with a single subcutaneous dose of plerixafor (10 mg / kg) on murine bone marrow HSCs. FIG. 4A depicts the number of bone marrow HSCs per femur. FIGs. 4B and 4C depict the bone marrow LSKF (Lin−Sca1+ckit+CD48−CD150+) as a percentage of live cells and Lin- cells. Each data point represents a single mouse. DETAILED DESCRIPTION

[0016] Bone marrow harvesting was the initial approach for hematopoietic stem and progenitor cell (HSPC) collection in sickle-cell disease (SCD), with evidence supporting its utility in both animal models and in vitro studies utilizing patients’ material. However, bone marrow harvesting results in suboptimal yields of high purity hematopoietic stem cells (HSC) at the end of collection and processing, along with substantial pain after each harvest, and most subjects required two or three harvests to yield sufficient cell doses for manufacturing.

[0017] The success of gene therapy in SCD relies on several key factors; these include the source, quality and number of transduced cells, the choice of the conditioning regimen, the level of therapeutic transgene expression, and the quality of the bone marrow (BM) microenvironment at the time of harvest and transplantation. Approximately 2 to 3 x106CD34+HSPC / kg are required for a successful outcome in autologous hematopoietic stem cell transplantation (HSCT). For gene therapy, considering cell processing and manufacturing losses, more than about 6x106CD34+cells / kg is typically required for the collected product.Attorney Docket No.58434-0039WO1 The recovery of HSPC from SCD patients’ bone marrow (BM) is peculiarly low, requiring multiple BM harvests (each requiring an exchange transfusion program before general anesthesia) to obtain enough cells to transduce and transplant.

[0018] Filgrastim- (or granulocyte colony-stimulating factor) based mobilization and apheresis is the standard method for HSPC collection in healthy adult donors, yet this approach in SCD is associated with high rates of adverse events requiring hospitalization, including vaso- occlusive crises, multi-organ failure, and even death, prompting calls for a moratorium on its use for HSPC mobilization in SCD.

[0019] As an alternative to filgrastim, CXCR4 antagonists (including, but not limited to plerixafor, motixafortide, burixafor (GPC-100), HF51116 ((S)-N-(4-(((3- (cyclohexylamino)propyl)amino)methyl)benzyl)-5-guanidino-2-((pyridin-2- ylmethyl)amino)pentanamide), POL5551, balixafortide (POL6326), motixafortide (BKT140), and ulocuplumab), CXCR2 agonists (including, but not limited to, N-terminal truncated GRO- β or MGTA-145), or VLA-4 antagonists (including, but not limited to, firategrast, BIO5192 ((S)-2-((S)-1-((3,5-dichlorophenyl)sulfonyl)pyrrolidine-2-carboxamido)-4-((S)-4-methyl-2- (N-methyl-2-(4-(3-(o-tolyl)ureido)phenyl)acetamido)pentanamido)butanoic acid), either of which can be PEG-conjugated, WU-106, SLU-2609, AVA4746 ((3S)-3-[[1-[(2- chlorophenyl)methyl]-4-hydroxy-5-methyl-2-oxopyridin-3-yl]carbamoylamino]-3-(4- methylphenyl)propanoic acid), CWHM-823, GW559090 ((2S)-3-[4-(4-carbamoylpiperidine- 1-carbonyl)oxyphenyl]-2-[[(2S)-4-methyl-2-[[2-(2- methylphenoxy)acetyl]amino]pentanoyl]amino]propanoic acid), and TBC3486 ((3S)-3-(1,3- benzodioxol-5-yl)-3-[[(2S)-1-[bis(thiophen-2-ylmethyl)amino]-1-oxohexan-2- yl]carbamoylamino]propanoic acid)) can effectively mobilize HSPC. CXCR4 antagonists directly inhibit the binding of stroma-cell-derived factor-1α to its CXCR4 chemokine receptor on HSPC, releasing HSPC from the BM niche. Granulocytes and monocytes are necessary for mobilization to occur, and CXCR2 is a receptor on granulocytes and monocytes. VLA-4 is another HSPC receptor integral for retention of HSPC in the BM. As provided herein, hematopoietic stem cell mobilizing agents include, but are not limited to, CXCR4 antagonists (e.g., any of the CXCR4 antagonists disclosed herein or known in the art), CXCR2 agonists (e.g., any of the CXCR2 agonists disclosed herein or known in the art), and VLA-4 antagonists (e.g., any of the VLA-4 antagonists disclosed herein or known in the art).Attorney Docket No.58434-0039WO1

[0020] However, these cell collection protocols do or may require prior red blood cell transfusion or exchange to achieve <30% sickle hemoglobin (HbS) prior to HSPC mobilization.

[0021] One problem associated with current protocols is inadequate HSPC mobilization with transfusion conditioning. Seventy-three percent of patients on red cell transfusion conditioning still require two to five days of apheresis, and 47% of patients still need at least two mobilization cycles. The disclosed conditioning regimen can, in some cases, improve the efficacy of HSPC mobilization compared to transfusion.

[0022] Another problem associated with current protocols is persistent vaso-occlusive events with transfusion conditioning. Twenty percent of transfused patients still have severe vaso-occlusive pain following HPC mobilization and collection. The disclosed conditioning regimen can, in some cases, increase the safety of HSPC mobilization and collection with regard to vaso-occlusive patient adverse events.

[0023] Another problem associated with current protocols are transfusion-specific patient adverse events. Red cell transfusion poses alloimmune risks such as delayed hemolytic transfusion reactions and hyperhemolysis. In some embodiments, the disclosed conditioning regimen does not involve transfusion and thus minimizes alloimmune risks.

[0024] Another problem associated with current protocols is transfusion availability. Sickle cell disease is most prevalent in sub-Saharan Africa but in these countries, transfusions are not easily available or accessible. In some embodiments, the disclosed conditioning regimen involves the patient taking oral tablets for the majority of the period of conditioning.

[0025] Yet another problem associated with current protocols is transfusion-related transmissible disease. In Sub-Saharan Africa, the safety of chronic red cell transfusion is challenged by the risks of infection, specifically transfusion-transmitted infections. In some cases, the disclosed conditioning regimen does not involve transfusion and thus has no risk of transfusion-transmitted infection.

[0026] Accordingly, provided herein are methods of mobilizing HSPCs using certain conditioning regimens. In some embodiments, the disclosed methods demonstrate improved safety (e.g., reduced vaso-occlusive events, alloimmune risks, or transfusion-transmitted infection) and efficacy (e.g., increased mobilization of hematopoietic stem and progenitor cells (HSPC) from the bone marrow to the peripheral blood) compared to mobilization methods that involve red cell transfusion. In some aspects, provided herein are methods of mobilizingAttorney Docket No.58434-0039WO1 hematopoietic stem and progenitor cells (HSPC) in a subject in need thereof, the method comprising administering one or both of (1) an inhibitor of the polymerization of hemoglobin S and (2) a P-selectin or E-selectin inhibitor, prior to administering a hematopoietic stem cell mobilizing agent. In some embodiments, one or both of (1) an inhibitor of the polymerization of hemoglobin S and (2) a P-selectin or E-selectin inhibitor is administered to the subject for pre-conditioning prior to HSPC mobilization, as an alternative to red cell transfusion.

[0027] In some embodiments, the disclosed methods include administering an inhibitor of the polymerization of hemoglobin S (e.g., as an alternative to red cell transfusion, for example, to improve the safety and efficacy of HSPC mobilization and collection) prior to administering a hematopoietic stem cell mobilizing agent. Exemplary inhibitors of polymerization of hemoglobin S include, but are not limited to, pyruvate kinase activators (PKAs), voxelotor (GBT440), ILX-002, GBT021601 (2-Hydroxy-6-[[(3S)-4-[2-(2-hydroxyethyl)pyridine-3- carbonyl]morpholin-3-yl]methoxy]benzaldehyde), and VZHE-039 (). In some embodiments, the inhibitor of polymerization ofa activator (PKA). Pyruvate kinase is an enzyme involved in the generation of adenosine triphosphate (ATP), a molecule that stores and provides energy in the form that cells can use for their survival and functions. In patients with pyruvate kinase deficiency, the specific isoform of pyruvate kinase (PK-R) that RBCs use for energy is mutated, leading to reduced ATP production, reduced RBC survival and chronic hemolysis. Pyruvate kinase activators bind to pyruvate kinase tetramer and can increase pyruvate kinase activity and ATP production and prolong RBC survival. Pyruvate kinase activators activate both mutated and normal pyruvate kinases.

[0028] In some embodiments, the disclosed methods include the administration of a P- selectin and / or an E-selectin inhibitor prior to administering a hematopoietic stem cell mobilizing agent. In some embodiments, the P-selectin inhibitor is an antibody which blocks the interaction between P-selectin and P-selectin glycoprotein ligand-1. In some embodiments, the P-selectin inhibitor is crizanlizumab or inclacumab. P-selectin inhibitors can be administered once, twice, or three times or more prior to administering the hematopoietic stem cell mobilizing agent. In some embodiments, a P-selectin inhibitor (e.g., crizanlizumab or inclacumab) is administered for about 1-7 days (e.g., for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) prior to the initiation of hematopoietic stem cell mobilizing agentAttorney Docket No.58434-0039WO1 therapy (e.g., administration of a hematopoietic stem cell mobilizing agent, such as plerixafor). For example, a P-selectin inhibitor (e.g., crizanlizumab or inclacumab) can be administered for about 1-4 days prior to the administration of a hematopoietic stem cell mobilizing agent. In some embodiments, a P-selectin inhibitor (e.g., crizanlizumab or inclacumab) is administered (e.g., as a single dose) about 12-48 hours prior to the initiation of hematopoietic stem cell mobilizing agent therapy (e.g., administration of a hematopoietic stem cell mobilizing agent such as plerixafor). For example, a P-selectin inhibitor (e.g., crizanlizumab) can be administered about 24 hours prior to administration of a hematopoietic stem cell mobilizing agent (e.g., plerixafor). In some embodiments, a P-selectin inhibitor (e.g., crizanlizumab or inclacumab) is administered (e.g., as a single dose) about 48-120 hours prior to the initiation of hematopoietic stem cell mobilizing agent therapy (e.g., administration of a hematopoietic stem cell mobilizing agent such as plerixafor).

[0029] As provided herein, the E-selectin inhibitor can be an E-selectin antagonist which blocks the interaction between E-selectin and E-selectin ligands. In some embodiments, the E- selectin ligand is GMI-1687. In some embodiments, an E-selectin inhibitor (e.g., GMI-1687) is administered for about 1-7 days or more (e.g., for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more) prior to the initiation of hematopoietic stem cell mobilizing agent therapy (e.g., administration of a hematopoietic stem cell mobilizing agent, such as plerixafor). In some embodiments, an E-selectin inhibitor (e.g., GMI-1687) is administered (e.g., as a single dose) about 1-48 hours (e.g., about 12-48 hours) prior to the initiation of hematopoietic stem cell mobilizing agent therapy (e.g., administration of a hematopoietic stem cell mobilizing agent such as plerixafor). For example, an E-selectin inhibitor (e.g., GMI-1687) can be administered about 24 hours prior to administration of a hematopoietic stem cell mobilizing agent (e.g., plerixafor). In some embodiments, an E-selectin inhibitor (e.g., GMI-1687) is administered (e.g., as a single dose) about 48-120 hours prior to the initiation of hematopoietic stem cell mobilizing agent therapy.

[0030] The methods disclosed herein can also include administration of aspirin prior to administering the hematopoietic stem cell mobilizing agent. For example, the disclosed methods can include (1) administering an inhibitor of the polymerization of hemoglobin S (e.g., a pyruvate kinase activator) and aspirin, (2) administering a P-selectin inhibitor (e.g., crizanlizumab or inclacumab) or E-selectin inhibitor (e.g., GMI-1687) and aspirin, or (3) administering an inhibitor of the polymerization of hemoglobin S (e.g., a pyruvate kinase activator), a P-selectin inhibitor (e.g., crizanlizumab or inclacumab) or E-selectin inhibitorAttorney Docket No.58434-0039WO1 (e.g., GMI-1687), and aspirin, prior to administering the hematopoietic stem cell mobilizing agent.

[0031] In some embodiments, the dose of aspirin is about 75-100 mg (e.g., about 75-85 mg, or about 81 mg), which can be administered daily. Aspirin can be administered (e.g., daily) for about 1 day or more (e.g., about 2 days or more, about 3 days or more, about 4 days or more, or about 5 days or more) prior to the initiation of hematopoietic stem cell mobilization therapy (i.e., prior to administering a hematopoietic stem cell mobilizing agent). For example, aspirin can be administered for about 3 days prior to the administration of a hematopoietic stem cell mobilizing agent, such as plerixafor. In some embodiments, aspirin is administered for about 1 to 30 days prior to administering a hematopoietic stem cell mobilizing agent. In some embodiments, aspirin is administered daily for at least about 10 days prior to the initiation of hematopoietic stem cell mobilization therapy (e.g., administration of a hematopoietic stem cell mobilizing agent). In some embodiments, aspirin is administered daily for at least about 14 days prior to the initiation of hematopoietic stem cell mobilization therapy (e.g., administration of a hematopoietic stem cell mobilizing agent). In some embodiments, aspirin is administered daily for at least about 20 days prior to the initiation of hematopoietic stem cell mobilization therapy (e.g., administration of a hematopoietic stem cell mobilizing agent). In some embodiments, aspirin is administered daily for at least about 30 days prior to the initiation of hematopoietic stem cell mobilization therapy (e.g., administration of a hematopoietic stem cell mobilizing agent).

[0032] In some embodiments, methods disclosed herein include administration of a pyruvate kinase activator and one or both of a low-dose aspirin (e.g., about 75-100 mg / day) and an inhibitor to P-selectin and / or E-selectin prior to administering a hematopoietic stem cell mobilizing agent (e.g., CXCR4 inhibitor, CXCR2 agonist, and / or VLA-4 inhibitor). Similar to red cell transfusions, a pyruvate kinase activator can increase hemoglobin concentrations and may thus improve the bone marrow microenvironment. Incorporation of a P-selectin and / or an E-selectin inhibitor and / or a low dose aspirin has an HSPC mobilizing effect as well as offers protection from vaso-occlusive complications. The disclosed methods involving administering a pyruvate kinase activator, a P-selectin and / or E-selectin inhibitor, and / or aspirin prior to administering a hematopoietic stem cell mobilizing agent offers a more efficient and safer method than transfusion for HSPC mobilization (e.g., prior to gene therapy in SCD).

[0033] Exemplary pyruvate kinase activators (PKAs) include, but are not limited to, mitapivat, etavopivat, and tebapivat (AG-946). As disclosed herein, PKAs can be administeredAttorney Docket No.58434-0039WO1 at a dose of about 50-1000 mg / day. In some embodiments, the dose of a pyruvate kinase activator (e.g., mitapivat) is about 50-400 mg / day (e.g., about 100-300 mg / day, about 150-250 mg / day, or about 100-200 mg / day). For example, the dose of a pyruvate kinase activator (e.g., mitapivat) can be about 200 mg / day. In some embodiments, the dose of a pyruvate kinase activator (e.g., etavopivat) is about 200-1,000 mg / day (e.g., about 200-600 mg / day, about 300- 500 mg / day, or about 350-450 mg / day). In some embodiments, the dose of a pyruvate kinase activator (e.g., AG-946) is about 5-2000 mg / day. For example, the dose of AG-946 can be about 5-50 mg / day, or about 5-30 mg / day.

[0034] As disclosed herein the pyruvate kinase activator (PKA) can be administered (e.g., daily) for at least about 2 weeks prior to initiation of hematopoietic stem cell mobilizing agent therapy (e.g., administration of a hematopoietic stem cell mobilizing agent, such as plerixafor). For example, PKA can be administered (e.g., daily) for at least about 3 weeks prior to administration of a hematopoietic stem cell mobilizing agent (e.g., plerixafor). In some embodiments, the PKA is administered daily for about 1-3 months prior to initiation of hematopoietic stem cell mobilizing agent therapy. In some embodiments, the PKA is administered daily for about 1 month prior to the initiation of hematopoietic stem cell mobilizing agent therapy. In some embodiments, the PKA is administered daily for about 2 months prior to the initiation of hematopoietic stem cell mobilizing agent therapy. In some embodiments, the PKA is administered daily for about 3 months prior to the initiation of hematopoietic stem cell mobilizing agent therapy.

[0035] In some embodiments, plerixafor is administered at a dose of about 80-480 µg / kg daily for up to four consecutive days. In some embodiments, the dose of plerixafor is about 100-400 µg / kg, about 200-400 µg / kg, or about 300-400 µg / kg. In some embodiments, the dose of plerixafor is about 250-350 µg / kg, or a range defined by any two of the foregoing values. In some embodiments, motixafortide is administered at a dose of about 1.25-2.50 mg / kg. In some embodiments, a CXCR2 agonist (e.g., N-terminal truncated GRO-β, MGTA-145) is administered at a dose of about 0.01 mg / kg-25 mg / kg. In some embodiments, the dose of MGTA-145 is about 0.01-25 mg / kg (e.g., about 0.01 to about 0.1 mg / kg, about 1 to about 5 mg / kg, about 0.03 mg / kg-10 mg / kg, or about 0.01 to about 10mg / kg).

[0036] In some embodiments, as a result of the pre-conditioning with the inhibitor of the polymerization of hemoglobin S (e.g., pyruvate kinase activators), and / or P-selectin inhibitor (e.g., crizanlizumab or inclacumab) or E-selectin inhibitor (e.g., GMI-1687), mobilization of HSPC from the bone marrow to the peripheral blood in the subject is increased compared toAttorney Docket No.58434-0039WO1 mobilization without pre-conditioning with these agents, or with pre-conditioning by other means (including e.g., red cell transfusion). In some embodiments, the mobilization is increased by 10%, by 20%, by 30%, by 40%, by 50%, by 60%, by 70%, by 80%, by 90%, by 100%, or by more than 100% compared to mobilization without pre-conditioning with these agents or with pre-conditioning by other means (including e.g., red cell transfusion). In some embodiments, as a result of the pre-conditioning with the inhibitor of the polymerization of hemoglobin S (e.g., pyruvate kinase activators), and / or P-selectin inhibitor (e.g., crizanlizumab or inclacumab) or E-selectin inhibitor (e.g., GMI-1687), the number of vaso-occlusive crises in the subject is reduced compared to mobilization without pre-conditioning with these agents, or with pre-conditioning by other means (including e.g., red cell transfusion). In some embodiments, the number of vaso-occlusive crises is decreased by 10%, by 20%, by 30%, by 40%, by 50%, by 60%, by 70%, by 80%, by 90%, or by 100%, compared to mobilization without pre-conditioning with these agents or with pre-conditioning by other means (including e.g., red cell transfusion).

[0037] The methods disclosed herein can be useful for mobilizing hematopoietic stem and progenitor cells (HSPC) in a subject for whom hematopoietic stem and progenitor cells (HSPC) are collected for gene therapy purposes. In some embodiments, the methods disclosed herein are useful for mobilizing hematopoietic stem and progenitor cells (HSPC) in a subject with sickle cell disease. For example, the subject may be receiving gene therapy for sickle cell disease after mobilization of hematopoietic stem and progenitor cells (HSPC).

[0038] Thus, disclosed herein are methods of increasing HSPC mobilization and recovery in subjects in need thereof (e.g., subjects with sickle cell disease) to (1) increase the degree of HSPC mobilization; (2) increase the safety of HSPC mobilization and collection in regard to vaso-occlusive patient adverse events; (3) decrease infectious disease risks; (4) remove alloimmunization risk; and / or (5) improve accessibility of gene therapy, all in comparison to the existing red blood cell transfusion regimen. EXAMPLES Example 1: Conditioning for hematopoietic progenitor cell mobilization in sickle cell disease

[0039] This study uses a SCD mouse model, SS Townes mice, to evaluate the effects of pyruvate kinase activators (PKA), to mobilize hematopoietic progenitor cells (HPC). PKA isAttorney Docket No.58434-0039WO1 administered to mice for a period of time and then the mice are administered a single dose of plerixafor (10 mg / kg) via subcutaneous injection. The PKA + plerixafor group (n=6) are compared to the following control groups: control + plerixafor (n=5), PKA alone (n=3 for each PKA to be studied), and control alone (n=3). Peripheral blood is collected in all groups prior to and two- and three-weeks post treatment with PKA (or control) to assess hemoglobin (Hgb), hematocrit (Hct), aged neutrophils, and circulating HPCs. One to two hrs after plerixafor, peripheral blood is collected for circulating HPC (LSKF) enumeration. Assay results in the groups are compared using ANOVA or Kruskal-Wallis testing.

[0040] Also evaluated are marrow HPCs and hematopoietic stem cells (HSCs) with and without plerixafor and peripheral blood inflammatory markers (aged neutrophils, sVCAM-1, P-selectin, and E-selectin) with and without plerixafor.

[0041] Gating with and without CD45+is performed due to the erythroid hyperplasia seen in SCD, where CD45+expression decreases along with erythroid maturation (Boulais et al. Immunity 49:627-639, 2018.

[0042] There is evidence that SCD is associated with a chronic inflammatory state. Therefore, inflammatory markers (sVCAM-1, P-selectin, and E-selectin) in the peripheral blood of mice treated with a PKA alone or with plerixafor is also evaluated. Example 2: Inclusion of anti-inflammatory agents

[0043] Using the methods disclosed in Example 1, PKA is administered in combination with low-dose aspirin and / or a P-selectin inhibitor.

[0044] The following groups are used for this experiment: plerixafor alone; PKA alone (different PKAs); (3) PKA pre-treatment followed by plerixafor; aspirin pre-treatment followed by plerixafor; (5) P-selectin inhibitor pre-treatment followed by plerixafor; PKA + aspirin pre-treatment followed by plerixafor; PKA + P-selectin inhibitor pre-treatment followed by plerixafor; PKA + aspirin + P-selectin inhibitor followed by plerixafor. Appropriate control groups are also used for each of these treatment groups.

[0045] The pre-treatment or control regimen is administered over the course of one month (daily for PKA and aspirin, and once, twice, or three times over the course of the pre-treatment for the P-selectin inhibitor). Peripheral blood is collected in all groups prior to, and two- and three-weeks post pre-treatment to assess hemoglobin (Hgb), hematocrit (Hct), aged neutrophils, and circulating HPCs. A subset of treatment and control mice are sacrificed without being given plerixafor to examine bone marrow HPC and HSC. A subset of treatmentAttorney Docket No.58434-0039WO1 and control mice are administered a single dose of plerixafor (10 mg / kg) via subcutaneous injection. One to two hours after plerixafor, peripheral blood and bone marrow is collected for HPC and HSC enumeration. Assay results in the groups are compared using ANOVA or Kruskal-Wallis testing. Example 3: Utility of anti-P selectin for hematopoietic progenitor cell mobilization

[0046] A SCD mouse model, SS Townes mice, was used to evaluate the effect of murine anti-P-selectin, the murine analog of crizanlizumab, to mobilize hematopoietic progenitor cells (HPC). A single dose of anti-P-selectin (clone RB40.34, 5 mg / kg intravenously) or isotype control was administered approximately 24 hr prior to a single dose of plerixafor (10 mg / kg subcutaneously). One to two hours after plerixafor, peripheral blood and bone marrow was collected for HPC (LSK cells) and HSC (LSKF cells) enumeration. The anti-P selectin group (n=5) was compared to the following control groups: isotype controls (n=5) and untreated SS controls (n=3). Assay results in the groups are compared using ANOVA or Kruskal-Wallis testing.

[0047] With the complete blood counts, there was an increase in the number of WBC (FIG. 1A) and WBC subtypes of neutrophils, lymphocytes, and monocytes (FIG. 1B-D), but no significant difference between the anti-P-selectin and isotype control groups. There were no significant differences between the groups in RBC, Hb, or PLT (FIG.1E-G).

[0048] In peripheral blood HPCs and HSCs, gating with and without CD45+ was performed due to the increase in peripheral blood erythroid progenitors seen in SCD, where CD45+ expression decreases along with erythroid maturation (Boulais et al. Immunity 49:627- 639, 2018) and the degree of erythroid hyperplasia in each mouse was unknown. The median percentage of CD45+ cells was 82% (range 53-94%).

[0049] Without CD45+ gating, the anti-P selectin group showed significant increases in the absolute number (FIG.2A) and percentage (FIG.2C) of peripheral blood HPCs (LSK cells) mobilized compared to both the isotype and non-treated SS controls. With CD45+ gating (gating out CD45- mature erythroid cells), the absolute number of LSK cells (FIG. 2B) were increased in the anti-P selectin group compared to both the isotype and non-treated SS controls, and the percentage of LSK cells was increased compared to the non-treated SS controls only.

[0050] Without CD45+ gating, the anti-P selectin group showed significant increases in the absolute number (FIG. 3A) and percentage (FIG. 3C) of peripheral blood HSCs (LSKF cells) mobilized compared to both the isotype and non-treated SS controls. With CD45+ gatingAttorney Docket No.58434-0039WO1 (gating out CD45- mature erythroid cells), the absolute number (FIG.3B) and percentage (FIG. 3D) were increased in the anti-P selectin group compared to only the non-treated SS controls.

[0051] In the bone marrow, as expected, the absolute number (FIG.4A) and percent (FIGS 4B-C) of HSC (regardless of Lin-negative gating) were decreased with plerixafor treatment, with no significant differences in the isotype versus anti-P selectin groups.

[0052] In summary, HPC mobilization with ant-P selectin appears safe as measured by the complete blood counts (FIGs. 1A-1G), with expected increases observed with plerixafor in leukocytes and no change in red cells or platelets. Significant increases in HPCs and HSCs with anti-P selectin were observed compared to isotype control (FIGs. 2 and 3). The expected decrease in bone marrow HSC was observed with anti-P selectin treatment (FIG.4).

[0053] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms "about" and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0054] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.Attorney Docket No.58434-0039WO1 The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0055] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0056] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0057] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0058] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.Attorney Docket No.58434-0039WO1

[0059] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described. Exemplary Embodiments

[0060] Embodiment 1: A method for pre-conditioning a subject with sickle cell disease in advance of hematopoietic stem cell mobilizing agent therapy, the method comprising: administering an inhibitor of the polymerization of hemoglobin S for one to three months prior to initiating plerixafor therapy; wherein the inhibitor of the polymerization of hemoglobin S is a pyruvate kinase activator; and wherein as a result of the pre-conditioning with an inhibitor of the polymerization of hemoglobin S, mobilization of hematopoietic stem and progenitor cells (HSPC) from the bone marrow to the peripheral blood in the subject is increased compared to mobilization without pre-conditioning with the inhibitor of the polymerization of hemoglobin S.

[0061] Embodiment 2: The method of Embodiment 1, further comprising administering a P-selectin inhibitor and / or an E-selectin inhibitor.

[0062] Embodiment 3: The method of Embodiment 2, wherein the P-selectin inhibitor is crizanlizumab or inclacumab.

[0063] Embodiment 4: The method of Embodiment 2, wherein the E-selectin inhibitor is GMI-1687.

[0064] Embodiment 5: The method of Embodiments 3 or 4, wherein the crizanlizumab or inclacumab and / or GMI-1687 is administered for 1-4 days

[0065] Embodiment 6: The method of any one of Embodiments 1-5, further comprising administering aspirin.

[0066] Embodiment 7: The method of Embodiment 6, wherein the aspirin is at a dose of about 81 mg / day.

[0067] Embodiment 8: The method of Embodiment 6, wherein the aspirin is administered for 1-30 days prior to hematopoietic stem cell mobilizing agent administration.Attorney Docket No.58434-0039WO1

[0068] Embodiment 9: The method of any one of Embodiments 1-8, further comprises administering both a P-selectin inhibitor and / or E-selectin inhibitor and aspirin.

[0069] Embodiment 10: The method of any one of Embodiments 1-9, further comprising administering hematopoietic stem cell mobilizing agent therapy after pre-conditioning with the inhibitor of the polymerization of hemoglobin S.

[0070] Embodiment 11: The method of any one of Embodiments 1-10, wherein the subject is not scheduled for red blood cell transfusion for the specific purpose of pre- conditioning for hematopoietic stem cell mobilizing agent therapy.

[0071] Embodiment 12: The method of any one of Embodiments 1-11, wherein the subject receives gene therapy for sickle-cell disease after completion of hematopoietic stem cell mobilizing agent therapy.

[0072] Embodiment 13: A method of mobilizing hematopoietic stem and progenitor cells (HSPC) in a subject with sickle cell disease comprising: administering an inhibitor of the polymerization of hemoglobin S for one to three months prior to initiating hematopoietic stem cell mobilizing agent therapy; wherein the inhibitor of the polymerization of hemoglobin S is a pyruvate kinase activator; and wherein as a result of the administration, the number of vaso- occlusive crises in the subject are reduced.

[0073] Embodiment 14: The method of Embodiment 13, further comprising administering a P-selectin inhibitor and / or an E-selectin inhibitor.

[0074] Embodiment 15: The method of Embodiment 14, wherein the P-selectin inhibitor is crizanlizumab or inclacumab.

[0075] Embodiment 16: The method of Embodiment 14, wherein the E-selectin inhibitor is GMI-1687.

[0076] Embodiment 17: The method of Embodiments 15 or 16, wherein the crizanlizumab or inclacumab or GMI-1687 is administered for 1-4 days.

[0077] Embodiment 18: The method of any one of Embodiments 13-17, further comprising administering aspirin.

[0078] Embodiment 19: The method of Embodiment 18, wherein the aspirin is at a dose of about 81 mg / day.Attorney Docket No.58434-0039WO1

[0079] Embodiment 20: The method of Embodiment 18, wherein the aspirin is administered for 1-30 days prior to hematopoietic stem cell mobilizing agent therapy.

[0080] Embodiment 21: The method of any one of Embodiments 13-20, further comprises administering both a P-selectin inhibitor and / or E-selectin inhibitor and aspirin.

[0081] Embodiment 22: The method of any one of Embodiments 13-21, further comprising administering hematopoietic stem cell mobilizing agent therapy after pre-conditioning with the inhibitor of the polymerization of hemoglobin S.

[0082] Embodiment 23: The method of any one of Embodiments 13-22, wherein the subject is not scheduled for red blood cell transfusion for the specific purpose of pre- conditioning for hematopoietic stem cell mobilizing agent therapy.

[0083] Embodiment 24: The method of any one of Embodiments 13-23, wherein the subject receives gene therapy for sickle-cell disease after completion of hematopoietic stem cell mobilizing agent therapy.

Claims

Attorney Docket No.58434-0039WO1 What is claimed is:

1. A method of mobilizing hematopoietic stem and progenitor cells (HSPC) in a subject in need thereof, the method comprising administering one or both of (1) an inhibitor of the polymerization of hemoglobin S and (2) a P-selectin or E-selectin inhibitor, prior to administering a hematopoietic stem cell mobilizing agent.

2. The method of claim 1, wherein the method comprises administering an inhibitor of the polymerization of hemoglobin S.

3. The method of claim 2, wherein the inhibitor of the polymerization of hemoglobin S is a pyruvate kinase activator, voxelotor, ILX-002, GBT021601, or VZHE-039.

4. The method of claim 2, wherein the inhibitor of the polymerization of hemoglobin S is a pyruvate kinase activator.

5. The method of claim 4, wherein the pyruvate kinase activator is mitapivat, etavopivat, or tebapivat.

6. The method of claims 4 or 5, wherein the pyruvate kinase activator is administered for at least about 3 weeks prior to administering the hematopoietic stem cell mobilizing agent.

7. The method of any one of claims 4-6, wherein the pyruvate kinase activator is administered at a dose of about 100-300 mg / day.

8. The method of any one of claims 4-6, wherein the pyruvate kinase activator is administered at a dose of about 300-500 mg / day.

9. The method of claim 1, wherein the method comprises administering a P-selectin inhibitor.

10. The method of claim 9, wherein the P-selectin inhibitor is crizanlizumab or inclacumab.

11. The method of claims 9 or 10, wherein the P-selectin inhibitor is administered about 12-48 hours prior to administering the hematopoietic stem cell mobilizing agent.

12. The method of claim 1, wherein the method comprises administering an E-selectin inhibitor.

13. The method of claim 12, wherein the E-selectin inhibitor is GMI-1687.Attorney Docket No.58434-0039WO1 14. The method of claims 12 or 13, wherein the E-selectin inhibitor is administered about 12-48 hours prior to administering the hematopoietic stem cell mobilizing agent.

15. The method of any one of the above claims, wherein the hematopoietic stem cell mobilizing agent is a CXCR4 antagonist.

16. The method of claim 15, wherein the CXCR4 antagonist is plerixafor.

17. The method of any one of claims 1-14, wherein the hematopoietic stem cell mobilizing agent is a CXCR2 agonist.

18. The method of any one of claims 1-14, wherein the hematopoietic stem cell mobilizing agent is a VLA-4 antagonist.

19. The method of any one of the above claims, further comprising administering to the subject aspirin prior to administering the hematopoietic stem cell mobilizing agent.

20. The method of claim 19, wherein aspirin is administered about 1-30 days prior to administering the hematopoietic stem cell mobilizing agent.

21. The method of claims 19 or 20, wherein aspirin is administered at a dose of about 75- 85 mg.

22. The method of claim 1, comprising administering an inhibitor of the polymerization of hemoglobin S and aspirin, prior to administering the hematopoietic stem cell mobilizing agent.

23. The method of claim 1, comprising administering a P-selectin or E-selectin inhibitor and aspirin, prior to administering the hematopoietic stem cell mobilizing agent.

24. The method of any one of the above claims, comprising administering an inhibitor of the polymerization of hemoglobin S, a P-selectin or E-selectin inhibitor, and aspirin, prior to administering the hematopoietic stem cell mobilizing agent.

25. The method of any one of the above claims, wherein mobilization of hematopoietic stem and progenitor cells (HSPC) from the bone marrow to the peripheral blood in the subject is increased compared to mobilization without the administration of the inhibitor of the polymerization of hemoglobin S and the P-selectin or E-selectin inhibitor.Attorney Docket No.58434-0039WO1 26. The method of any one of the above claims, wherein the number of vaso-occlusive crises in the subject is reduced compared to mobilization without the administration of the inhibitor of the polymerization of hemoglobin S and the P-selectin or E-selectin inhibitor.

27. The method of any one of the above claims, wherein the subject has sickle cell disease.

28. The method of claim 27, wherein the subject receives gene therapy for sickle cell disease after mobilization of hematopoietic stem and progenitor cells (HSPC).

Images