WO2025064402A1 - Method for treating sickle cell disease by targeting senescent cells - Google Patents

Abstract

Disclosed are methods for treating, preventing, reducing, or eliminating a manifestation or complication associated with sickle cell disease in a subject and improving the preparation of hematopoietic stem cells from a subject using senolytic agents such as Bcl inhibitors.

Description

METHOD FOR TREATING SICKLE CELL DISEASE BY TARGETING SENESCENT CELLS

Introduction

[0001] This application claims benefit from U.S. Provisional Patent Application Serial No. 63/584,516, filed September 22, 2023, the content of which is incorporated herein by reference in its entirety.

[0002] This invention was made with government support under grant nos. HL168893 and HL164095 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background

[0003] Senolytics are a class of drugs that selectively clear senescent cells. The first senolytic drugs Dasatinib, Quercetin, Fisetin and Navitoclax (ABT-263) were discovered using a hypothesis-driven approach. Senescent cells accumulate with ageing and are often resistant to controlled cell death or apoptosis. Senescent cells exhibit an upregulation of anti-apoptotic pathways, which defend these senescent cells against their own inflammatory senescence- associated secretory phenotype, allowing them to survive, despite death of neighboring cells due to these secreted proteins. Senolytics transiently disable these senescence- associated secretory phenotypes and also target the anti- apoptotic pathways that protect senescent cells from death, causing apoptosis or death of the senescent cells, allowing healthy cells to grow. Because senescent cells take weeks to reaccumulate, senolytics may be administered intermittently. Early pilot trials of senolytics suggest they decrease senescent cells, reduce inflammation, and alleviate frailty in humans. The use of senolytics in the treatment of diabetes, pulmonary conditions, COVID-19, ophthalmic conditions, atherosclerosis, and age-related pathologies such as Alzheimer's disease, osteoarthritis, and osteoporosis have been described. See US 2017/0056421 Al, US 2019/0151337 Al, US 2020/0360386 Al, US 2021/0379078 Al.

Summary of the Invention

[0004] Provided herein is a method for treating, preventing, reducing, or eliminating a manifestation or complication associated with sickle cell disease in a subject by administering to the subject an effective amount of at least one senolytic agent thereby treating, preventing, reducing, or eliminating a manifestation or complication associated with sickle cell disease sickle in the subject.

[0005] Also provided is a method for improving the quality of hematopoietic stem cells of a subject by administering at least one senolytic agent to the subject. In some aspects, the subject is further administered a mobilizing agent. In some aspects, the hematopoietic stem cells are collected or harvested from the subject, e.g., from peripheral blood or bone marrow of a healthy subject or subject with a sickle cell disease. In some aspects, the hematopoietic stem cells are genetically modified and/or transplanted.

Brief Description of the Drawings

[0006] FIGS. 1A-1I show that sickle cell disease (SCD) perturbs HSC numbers and induces DNA damage and oxidative stress in mice. Percent long-term HSCs (LT-HSCs; FIG. 1A) or percent short-term HSCs (ST-HSCs; FIG. IB) with high yH2AX in middle-aged SCD (n=6) and non-SCD (n=6) littermates. FIG. 1C, Co-localization of y^2AX and 53BP1 and y^2AX puncta numbers/cell (n~38 cells for non-SCD and n=37 cells for SCD). FIG. ID, Flow cytometry of CellRox®-Green in LT-HSCs of middle-aged non-SCD (left) and SCD mice (right). Percent reactive oxygen species (ROS)-positive (FIG. IE) LT-HSC and (FIG. IF) ST-HSCs in middle-aged non-SCD and SCD mice (n=4-7 per cohort). FIG. 1G, Representative flow cytometry (top) and quantification (bottom) of senescence associated fJ-gal- positive (SA-p-gal⁺) LT-HSCs and ST-HSCs isolated from middle-aged SCD (n=ll) and non-SCD (n=9) mice. Extreme limiting dilution analysis to estimate numbers of repopulating HSCs per whole bone marrow (WBM) cells in young (FIG. 1H) and middle-aged (FIG. II) SCD mice and non-SCD mice. [0007] FIGS. 2A-2D show that sickle cell disease induces hallmarks of senescence in HSCs from individuals with SCD. Representative flow cytometry plots (left) and quantification (right) of DNA damage (FIG. 2A) and SA-p-gal activity (FIG. 2B) in bone marrow Lineage-CD34+CD38- cells from individuals with SCD or age-matched non-SCD controls (n-16 and 12, respectively) . Representative flow cytometry plots (left) and quantification (right) of intracellular pl6 (FIG. 2C) and p21 (FIG. 2D) protein levels in bone marrow Lineage-CD34+CD38- cells from individuals with SCD or age-matched non-SCD controls (n=9 and 10, respectively). Isotype controls are shown at the bottom. OPM2 cells expressing pl6 and irradiated OPM2 cells were positive controls for pl6 and p21 content, respectively .

[0008] FIGS. 3A-3G show that Navitoclax increases hematopoietic stem and progenitor cells (HSPCs) and restores hematopoietic stem cell (HSC) function in mice with sickle cell disease (SCD). FIG. 3A, Treatment regimen of young SCD or non-SCD mice with Navitoclax (i.e. ABT-263) followed by bone marrow analysis and transplant to assess HSPC numbers and function. Quantification of LT-HSCs (FIG. 3B), ST-HSCs (FIG. 3C), and c-kit⁺ hematopoietic progenitors (FIG. 3D) in SCD and non-SCD mice treated with vehicle or ABT-263 (n=6-8 for all groups). FIG. 3E, Quantification of DNA damage in HSPCs (Lineage-Scal⁺c-kit⁺ cells) isolated from SCD and non- SCD mice treated with vehicle or ABT-263 (n=4 for all groups). FIG. 3F, %CD45.2⁺ peripheral blood of recipients of bone marrow from non-SCD or SCD mice treated with vehicle or ABT- 263 over time post-transplant (n=7-12 for non-SCD groups and n-5-9 for SCD groups). FIG. 3G, %CD45.2⁺ peripheral blood of recipients of bone marrow from non-SCD and SCD mice treated with vehicle or ABT-263 at 16-weeks post transplantation (n=12 and 10 for recipients of cells isolated from non-SCD mice treated with vehicle or ABT-263, respectively. N=9 for recipients of cells isolated from SCD mice treated with vehicle or ABT-263.

Detailed Description of the Invention

[0009] Hematopoietic stem cells (HSCs) reside in the bone marrow and are responsible for life-long blood cell production. They are also the critical therapeutic cell population used in HSC transplantation therapies, which can involve transplantation of bone marrow- or mobilized peripheral blood-derived stem cells. To encourage HSCs to migrate from the bone marrow and into the peripheral blood where they can be easily collected and used for gene therapy or transplantation, individuals may be treated with mobilizing agents. A major challenge is that many individuals with sickle cell disease cannot mobilize enough HSCs into the peripheral blood for collection, especially as they get older. Further, many individuals reguire several rounds of mobilization and collection, which take times, are challenging for the patients, and is very expensive. [0010] It has now been shown that HSCs in individuals and mice with sickle cell disease are enriched for cells with molecular programs and biomarkers of senescence. In particular, elevated cycling, DNA damage, reactive oxygen species, and hallmarks of senescence were observed in bone marrow hematopoietic stem and progenitor cells (HSPCs) from sickle cell disease (SCO) mice, which correlated with a loss of long-term repopulating HSPCs. Bone marrow HSPCs from individuals with SCD also display hallmarks of senescence and diminished function. Transcriptomic profiling of mouse and human HSPCs revealed reduced expression of genes regulating cell cycle, DNA replication, and DNA repair, consistent with senescence. Treatment of SCD mice with the senolytic, ABT- 263 (Navitoclax), increased HSPC frequency, restored HSPC transplantation activity, and decreased numbers of HSPCs with DNA damage. Not wishing to be bound by theory, it is believed that chronic exposure to complex SCD pathophysiology, including hemolysis and chronic systemic inflammation, and the rapid turnover of blood forming cells in the bone marrow due to chronic anemia, directly drives acquisition of senescence in HSCs. As such, the results herein indicate that ABT-263, and other senolytic agents, can be used to treat individuals with sickle cell disease and restore function to, and the quality of, the HSC pool in this context. Accordingly, provided herein is a method for treating, preventing, reducing, or eliminating a manifestation or complication associated with sickle cell disease in a subject by administering to the subject an effective amount of at least one senolytic agent. Use of a senolytic agent in the treatment of a healthy subject or an individual with sickle cell disease can eliminate senescent HSCs from their bone marrow, improve the quality and function of the subject's HSC pool, increase the total HSC yield following mobilization and apheresis, or via other collection methods, thereby improving the quality of 'product' for gene-editing/gene-therapy and autologous HSC transplantation or allogeneic transplantation.

[0011] As used herein, the term "sickle cell disease" or "SCD" refers to a group of inherited red blood cell disorders in which affected persons have an abnormal protein in their red blood cells. More common types of sickle cell disease include Hemoglobin SS, (HbSS; also called sickle cell anemia, is usually the most severe type of this disorder); Hemoglobin SC (HbSC; usually mild); Hemoglobin Sp thalassemia (of which there are two types: "0" and

HbS beta 0-thalassemia is usually more severe; HbS betad— thalassemia is usually less severe) . Some other types of sickle cell disease include Hemoglobin SD, Hemoglobin SE, Hemoglobin SO, etc. Manifestations and complications of sickle cell disease depend on the patient and severity of the disease. Early manifestations and complications may include painful swelling of the hands and feet, dark urine, symptoms of anemia such as fatigue and paleness, and jaundice. Over time, sickle cell disease can lead to additional manifestations and complications such as infections, delayed and/or stunted growth, and episodes of pain (e.g., vaso-occlusive pain events or vaso-occlusive pain crises). Younger children who have sickle cell disease may be pain-free or have reduced pain between crises. Adolescents and adults (including young adults) may suffer chronic, ongoing pain. Additional manifestations and complications that may be present in patients include, but are not limited to, fatigue, hospitalization, poor sleep quality, opiate use, acute blood transfusion therapy given for disease manifestations, chronic blood transfusion therapy given to prevent disease complications, acute chest syndrome, bone infarcts, avascular necrosis, osteonecrosis, stroke (e.g., ischemic stroke or hemorrhagic stroke), priapism, painful unwanted erection of penis, low body weight, growth delays, low body-mass index, slowed growth, and cardiovascular disorders. Over time, sickle cell disease may harm a patient's organs including the spleen, brain, eyes, lungs, liver, heart, kidneys, penis, joints, bones, and/or skin. Individuals with sickle cell disease may also be at a higher risk of developing leukemia. Individuals with sickle cell disease also could die prematurely due to all of these complications.

[0012] A "senolytic, " "senolytic agent," or "senolytic compound" refers to a compound that selectively (preferentially or to a greater degree) destroys, kills, removes, or facilitates selective destruction of senescent cells, i.e., the compound destroys or kills a senescent cell in a biologically, clinically, and/or statistically significant manner compared with its capability to destroy or kill a non-senescent cell. The senolytic compound is used in an amount and for a time sufficient to selectively kill established senescent cells, but which is insufficient to kill non-senescent cells in a clinically significant or biologically significant manner.

[0013] In some aspects, a senolytic agent described herein alters at least one signaling pathway in a manner that induces (initiates, stimulates, triggers, activates, promotes) and results in death of the senescent cell. A senolytic agent may alter one or more signaling pathways in a senescent cell by interacting with one or more target proteins, which results in removing or reducing suppression of a cell death pathway, such as an apoptotic pathway. For example, contacting or exposing a senescent cell to a senolytic agent may restore the cell's mechanisms and pathways for initiating apoptosis. In one aspect, the senolytic agent induces apoptosis.

[0014] Senolytic agents targeting Bel (B-cell lymphoma) protein family members, protein kinase B (Akt), and/or MDM2 (MDM2 Proto-Oncogene, E3 Ubiquitin Protein Ligase) have been described. In addition, FAK inhibitors, HMG-CoA reductase inhibitors, HSP90 inhibitors, Src kinase inhibitors, PI3- kinase inhibitors, proteasome inhibitors, HDAC inhibitors, and/or p97 pathway inhibitors may be used as senolytic agents. See, e.g., Kirkland & Tchkonia (2020) J. Int. Med. 288:518- 536; Chaib, et al. (2022) Nat. Med. 28:1556-1568. In some aspects, the senolytic agent of use in the methods of this invention may be a Bel inhibitor, MDM2 inhibitor, and/or Akt inhibitor. In some aspects, the senolytic agent is a Bel inhibitor .

[0015] The Bel protein family includes evolutionarily conserved proteins that share Bcl-2 homology (BH) domains. Bel proteins are most notable for their ability to up- or down-regulate apoptosis, a form of programmed cell death, at the mitochondrion. In the context of this invention, the Bel proteins of particular interest are those that downregulate apoptosis. All proteins belonging to the Bcl-2 family contain either a BH1, BH2, BH3, or BH4 domain. All anti-apoptotic proteins contain BH1 and BH2 domains, some of them contain an additional N-terminal BH4 domain. Proteins that are known to contain these domains include vertebrate Bcl-2 (alpha and beta isoforms), Bcl-x (isoforms (Bcl-xL and Bcl-xS), Bcl-w, and human induced myeloid leukemia cell differentiation protein Mell. Inhibiting these proteins increases the rate or susceptibility of cells to apoptosis. Thus, an inhibitor of such proteins may be used to help eliminate cells in which the proteins are expressed. [0016] Senolytic agents that act as Bel inhibitors may be characterized as a benzothiazole-hydrazone, an amino pyridine, a benzimidazole, a benzylpiperazine, a tetrahydroquinolin, or a phenoxyl compound. Exemplary compounds of use in inhibiting Bcl-2, Bcl-xL, Bcl-xS, Bcl-w, and/or Mell include, but are not limited to, ABT-737 (CAS No. 852808-04-9), Navitoclax (ABT-263; CAS No. 923564-51-6), Pelcitoclax (APG-1252-M1, BM-1244; CAS No. 1619923-32-8), Obatoclax (GX15-070; CAS No. 803712-67-6), A-1155463 (CAS No. 1235034-55-5), Lisaftoclax (APG-2575; CAS No. 2180923-05-9), Venetoclax (ABT-199, GDC-0199, RG7601; CAS No. 1257044-40-8) and its prodrug ABBV-167 (CAS No. 1351456-78-4), Sabutoclax (BI-97C1; CAS No. 1228108-65-3), Maritoclax (Marinopyrrole A; CAS No. 1227962-62-0), Lacutoclax (CAS No. 2291166-56-6), Tapotoclax (AMG-176; CAS No. 1883727-34-1), Pyridoclax (MR- 29072; CAS No. 1651890-44-6), AB141523 (2-methoxy-antimycin A3), A-1331852 (CAS No. 1430844-80-6), A-385358 (CAS No. 406228-55-5), A-1210477 (CAS No. 1668553-26-1), TW-37 (CAS No. 877877-35-5), oblimersen (G3139; CAS No. 190977-41-4), LP-118, LP-108, gambogic acid (CAS No. 2752-65-0), UMI-77 (CAS No. 518303-20-3), S6345 (CAS No. 1799633-27-4), S55746 (CAS No. 1448584-12-0), MIK665 (S64315; CAS No. 1799631-75- 6), AZD5991 (CAS No. 2143010-83-5), VU661013 (CAS No. 2131184-57-9), YC137 (CAS No. 810659-53-1), Antimycin A (CAS No. 1397-94-0), BH3I-1 (CAS No. 300817-68-9), BM-957 (CAS No. 1391107-54-2), WEHI-539 (CAS No. 2070018-33-4), AZD4320 (CAS No. 1357576-48-7), BH3I-1 (CAS No. 300817-68-9), BM-1074 (CAS No. 1391108-10-3), BM-1197 (UBX1967; CAS No. 1391107-89-3), HA14-1 (CAS No. 65673-63-4), BXI-72 (NSC334072; CAS No. 23491-52-3), EU5346 (ML311; CAS No. 315698-17-0), (-)BI97D6 (BI112D1), BDA-366 (NSC639366; CAS No. 1909226-00-1), S63845 (CAS No. 1799633-27-4), SW076956, SW063058, gossypolone (CAS No. 4547-72-2), apogossypol (NSC 736630; CAS No. 66389-74- 0), apogossypolone (CAS No. 886578-07-0), R-(-)-gossypol (AT- 101; CAS No. 866541-93-7), S-(-)-gossypol (CAS No. 1189561- 66-7), gossypol (BL 193; CAS No. 303-45-7), 2,3-DCPE (2-[[3- (2,3-dichlorophenoxy)propyl]amino]ethanol; CAS No. 1009555- 55-8), Compound 21 ((R) 4-(4-chlorophenyl)-3-(3-(4-(4-(4-

( (4- (dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl) phenyl)-5- ethyl-l-methyl-lH-pyrrole-2-carboxylic acid) , Compound 14 ((R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)- 1- (phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl) phenyl)-1- ethyl-2-methyl-lH-pyrrole-3-carboxylic acid) , Compound 15 ((R)-5-(4-Chlorophenyl)-4-(3- (4-(4-(4((4-(dimethylamino)-1- (phenylthio)butan-2-yl)amino)-3- nitrophenylsulfonamido)phenyl)piperazin-l-yl) phenyl)-1- isopropyl-2-methyl-lH-pyrrole-3-carboxylic acid), and pharmaceutically acceptable salts thereof. See also US 8,691,184, US 9,096,625, US 9,403,856, and WO 2016/127135, the disclosure of which pertaining to inhibitors is incorporated herein by reference.

[0017] In some aspects, the methods of the invention provide for the administration of a Bcl-2 inhibitor. In some aspects, the Bcl-2 inhibitor used herein is Navitoclax, Venetoclax, ABT-737, Obatoclax, oblimersen, Pelcitoclax, Lisaftoclax, LP- 118, LP-108, HA14-1, TW-37, and pharmaceutically acceptable salts thereof.

[0018] In some aspects, the methods of the invention provide for the administration of a Bcl-xL inhibitor. In some aspects, the Bcl-xL inhibitor used herein is A-1155463, A-1331852, A- 385358, AB141523, BH3I-1, Pelcitoclax, Lisaftoclax, gossypol (BL 193), R- (-)-gossypol, S-(-)-gossypol, apogossypol, gossypolone, Sabutoclax, WEHI-539, 2,3-DCPE, and pharmaceutically acceptable salts thereof.

[0019] Senolytic agents that act as MDM2 inhibitors may be characterized as a cis-imidazoline, a dihydroimidazothiazole, a spiro-oxindole, a benzodiazepine, or a piperidinone . compound. Exemplary compounds of use in inhibiting MDM2 include, but are not limited to, a nutlin compound (e.g., Nutlin-1 (CAS No. 548472-58-8), Nutlin-2 (CAS No. 548472-76- 0), or Nutlin-3a (CAS No. 548472-68-0)) or nutlin derivative (e.g., Idasanutlin (RG7388, R05503781; CAS No. 1229705-06- 9)), Milademetan (DS-3032b, CAS No. 2095625-97-9), Navtemadlin (AMG 232; CAS No. 1352066-68-2), Sulanemadlin (ALRN-6924; CAS No. 1451199-98-6), Siremadlin (NVP-HDM201; CAS No. 1448867-41-1), Alrizomadlin (APG-115; CAS No. 1818393-16-6), Brigimadlin (BI 907828; CAS No. 2095116-40- 6), RG-7112 (R05045337; CAS No. 939981-39-2), NVP-CGM097 (CAS No. 1313363-54-0), MDM2-IN-1 (CAS No. 1410737-09-5), NSC 66811 (CAS No. 6964-62-1), 3- (4-chlorophenyl)-3- ((1-

(hydroxymethyl)cyclopropyl)methoxy)-2-(4- nitrobenzyl)isoindolin-l-one, BI-0282 (Compound 1; CAS No. 1883383-48-9), MI-773 (CAS No. 1303607-07-9), SAR405838 (MI- 77301; CAS No. 1303607-60-4), and pharmaceutically acceptable salts thereof.

[0020] Senolytic agents that act as inhibitors of Akt are the competitive Akt inhibitors may include, e.g., CCT128930, GDC-0068, GSK2110183 (afuresertib), GSK690693, and AT7867; the lipid-based Akt inhibitors Calbiochem Akt Inhibitors I, II and III, PX-866, and Perifosine (KRX-0401); the pseudosubstrate inhibitors vKTide-2 T and F0XO3 hybrid; allosteric inhibitors of the Akt kinase domain including MK- 2206 (8-[4- (1-aminocyclobutyl)phenyl]-9-phenyl-2H-

[1,2,4]triazolo [3,4-f][1,6]naphthyridin-3-one; dihydrochloride); the antibody GST-anti-Aktl-MTS; the compounds that interact with the PH domain of Akt Triciribine and PX-316; and other compounds exemplified by GSK-2141795, VQD-002, miltefosine, AZD5363, GDC-0068, and API-1.

[0021] Other suitable senolytic agents include, e.g., cardiac glycoside or aglycone, JFD00244, Cyclosporine, Tyrphostin AG879, Cantharidin, Diphenyleneiodonium chloride, Rottierin, 2,3-Dimethoxy-1,4-naphthoquinone, LY-367,265, Rotenone, Idarubicin, Dequalinium chloride, Vincristine, Nitazoxanide, Nitrofurazone, Temsirolimus, Eltrombopag, Adapalene, Azacyclonol, Enoxacin, dasatinib, Fisetin Quercetin, piperlongumine and Raltegravir, and pharmaceutically acceptable salts thereof.

[0022] Senolytic agents disclosed herein can be used alone or in combination with each other or other agents used in the treatment of a sickle cell disease. In some aspects, a synergy may be obtained by administering a Bcl-2 inhibitor with a Mcl-1 inhibitor or another agent. See, e.g., US 2021/0379078 Al.

[0023] Provided herein is a method of treating, preventing, reducing, or eliminating a manifestation or complication associated with sickle cell disease in a subject in need thereof, optionally wherein the manifestation or complication is selected from pain, fatigue, hospitalization, poor sleep quality, opiate use, acute blood transfusion therapy given for disease manifestations, chronic blood transfusion therapy given to prevent disease complications, acute chest syndrome, bone infarct, avascular necrosis, osteonecrosis, stroke, priapism, a cardiovascular disorder, growth delay, stunted growth, low body mass index (BMI), low body weight, organ damage (e.g,, end organ damage), cognitive dysfunction, chronic systemic inflammation, and hemolysis-associated endothelial dysfunction. In some aspects, the method of treating, preventing, reducing, or eliminating a manifestation or complication associated with sickle cell disease in a subject in need thereof will result in improved HSC function.

[0024] The effects herein for treating, preventing, reducing, or eliminating a manifestation or complication associated with sickle cell disease in a subject in need thereof may be demonstrated after a period of administering a treatment regimen described herein. For example, in one aspect, the treatment, reduction, or elimination of a manifestation or complication of sickle cell disease after treatment may be achieved by the administration of the senolytic agent at a certain dose and/or a certain administration schedule, optionally in combination with an additional therapeutic agent as described herein.

[0025] The term "administering" in relation to a compound, e.g., a senolytic agent, is used to refer to delivery of that compound to a patient by any route.

[0026] As used herein, the term "patient" and "subject" are interchangeable. In some aspects, a subject in need of treatment with a senolytic agent in accordance with the method of this invention may be a subject who has poor control of their disease manifestations and requires an improved quality of their stem cells, e.g., to directly help with blood formation and allow for hematopoietic cell transplant, gene therapy, or gene editing. Such a subject may be identified using biomarkers of senescence to evaluate the quality of their bone marrow HSC pool.

[0027] In some aspects, treatment of the manifestations or complications of sickle cell disease may include preventing or reducing severity and/or duration of the one or more manifestation (s) or complication (s) of sickle cell disease, wherein each of the manifestation (s) or complication (s) may be selected from pain, fatigue, hospitalization, poor sleep quality, opiate use, acute blood transfusion therapy given for disease manifestations, chronic blood transfusion therapy given to prevent disease complications, acute chest syndrome, bone infarct, avascular necrosis, osteonecrosis, stroke, cognitive dysfunction, priapism, infarction of penis, a cardiovascular disorder, growth delay, stunted growth, low body mass index (BMI), low body weight, and organ damage. In some aspects, treatment of the manifestations or complications of sickle cell disease may include reducing the number or percentage of non-functional and senescent HSCs and/or restoring or improving function of the HSC pool in bone marrow.

[0028] In some aspects, the subject may be a pediatric patient or pediatric subject. In some aspects, the pediatric patient is under 18 years old. In some aspects, the pediatric patient may be an adolescent of between 12 and 18 years. In some aspects, the pediatric patient may be a child of under 12 years. In some aspects, the subject may be an adult patient. In some aspects, the adult patient is 18 years old or older. In some aspects, the adult patient is 21 years old or older.

[0029] In some aspects, the cardiovascular disorder is any cardiovascular disorder associated with sickle cell disease. In certain aspects, the cardiovascular disorder is selected from hypertension, peripheral vascular disease, heart failure, coronary artery disease (CAD), ischemic heart disease (IHD), mitral stenosis and regurgitation, angina, hypertrophic cardiomyopathy, diabetic cardiomyopathy, supraventricular and ventricular arrhythmias, cardiac dysrhythmia, atrial fibrillation (AF), new onset of atrial fibrillation, recurrent atrial fibrillation, cardiac fibrosis, atrial flutter, detrimental vascular remodeling, plaque stabilization, and myocardial infarction (MI).

[0030] In some embodiments, the organ damage is damage to one or more organs selected from spleen, brain, eyes, lungs, liver, heart, kidneys, penis, joints, bones, and skin. Such organ damage may be any type known to be caused by sickle cell disease. In some embodiments the organ damage is stroke (e.g., ischemic stroke or a hemorrhagic stroke).

[0031] By "an effective amount" is meant the amount of a required agent or composition comprising the agent to ameliorate or eliminate symptoms of a disease relative to an untreated patient. The effective amount of composition (s) used to practice the methods described herein for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

[0032] In one aspect, at least one senolytic agent is administered. For example, 1, 2, 3, 4, 5 or more senolytic agents may be administered. Each senolytic agent administered may target the same or different anti-apoptotic protein in the Bcl~2 family. In one aspect, two inhibitors of one or more anti-apoptotic proteins in the Bcl-2 family may be administered. In another aspect, one inhibitor of one or more anti-apoptotic proteins in the Bcl-2 family may be administered.

[0033] Dosages of the senolytic agent may vary between wide limits, depending upon the severity of the sickle cell disease to be treated, the age and the condition of the subject to be treated. In one aspect, the senolytic agent may be administered to a subject at a dose from about 0.1 mg/kg to about 500 mg/kg. For example, the dose of the senolytic agent may be about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, or about 25 mg/kg. Alternatively, the dose of the senolytic agent may be about 25 mg/kg, about 50 mg/kg, about 75 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 225 mg/kg, or about 250 mg/kg. Additionally, the dose of the senolytic agent may be about 300 mg/kg, about 325 mg/kg, about 350 mg/kg, about 375 mg/kg, about 400 mg/kg, about 425 mg/kg, about 450 mg/kg, about 475 mg/kg or about 500 mg/kg. A composition comprising at least one senolytic agent (e.g., a pharmaceutical composition) may be administered to a subject at various frequencies, intervals, and durations by various routes (topical application, enteral, or parenteral administration).

[0034] In some aspects, the step of administering the senolytic agent to the subject may be carried out daily, multiple times a week, once every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, or every twelve weeks in order to reduce or eliminate senescent HSCs, restore function to the HSC pool, and/or reduce the severity and/or duration of one or more manifestation (s) or complication (s) of sickle cell disease described herein.

[0035] The senolytic agent may be administered in the form of a pharmaceutical composition comprising at least one senolytic agent in admixture with a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a diluent, a binder, a filler, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant, taste-masking agent, a flavoring agent, or a coloring agent. The amount and types of excipients used to form the pharmaceutical composition may be selected according to known principles of pharmaceutical science. [0036] In one aspect, the excipient may be a diluent. The diluent may be compressible (i.e., plastically deformable) or abrasively brittle. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol, xylitol, maltodextrin, and trehalose. Non-limiting examples of suitable abrasively brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), calcium phosphate tribasic, calcium carbonate, and magnesium carbonate .

[0037] In another aspect, the excipient may be a binder. Suitable binders include, but are not limited to, starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, Ci2“Cis fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof. [0038] In another aspect, the excipient may be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. By way of non-limiting example, the filler may be calcium sulfate, both di- and tri-basic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, lactose, sucrose, mannitol, or sorbitol.

[0039] In still another aspect, the excipient may be a buffering agent. Representative examples of suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., Tris-buffered saline or phosphate-buffered saline).

[0040] In various aspects, the excipient may be a pH modifier. By way of non-limiting example, the pH modifying agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

[0041] In a further aspect, the excipient may be a disintegrant. The disintegrant may be non-effervescent or effervescent. Suitable examples of non-ef fervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0042] In yet another aspect, the excipient may be a dispersant or dispersing enhancing agent. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.

[0043] In another aspect, the excipient may be a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; and antimicrobials, such as parabens, chlorobutanol, or phenol.

[0044] In a further aspect, the excipient may be a lubricant. Non-limiting examples of suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate or stearic acid.

[0045] In yet another aspect, the excipient may be a tastemasking agent. Taste-masking materials include cellulose ethers; polyethylene glycols; polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers; monoglycerides or triglycerides; acrylic polymers; mixtures of acrylic polymers with cellulose ethers; cellulose acetate phthalate; and combinations thereof.

[0046] In another aspect, the excipient may be a flavoring agent. Flavoring agents may be chosen from synthetic flavor oils and flavoring aromatics and/or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof .

[0047] In still a further aspect, the excipient may be a coloring agent. Suitable color additives include, but are not limited to, food, drug, and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C). [0048] The weight fraction of the excipient or combination of excipients in the pharmaceutical composition may be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the pharmaceutical composition,

[0049] The pharmaceutical composition may be formulated into various dosage forms and administered by a number of different means that will deliver a therapeutically effective amount of the active ingredient. Such pharmaceutical compositions may be administered orally (e.g., by mouth or inhalation), parenterally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-osseous, intra-articular, or intrasternal injection, or infusion techniques. In one aspect, a composition may be a food supplement or a cosmetic.

[0050] Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets, and granules. In such solid dosage forms, the active ingredient is ordinarily combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above. Oral preparations may also be administered as aqueous suspensions, elixirs, or syrups. For these, the active ingredient may be combined with various sweetening or flavoring agents, coloring agents, and, if so desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0051] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular, intra-osseous, intra-articular and intraperitoneal) , the preparation may be an aqueous or an oil-based solution. Aqueous solutions may include a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents; an antibacterial and/or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and/or an agent for the adjustment of tonicity such as sodium chloride, dextrose, or a polyalcohol such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Oil-based solutions or suspensions may further comprise sesame, peanut, olive oil, or mineral oil. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0052] For topical (e.g., transdermal or transmucosal) administration, penetrants appropriate to the barrier to be permeated are generally included in the preparation. Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some aspects, the pharmaceutical composition is applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes. Transmucosal administration may be accomplished through the use of nasal sprays, aerosol sprays, tablets, or suppositories, and transdermal administration may be via ointments, salves, gels, patches, or creams as generally known in the art.

[0053] In certain aspects, a pharmaceutical composition including at least one senolytic agent is encapsulated in a suitable vehicle to either aid in the delivery of the compound to target cells, to increase the stability of the composition, or to minimize potential toxicity of the composition. As will be appreciated by a skilled artisan, a variety of vehicles are suitable for delivering a composition of the present invention. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers, and other phospholipidcontaining systems. Methods of incorporating compositions into delivery vehicles are known in the art.

[0054] In one aspect, a liposome delivery vehicle may be utilized. Liposomes are spherical vesicles with a phospholipid bilayer membrane. The lipid bilayer of a liposome may fuse with other bilayers (e.g., the cell membrane), thus delivering the contents of the liposome to cells. In this manner, at least one senolytic agent may be selectively delivered to a cell by encapsulation in a liposome that fuses with the targeted cell's membrane.

[0055] Liposomes may be composed of a variety of different types of phospholipids having varying hydrocarbon chain lengths. Phospholipids generally comprise two fatty acids linked through glycerol phosphate to one of a variety of polar groups. Suitable phospholipids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fatty acid chains comprising the phospholipids may range from about 6 to about 26 carbon atoms in length, and the lipid chains may be saturated or unsaturated. Suitable fatty acid chains include (common name presented in parentheses) n- dodecanoate (laurate), n-tretradecanoate (myristate), n- hexadecanoate (palmitate), n-octadecanoate (stearate), n- eicosanoate (arachidate), n-docosanoate (behenate), n- tetracosanoate (lignocerate), cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate), cis,cis-9,12- octadecadienoate (linoleate), all cis-9, 12, 15- octadecatrienoate (linolenate), and all cis-5,8,11,14- eicosatetraenoate (arachidonate). The two fatty acid chains of a phospholipid may be identical or different. Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS, distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl, oleoyl PS, palmitoyl, linolenyl PS, and the like.

[0056] The phospholipids may come from any natural source, and, as such, may include a mixture of phospholipids. For example, egg yolk is rich in PC, PG, and PE; soybeans contain PC, PE, PI, and PA; and animal brain or spinal cord is enriched in PS. Phospholipids may come from synthetic sources too. Mixtures of phospholipids having a varied ratio of individual phospholipids may be used. Mixtures of different phospholipids may result in liposome compositions having advantageous activity or stability of activity properties. The above mentioned phospholipids may be mixed, in optimal ratios with cationic lipids, such as N-(l-(2,3- dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride, 1,1'- dioctadecyl-3,3,3 ',3'-tetramethylindocarbocyanine perchlorate, 3,3'-deheptyloxacarbocyanine iodide, 1,1'- dedodecyl-3,3,3 ',3'-tetramethylindocarbocyanine perchlorate, 1,1'-dioleyl-3,3 ,3',3'-tetramethylindo carbocyanine methanesulfonate, N-4- (delinoleylaminostyryl)-N- methylpyridinium iodide, or 1,1,-dilinoleyl-3, 3,3',3'- tetramethylindocarbocyanine perchlorate.

[0057] Liposomes may optionally include sphingolipids, in which spinosine is the structural counterpart of glycerol and one of the one fatty acids of a phosphoglyceride, or cholesterol, a major component of animal cell membranes. Liposomes may optionally contain pegylated lipids, which are lipids covalently linked to polymers of polyethylene glycol (PEG). PEGs may range in size from about 500 to about 10,000 daltons.

[0058] Liposomes may further include a suitable solvent. The solvent may be an organic solvent or an inorganic solvent. Suitable solvents include, but are not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone, N- methylpyrrolidone, acetronitrile, alcohols, dimethylformamide, tetrahydrofuran, or combinations thereof. [0059] Liposomes carrying at least one senolytic agent may be prepared by any known method of preparing liposomes for drug delivery, such as, for example, detailed in US 4,241,046, US 4,394,448, US 4,529,561, US 4,755,388, US 4,828,837, US 4,925,661, US 4,954,345, US 4,957,735, US 5,043,164, US 5,064,655, US 5,077,211 and US 5,264,618, the disclosures of which are hereby incorporated by reference in their entirety. For example, liposomes may be prepared by sonicating lipids in an aqueous solution, solvent injection, lipid hydration, reverse evaporation, or freeze drying by repeated freezing and thawing. In one aspect, the liposomes may be formed by sonication. The liposomes may be multilamellar, which have many layers like an onion, or unilamellar. The liposomes may be large or small. Continued high-shear sonication tends to form smaller unilamellar liposomes.

[0060] As would be apparent to one of ordinary skill, all of the parameters that govern liposome formation may be varied. These parameters include, but are not limited to, temperature, pH, concentration of methionine compound, concentration and composition of lipid, concentration of multivalent cations, rate of mixing, presence of and concentration of solvent.

[0061] In another aspect, a pharmaceutical composition of the invention may be delivered to a cell as a microemulsion. Microemulsions are generally clear, thermodynamically stable solutions comprising an aqueous solution, a surfactant, and "oil." The "oil" in this case, is the supercritical fluid phase. The surfactant rests at the oil-water interface. Any of a variety of surfactants are suitable for use in microemulsion formulations including those described herein or otherwise known in the art. The aqueous microdomains suitable for use in the invention generally will have characteristic structural dimensions from about 5 nm to about 100 nm. Aggregates of this size are poor scatterers of visible light and hence, these solutions are optically clear. As will be appreciated by a skilled artisan, microemulsions can and will have a multitude of different microscopic structures including sphere, rod, or disc shaped aggregates. In one embodiment, the structure may be micelles, which are the simplest microemulsion structures that are generally spherical or cylindrical objects. Micelles are like drops of oil in water, and reverse micelles are like drops of water in oil. In some aspects, the microemulsion structure is the lamellae. It may include consecutive layers of water and oil separated by layers of surfactant. The "oil" of microemulsions optimally includes phospholipids. Any of the phospholipids detailed above for liposomes are suitable for embodiments directed to microemulsions. At least one senolytic agent may be encapsulated in a microemulsion by any method generally known in the art.

[0062] In yet another aspect, at least one senolytic agent may be delivered in a dendritic macromolecule, or a dendrimer. Generally speaking, a dendrimer is a branched tree-like molecule, in which each branch is an interlinked chain of molecules that divides into two new branches (molecules) after a certain length. This branching continues until the branches (molecules) become so densely packed that the canopy forms a globe. Generally, the properties of dendrimers are determined by the functional groups at their surface. For example, hydrophilic end groups, such as carboxyl groups, would typically make a water-soluble dendrimer. Alternatively, phospholipids may be incorporated in the surface of a dendrimer to facilitate absorption across the skin. Any of the phospholipids detailed for use in liposome embodiments are suitable for use in dendrimer embodiments. Any method generally known in the art may be utilized to make dendrimers and to encapsulate compositions of the invention therein. For example, dendrimers may be produced by an iterative sequence of reaction steps, in which each additional iteration leads to a higher order dendrimer. Consequently, they have a regular, highly branched 3D structure, with nearly uniform size and shape. Furthermore, the final size of.a dendrimer is typically controlled by the number of iterative steps used during synthesis. A variety of dendrimer sizes are suitable for use in the invention. Generally, the size of dendrimers may range from about 1 nm to about 100 nm.

[0063] The treatment described herein may be combined with other treatment partners or therapeutic agents such as the current standard of care for a disease associated with sickle cell disease. For example, the senolytic agent may be combined with one or more of an antibody or antigen binding fragment thereof that specifically binds to P-selectin, L-glutamine oral powder, an agent that increases fetal hemoglobin, and combinations thereof. In some aspects, the agent that increases fetal hemoglobin is hydroxyurea, an antibody or antigen binding fragment thereof that specifically binds to P-selectin, L-glutamine oral powder, voxelotor, or stem cells (e.g., blood-producing hematopoietic stem cells (HSCs)) comprising a lentiviral vector which inserts a functioning version of the HBB or the HBG genes (e.g., lovotibeglogene autotemcel (lovo-cel), or betibeglogene autotemcel, or LentiGlobin BB305) or a modified version thereof. In some aspects, the subject has already been treated with one or more gene replacement or gene editing therapies (e.g., exagamglogene autotemcel (exa-cel), or CTX-001, ST-100, or RVT-1801) but may still have one or more manifestations or complications of sickle cell disease (e.g., one or more of the manifestations or complications described herein). In such cases, the subject may additionally benefit from one or more of the treatment regimens described herein. In some cases, treatment described herein may be administered to the subjects prior to collection of their hematopoietic stem cells for genetic modification. In other cases, treatment described herein may be administered to the subjects before they are administered drug products for ex vivo or in vivo genetic modification. Alternatively, the treatment described herein may be administered to allogeneic donors before they donate hematopoietic stem cells for transplantation into another individual.

[0064] As another set of non-limiting examples, the senolytic agent may be combined with one or more of an IL-18 inhibitor and/or other inflammasome pathway inhibitor (e.g., inhibitors of NLRP3, gasdermin D, or other inflammasome pathway members), an agent preventing red cell alterations (e.g., Hb polymerization, dehydration, microparticle generation, or mobilization of Weibel-Palade bodies), an ATP or ATP receptor inhibitor, a Bachl inhibitor, a CD40 pathway inhibitor, a P- selectin pathway inhibitor, an inhibitors of an adhesion molecule (e.g., ICAM or VCAM), a compound preventing platelet activation, a platelet stabilizing compound (e.g., multimerized IgGlFc or IVIG), a TLR antagonist (e.g., an antagonist of TLR4, TLR7, TLR8, and/or TRL9), a ROS inhibitor, an agent for regulation of oxygen regulated genes (e.g., HIF2a), an agent that prevents or disrupts NET formation, a complement inhibitor, a heme oxygenase-l/HO-1 pathway modulator, a CXCR4 pathway modulator, a phosphodiesterase inhibitor, and agent that acts in an anti-angiogenic manner, EPO, a leukotriene inhibitor (e.g., Leukotriene A-4 hydrolase), or combinations thereof.

[0065] As indicated herein, senolytic agents can restore function to the HSC pool. Therefore, this invention also provides a method for improving the quality of HSCs of a subject by administering to the subject at least one senolytic agent, e.g., to eliminate non-functional and senescent HSCs and improve the quality of the subject's HSCs. Treatment of a subject with at least one of the senolytic agents described herein allows for mobilization and/or collection of a healthier pool of HSCs for transplantation and/or increases the total HSC yield following mobilization.

[0066] In some aspects, the subject may be further administered at least one mobilizing agent during or after treatment with the at least one senolytic agent. In some aspects, the at least one mobilizing agent is plerixafor, with or without granulocyte colony-stimulating factor (G-CSF, e.g., filgrastim or GRANIX) or another mobilizing agent. In some aspects, a single dose of plerixafor, IUPAC name: l-{[4- (1,4,8,11-tetrazacyclotetradec-l-ylmethyl)phenyl]methyl}- 1,4,8,11 tetrazacyclotetradecane) is used as the mobilizing agent. Plerixafor is well known in the art and disclosed in, e.g., US 2014/0219952, US 2018/0207202 and US 2014/0030308. In another aspect, the mobilizing agent is motixafortide with or without filgrastim. In another aspect, two or more doses of human G-CSF are used as the mobilizing agent. G-CSF is well known in the art and disclosed in US 2004/0028649, US 2011/0135651, US 2007/0036747, US 2005/0186182 and US 2014/0065706.

[0067] In some aspects, HSCs from the subject treated with the at least one senolytic agent (and optionally at least one mobilizing agent) are harvested or collected. Any one of a variety of apheresis methodologies known in the art may be used to collect or harvest HSCs. Exemplary methods are disclosed in, e.g., US 2016/0184361, US 2018/0043082, US 2017/0021083, US 2006/0116271, US 2005/0155932, US 2005/0143684 and US 2003/0195455.

[0068] In some aspects of this invention, HSCs are collected and isolated from the peripheral blood of the subject. In some aspects, the identification and isolation of HSCs is determined by the presence of cell surface markers. Cell surface markers useful in the identification and isolation of HSCs include, e.g., CD34+, CD59+, CD90/Thyl+, CD38^low/", CD49f, CD45RA, c-Kit^_/low, and Lin-. Detecting the expression of these marker panels allows separation of specific cell populations via techniques like fluorescence-activated cell sorting (FACS). In some aspects, the HSCs are isolated to about 90% to 95% purity, i.e., the cell population contains less than 10% of other cells types, e.g., mesenchymal stem cells, CD133+ stem/progenitor cells, CD4⁺ helper T cells, CD8⁺ cytotoxic T cells, CD14⁺ monocytes, CD19⁺ B cells, CD56⁺ NK cells, dendritic cells, macrophages, mononuclear cells, granulocytes, erythrocytes and platelets.

[0069] In some aspects, at least 1.0*10⁶, 5><10⁶, l.QxlO⁷, 5*10⁷, lxl0^e or 2*10⁸ CD34⁺ cells are isolated using the method of this invention. In some aspects, the amount of CD34+ cells isolated is sufficient to meet the threshold requirement for stem cell transplant (e.g., a target dose of 2><10⁶ CD34+ cells/kg recipient weight).

[0070] In some aspects, the subject treated with the at least one senolytic agent (and optionally at least one mobilizing agent) is a healthy donor. HSCs collected or harvested from a healthy donor treated with the at least one senolytic agent may be used in allogeneic hematopoietic cell transplant therapies. In some aspects, HSCs from a healthy donor may or may not be genetically modified. Wherein the HSCs are for allogeneic transplantation, treatment of the donor with at least one senolytic agent may lead to better long-term outcomes for the recipients of the HSCs. In some aspects, recipients of such HSCs may have a sickle cell disease.

[0071] In other aspects, the subject treated with the at least one senolytic agent (and optionally at least one mobilizing agent) has sickle cell disease. In some aspects, the subject is treated with the at least one senolytic agent and HSCs within the body (in vivo) are genetically modified by administering one or more agents, e.g., gene therapy agents described herein, to induce the genetic change in the HSCs in vivo. In other aspects, a subject with a sickle cell disease is administered at least one senolytic agent and optionally at least one mobilizing agent, HSCs are collected or harvested from the subject, and the collected or harvested HSCs are subsequently transplanted back into the subject, i.e., autologous transplantation. Wherein the HSCs are for autologous transplantation in a subject with sickle cell disease, treatment of the subject with at least one senolytic agent may lead to better long-term outcomes for these patients and expands the pool of patients that may benefit from such therapy. In some aspects, a subject with a sickle cell disease is administered at least one senolytic agent and optionally at least one mobilizing agent, HSCs are collected or harvested from the subject, and the collected or harvested HSCs are genetically modified and subsequently transplanted back into the subject. Use of a senolytic agent increases the total HSC yield from a subject with sickle cell disease leading to the collection of better quality 'product¹ for gene-editing and autologous HSC transplantation protocols used to treat the sickle cell disease and improve gene-editing treatment outcomes.

[0072] The following non-limiting examples are provided to further illustrate the present invention.

Example 1: Materials and Methods

[0073] Mice. B6;129-Hbbtm2(HBG1,HBB*) Tow/Hbbtm3 (HBG1,HBB) TowHbatml (HBA)Tow/J (Townes model) mice have been described (Ryan et al. (1990) Science 247:566-568). C57BL/6J, C57BL/6 .SJL-PtprcaPep3b/BoyJ, C57BL/6J-Ptprcem6Lutzy/J (l.e. JaxBoy) mice were obtained from Jackson Laboratory (Bar Harbor, Maine). All animals were housed in a pathogen-free facility and all experiments were carried out according to procedures approved by the St. Jude Children's Research Hospital Institutional Animal Care and Use Committee.

[0074] Bone Marrow Samples and Mononuclear Cell (MNC) Isolation from Normal and SCD Individuals. Bone marrow aspirates from children and young adults with SCD were acquired from the participants of an institutional bone marrow transplant protocol (NCT04362293) before undergoing conditioning and after obtaining written informed consent from the participant or their parent/guardian. All individuals with SCD who donated bone marrow were receiving hydroxyurea for a variable duration prior to undergoing a bone marrow aspirate. Some of them were receiving regular blood transfusions as well. This study protocol was approved by the Institutional Review Board at St. Jude Children's Research Hospital and all study related activities were performed in accordance with the Declaration of Helsinki. Bone marrow from non-SCD individuals were acquired as clinical discard from individuals who were undergoing an orthopedic surgery for any reason.

[0075] Bone marrow mononuclear cells (MNCs) were isolated by density gradient centrifugation using Ficoll-Paque® PLUS (Cytiva, Marlborough, MA) and centrifuging at 450 g for 30 minutes at room temperature with no brake. The MNC layer was then collected and washed twice in phosphate buffered saline (PBS) containing 2% fetal bovine serum (PBS). Total bone marrow MNCs were then resuspended in 90% FBS/10% dimethyl sulfoxide and aliquoted to a cell concentration of 1-3 x 10⁶ cells/vial and stored in liquid nitrogen.

[0076] Isolation of Mouse Blood and Bone Marrow, For animal studies, bone marrow was liberated from the tibias, femurs, pelvic bones, and spines of mice by crushing followed by filtration with a 70 pM cell strainer. Bone marrow cell suspensions were then incubated in red blood cell (RBC) lysis buffer (Sigma-Aldrich, St. Louis, MO) for 10 minutes on ice. Mouse peripheral blood (PB) was collected from the retro- orbital sinus and RBC lysis performed as previously described (Holmfeldt et al. (2016) J. Exp. Med. 213:433-449). For studies assessing HSPC frequency, DNA damage by yH2AX staining, ROS burden, and SA-(3-gal activity, bone marrow was isolated from mice as described but instead of RBC lysis, bone marrow was enriched for c-Kit⁺ cells (i.e._z total HSPCs) via magnetic enrichment using anti-CD117 microbeads (Miltenyi Biotec, Carlsbad, CA) and an autoMACs magnetic cell separator (Miltenyi Biotec, Carlsbad, CA) per manufacturer's instructions.

[0077] Preparation of Mouse and Human Bone Marrow for Flow Cytometry Analysis. Bone marrow HSPCs were visualized by flow cytometry after staining for 20 minutes on ice with the following antibodies: Lineage (Lin) cocktail [B220-BV605 (RA3-6B2), CD4-BV605 (GK1.5), CD8-BV605 (53-6.7), Gr-l-BV605 (RB6-8C5), Terll9-BV605 (TER-119)], Sca-l-PerCP-Cy5.5 (E13- 161.7), c-Kit-APC-780 (2B8), CD150-PE-Cy7 (TC15-12F12.2), CD48-Alexa Fluor 700 (HM48-1) (all antibodies used at 1:200 dilution, BD Biosciences, San Jose, CA). 4’,6-diamidino-2- fenilindol (DAPI, Sigma-Aldrich, St. Louis, MO) was used for dead cell exclusion. Immunophenotypic definitions of each mouse HSPC population used throughout the manuscript are as follows: LT-HSC (Lin~Sca-l⁺c-kit⁺CD150⁺CD48”), ST-HSC (Lirr Sca-l⁺c-kit⁺CD150-CD48-), MPP2 (Lin”Sca-l⁺c-kit⁺CD150⁺CD48+), and MPP3/MPP4 (Lin-Sca-l⁺c-kit⁺CD150”CD48+).

[0078] Human cryopreserved bone marrow mononuclear cells were rapidly thawed in a 37°C water bath, washed twice and resuspended in PBS with 2% FBS and 0.1 mg/mL DNasel. Bone marrow HSPCs were visualized by flow cytometry for HSCs, MPPs, and MLPs after staining for one hour on ice with the following antibodies: CD45-BV711 (HI30), Lineage-FITC (UCHT1; HCD14; 3G8; HIB19; 2H7; HCD56), CD34-APC-Cy7 (581), CD38-PE-Cy7 (HIT2), CD90-APC (5E10), CD45RA-PE-CF594 (HI100). All antibodies used at 1:200 dilution except CD34 antibody, which was used at 1:100 (BioLegend, San Diego CA). DAPI was used for dead cell exclusion. Immunophenotypic definitions of human HSPCs used throughout the manuscript are as follows: HSCs (Lin-CD45⁺CD34+CD38-CD90⁺CD45RA-), MPP (Lin” CD45+CD34+CD38-CD90-CD45RA”), and MLP (Lin”CD45⁺CD34⁺CD38”CD90” CD45RA+). [0079] Flow Cytometry Data Acquisition and Analysis. For all experiments in this study, data were acquired using a 4-laser LSR Fortessa or a 5-laser FACSymphony™ A3 (BD Biosciences, San Jose, CA) and data analysis was performed using FloJo Version 10.8 (LLC, Ashland, OR).

[0080] Analysis of Mouse Bone Marrow HSPCs for Apoptosis. Bone marrow cells were isolated as described above and following RBC lysis, cells were incubated on ice for 20 minutes with the following antibodies: B220-BV605 (RA3-6B2), CD4-BV605 (GK1.5), CD8-BV605 (53-6.7), Gr-l-BV605 (RB6-8C5), Terll9-BV605 (TER-119), Sca-l-PerCP-Cy5.5 (E13-161.7), c- Kit-APC-780 (2B8), CD150-PE-Cy7 (TC15-12F12.2), CD48-Alexa Fluor 700 (HM48-1). All antibodies were used at 1:200 dilutions and were from BD Biosciences (San Jose, CA). Stained cells were resuspended in Annexin V binding buffer (BD Biosciences, San Jose, CA) and then incubated with an Annexin V-FITC antibody (1:100 dilution, BioLegend, San Diego, CA) and DAPI for 20 minutes on ice before analysis by flow cytometry. Dying cells were defined as Annexin V⁺DAPI~.

[0081] Cell Cycle Analysis of Mouse Bone Marrow HSPCs. Bone marrow cells were isolated as described above and following RBC lysis, cells were incubated on ice for 20 minutes with the following antibodies: B220-BV605 (RA3-6B2), CD4-BV605 (GK1.5), CD8-BV605 (53-6.7), Gr-l-BV605 (RB6-8C5), Terll9- BV605 (TER-119), Sca-l-PerCP-Cy5.5 (E13-161.7), c-Kit-APC- 780 (2B8), CD150-PE-Cy7 (TC15-12F12.2), CD48-Alexa Fluor® 700 (HM48-1). All antibodies were used at 1:200 dilution and were from BD Biosciences (San Jose, CA). Stained cells were then fixed via incubation in 4% paraformaldehyde (PFA) in PBS for 15 minutes on ice. After thorough washing, fixed cells were permeabilized using Perm/Wash buffer (BD Biosciences, San Jose, CA) according to the manufacturer's instructions followed by staining with a K167-FITC (SolA15) antibody (1:50 dilution, Invitrogen, Carlsbad, CA) and DAPI. GO cells were defined as Ki67~DAPI~, G1 cells were defined as Ki67⁺DAPI~ and G2/S cells were defined as Ki67⁺DAPI⁺.

[0082] In vivo 5-Ethynyl-2 '-deoxyuridine (EdU) Uptake in Mouse HSPCs. Assessment of steady state mouse HSPCs that had transited through the S phase in middle-aged SCD and non-SCD mice was performed by in vivo administration of 200 mg/kg EdU by tail vein injection. 24 hours post-EdU administration, mice were euthanized and bone marrow was isolated and enriched for c-Kit⁺ cells. Cells were then incubated on ice for 20 minutes with the following antibodies: B220-BV605 (RA3-6B2), CD4-BV605 (GK1.5), CD8-BV605 (53-6.7), Gr-l-BV605 (RB6-8C5), Terll9-BV605 (TER-119), Sca-l-PerCP-Cy5.5 (E13-161.7), c- Kit-APC-780 (2B8), CD150-PE-Cy7 (TC15-12F12.2), CD48-Alexa Fluor® 700 (HM48-1). All antibodies were used at 1:200 dilution and were from BD Biosciences (San Jose, CA). EdU incorporation into DNA was visualized by click chemistry using the In Vivo EdU Flow Cytometry Kit 488 (Sigma-Aldrich, Burlington, MA) per manufacturer's instructions and HSPC EdU uptake was analyzed by flow cytometry as described above.

[0083] Purification of Mouse and Human HSPCs by Cell Sorting. Bone marrow was isolated from mice and enriched for c-Kit⁺ cells as described above. For collection of LT-HSCs, cells were stained with B220-BV605 (RA3-6B2), CD4-BV605 (GK1.5), CD8-BV605 (53-6.7), Gr-l-BV605 (RB6-8C5), Terll9-BV605 (TER- 119), Sca-l-PerCP-Cy5.5 (E13-161.7), c-Kit-APC-780 (2B8), CD150-PE-Cy7 (TC15-12F12.2), CD48-Alexa Fluor® 700 (HM48-1). DAPI (Sigma-Aldrich, St. Louis, MO) was used for dead cell exclusion .

[0084] To collect human HSPCs, cryopreserved bone marrow MNCs were rapidly thawed in a 37°C water bath, washed twice, and then resuspended in PBS with 2% BBS. For collection of HSCs, MPPs, and MLPs, cells were stained for one hour on ice with CD45-BV711 (HI30), Lineage-FITC (UCHT1; HCD14; 3G8; HIB19; 2H7; HCD56), CD34-Alexa Fluor® 700 (581), CD38-PE-Cy7 (HIT2). All antibodies were used at 1:200 dilution except CD34, which was used at 1:100. All antibodies came from BioLegend (San Diego, CA). DAPI was used for dead cell exclusion.

[0085] For all experiments in this study, fluorescence- activated cell sorting cell sorting (FACS) was performed using a FACSAria III or FACSymphony S6 (BD Biosciences, San Jose, CA).

[0086] Small Cell Number Input Western Blot. Briefly, 5000 mouse LT-HSCs were isolated from middle-aged SCD and non-SCD mice by FACS (as detailed above) directly into 20 pL of PBS with 2% FBS containing protease and phosphatase inhibitors (Invitrogen, Carlsbad, CA). Next, an appropriate volume of 4X LDS sample buffer containing reducing agent was added to each sample such that the final concentration was IX, and cell lysis and protein linearization were achieved by heating samples to 95°C for 5 minutes. Polyacrylamide gradient gel electrophoresis, immunoblotting, and analysis were performed as previously described (Barve et al. (2019) Cell 8(11):1322; Saforo et al. (2019) Sci. Rep. 9:4177) using primary antibodies against RPL21 (A305-031A, Bethyl Laboratories, Montgomery, TX), H2A (D6O3A, Cell Signaling Technologies, Danvers, MA), and p-actin (13E5, Cell Signaling Technologies, Danvers, MA) at a 1:1000 dilution and anti-rabbit HRP conjugated secondary antibody (#7074, Cell Signaling Technologies, Danvers, MA) at 1:10,000 dilution.

[0087] Bone Marrow Transplantation. For quantitative assessment of numbers of functional HSCs, whole bone marrow (WBM) was isolated as described above from CD45.2+ SCD (i.e., Townes) mice or non-SCD littermate controls and injected at limiting dilution (i.e., 5000-100000 cells per recipient) into cohorts of CD45.2⁺CD45.1⁺ recipient mice via tail vein along with 100000 CD45.1⁺ competitor WBM cells, which were isolated from JaxBoy mice. Prior to injection, recipients were subjected to lethal irradiation of two doses of 5.5 Gy separated by 2-3 hours. Recipients were treated with 1 mL/9.1 kg enrofloxacin in drinking water for 10 days posttransplant. To minimize biological variation, WBM was collected and pooled from at least three donors for all transplants. Engraftment was defined as >2% total CD45.2+ PB and >1% CD45.2⁺ cells in myeloid, B-cell, and T-cell compartments. Extreme limiting dilution analysis and estimation of long-term blood repopulating cells was performed using web based software as previously described (Hu & Smyth (2009) J. Immunol. Methods 347:70-78), and this webtool also subjects the resultant data to tests for goodness of fit and heterogeneity. The likelihood ratio test of single model hit on our data from young SCD and non-SCD WBM resulted in x²=0.0677, P-0.795, df=l and _X ²=2.35, P= 0.125, df=l for the data from aged SCD and non-SCD WBM, indicating that we cannot reject the single-hit model.

[0088] To assess hematopoietic repopulating potential of mouse WBM following treatment with the senolytic, ABT-263, WBM was recovered from mice treated with drug or vehicle two weeks post final dosing. Bone marrow was pooled from three individuals from each treatment cohort and transplanted via tail vein at 2x10^s WBM cells/recipient into CD45.1+CD45.2⁺ recipients subjected to lethal irradiation along with IxlO⁵ WBM competitor cells (CD45.1+).

[0089] For all transplants, PB was sampled every four weeks for at least 16 weeks post-transplant and assessed for CD45.2+ and CD45.1+ PB reconstitution, as well as myeloid, B-cell, and T-cell reconstitution via staining for 20 minutes on ice with Grl-PerCP Cy5.5 (RB6-8C5), B220- PerCP Cy5.5 (RA3-6B2), CDllb PerCP Cy5.5 (MI/70), B220-PE Cy7 (RA3-6B2), CD4-PE Cy7 (RM4-5), CD8-PE Cy7 (53-6.7), anti-CD45.2 V500 (104) and anti-CD45.1 FITC (A20). All were used at 1:200 dilution and acquired from BD Biosciences (San Jose, CA). DAPI (Sigma- Aldrich, St. Louis, MO) was used for dead cell exclusion. [0090] Treatment of SCD Mice with the Senolytic, ABT-263. Cohorts of two-month-old SCD or non-SCD littermates (i.e., Townes mice) were administered 50 mg/kg of ABT-263 (Biovalley, Nanterre, France) dissolved in 60% Phosal® 50/30% PEG400/10% EtOH or vehicle by daily oral gavage for one week. Mice were then rested for two weeks, followed by another week of daily ABT-263 or vehicle via oral gavage. Two weeks later, mice were euthanized, and bone marrow collected for analysis of HSPCs and transplantation studies (detailed above).

[0091] CFU Potential of Human HSPCs. Cryopreserved bone marrow MNCs were thawed in a 37°C water bath and transferred to a 15 mL conical tube. 5 mL of PBS with 2% FBS 1%. PEST was then added dropwise. MNCs were washed twice and resuspended in PBS with 2% FBS 1% PEST. Cells were stained with CD45- BV711 (HI30), Lineage-FITC (UCHT1; HCD14; 3G8; HIB19; 2H7; HCD56), CD34-APC-Cy7 (581), CD38-PE-Cy7 (HIT2) at 1:200 dilutions. All antibodies came from BioLegend (San Diego CA). DAPI (Sigma-Aldrich, St. Louis, MO) was used for dead cell exclusion. 500 CD45⁺Lineage^_CD34⁺ cells were sorted into each well of a 96-well U-bottom plate (Corning) containing 100 pL of X Vivo-10 media (Lonza) with 1% BSA, 1% PSG, hGM-CSF (10 ng/pL), hTPO (15 ng/pL), hIL-6 (10 ng/pL), hFlt3L (100 ng/pL), and hSCF (100 ng/pL). All cytokines were purchased from Peprotech (Cranbury, NJ). Wells were then transferred to 2 mL aliquots of H4435 methylcellulose (StemCell Technologies). Each well of the 96-well plate was washed with 20 pL PBS 2% FBS 1% PEST and added to the corresponding aliquot of methylcellulose. Methylcellulose and cell suspensions were vortexed and allowed to settle. Methylcellulose was then dispensed into SmartDish 6-well plates (StemCell Technologies) . After 14 days, wells were imaged with the STEMvision imager (StemCell Technologies) and colonies scored .

[0092] Analysis of Mouse and Human Cells for [3-Galactosidase Activity . Senescence-associated fi-galactosidase (SA-p-Gal) activity in mouse and human HSCs and HSPCs was assessed via a live cell flow cytometry-based assay kit (Cell Signaling Technology, Danvers, MA) according to the manufacturer's instructions. Briefly, c-~kit-enriched mouse bone marrow or thawed cryopreserved human bone marrow MNCs were incubated with 100 nM bafilomycin A for one hour at 37°C and 5% CO2 to alkalize cellular lysosomes and inhibit autophagy. Next, cells were treated with 33 pM of the provided cell permeable fluorogenic substrate (which fluoresces when hydrolyzed by p-galactosidase) for 3-4 hours followed by two washes of PBS with 2% FBS. Cells were then stained with appropriate antibodies for HSC or HSPC visualization, as described above. Dead cells were excluded using either Live/Dead™ Aqua or Live/Dead™ Violet (Invitrogen, Carlsbad, CA). Mouse LT-HSC SA-[3-Gal activity was also assayed and confirmed by using FACS to isolate LT-HSCs from middle-aged SCD and non-SCD mice which were then mounted on microscopy slides by cytospin at 500 g for 5 minutes at room temperature. LT-HSCs were then stained for SA-p-Gal activity using the Senescence Detection Kit (Millipore Sigma, Burlington, MA) per manufacturer'’s instructions. SA-^-Gal⁺ cells were then assessed visually by bright field microscopy of 10 random fields of view per biological replicate.

[0093] Analysis of HSPCs for Oxidative Stress and DNA Damage. Cellular radical oxidative species (ROS) content was measured using the CellRox® Green ROS detection reagent (Invitrogen, Carlsbad, CA) according to manufacturer''s instruction. Briefly, c-kit-enriched mouse bone marrow cells were incubated for 30 minutes at 37°C 5% CO2 with 5 pM CellRox® reagent followed by three washes and resuspension in PBS with 2% FBS. Cells were then stained for visualization of HSPCs by flow cytometry, as described above.

[0094] DNA damage was assessed in human and mouse cells by flow cytometry for phosphorylated histone Y^H2AX, which marks double-stranded DNA breaks (Rossi et al. (2007) Nature 447:725-729; Rube et al. (2011) PLoS One 6:el7487). C-kit- enriched mouse bone marrow or thawed cryopreserved human bone marrow MNCs were stained for HSPC visualization, as described above. Cells were then fixed in 4% paraformaldehyde (PFA) in PBS for 10 minutes on ice followed by two washes and resuspension in lOOpL of PBS with 2% FBS. Cells were then stained with an anti-YH2AX-PE (2F3, BioLegend, San Diego, CA) at 1:100 dilution for 20 minutes on ice followed by two washes and resuspension in PBS with 2% FBS. Dead cells were excluded using Live/Dead™ Aqua or Live/Dead™ Violet and cells were analyzed by flow cytometry.

[0095] Immunofluorescence and Microscopy to Assess DNA Damage in Mouse LT-HSCs. LT-HSCs were isolated from six- month-old SCD (i.e._z Townes) or non-SCD littermates as described above. Coverslips were coated with 10 pg/mL CD44 in PBS for one hour at room temperature. After a gentle PBS wash, approximately 700 LT-HSCs were added to coverslips and incubated at 37°C for one hour. Cells were gently washed again and crosslinked with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in HEPES-KOH (pH 7.5) at room temperature for 15 minutes. After three washes, cells were permeabilized and blocked simultaneously with 0.3% Triton™ X-100 in blocking buffer (0.5% bovine serum albumin, 50 mM ammonium chloride in PBS) for 30 minutes. Cells were then washed three times and stained sequentially with mouse anti- gH2AX (Serl39, 1:200 dilution, Sigma, Burlington, MA) and rabbit anti-53BPl (1:200, Novus Biologicals, Centennial, CO) in blocking buffer at room temperature for one hour each. After staining, cells were washed three times and then incubated with goat anti-mouse Alexa Fluor® 647 (1:300 dilution, Abeam, Waltham, MA) and donkey anti-rabbit Alexa Fluor® 488 (1:300, Abeam, Waltham, MA) in blocking buffer at room temperature for one hour. Coverslips were mounted on slides using Prolong Diamond with DAPI (ThermoFisher, Waltham, MA). Images were captured using a Leica SP8 confocal microscope at 63X objective lens and 4X optical zoom using hybrid (HYD) detectors. The acquired images were analyzed using Fiji software and the quantitation was done manually by counting the yH2Ax puncta in each cell.

[0096] Bulk RNA-Sequencing of Mouse LT-HSCs and Human HSPCs. Mouse bone marrow LT-HSCs or human bone marrow HSPCs (CD45⁺Lineage^_CD34⁺CD38^_) were collected by FACS directly into the provided RNA lysis buffer for isolation of total RNA (RNeasy Micro kit; QIAGEN, Germantown, MD). Cells were collected from either SCD and non-SCD littermates (i.e., Townes mice) or cryopreserved SCD and non-SCD patient bone marrow samples. The Ovation RNA Seq System V2 kit (Tecan, Mannedorf, Switzerland) was used for library preparation for mouse cells, while the SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio, Kusatsu, Japan) was used for human cells. 150 bp paired-end sequencing was performed on the Illumina NovaSeq 6000, targeting an average of 50 million reads/sample by the Vanderbilt Technologies for Advanced Genomics Genomics core laboratory (Vanderbilt University Medical Center, Nashville, TN).

[0097] Gene Expression Data Analysis for Bulk RNA-Seq. Human and mouse datasets were analyzed separately via similar strategies. For both mouse and human bulk RNA-Seq datasets, technical quality of reads was checked with FastQC (version 0.12.0) before and after read trimming. Reads were trimmed and filtered with fastp (version 0.23.4) to remove adapter sequence, low-quality bases near the ends of reads, and to remove reads with fewer than 40 high-quality bases. Library quality was assessed with BBMap (version 38.86) and RSeQC (version 3.0.1). Filtered reads were mapped to their respective genome with STAR (version 2.7.11). For human, the T2T-CHM13v2 .0 genome assembly with annotation from Ensembl (release 110.1) was used, and for mouse the GRCm39 genome assembly with annotation from Gencode (version M35) was used. Gene-level counts were obtained with featurecounts from Subread (version 2.0.5) and transcript-level expression was quantified with RSEM (version 1.3.3).

[0098] Downstream analysis was performed primarily in R (version 4.3.2) with packages from the Bioconductor repository (version 3.18). For both mouse and human datasets, features with 10 or more raw counts in any sample within the respective dataset were retained; these were further filtered to exclude gene types which have not been reported to exhibit polyadenylation, leaving 14012 coding genes for mouse (18245 total expressed features), and 15699 coding genes for human (19320 total expressed features). Exploratory analysis and visualization (PCA, clustering, sample-sample correlation, expression heatmaps) was performed using normalized counts after variance-stabilizing transformation (VST) as implemented in DESeq2, or in transcript per million (TPM) estimates from RSEM. The factoextra R package was used for PCA plots with confidence ellipses, with the ellipses representing the 95% confidence interval for the indicated PCs and sample groups based on a normal distribution. Differential gene expression analysis was facilitated by DESeq2, using raw counts as input. The resulting loga foldchange estimates were moderated to reduce the apparent effect size of genes with low or highly variable expression with the apeglm fold-change shrinkage method.

[0099] For the mouse dataset, one sample with low library complexity was identified as an outlier from PCA and removed, leaving n=4 SCD and n-5 non-SCD samples for differential expression. For the human dataset, biological sex was included as a blocking factor in the design formula for differential expression (design ~ sex + Condition) and DE analysis was run first with all samples (n=7 SCD, n = 4 non- SCD), and then on a subset of samples (n=3 SCD and n=3 non- SCD) found to separate by condition on PC3 in the PCA with all 11 samples. Genes with Benjamini-Hochberg FDR-adjusted p-values <0.05 and |log2 fold-changel ^0.5 were considered as differentially expressed genes, except for the human analysis with all 11 samples, where only the FDR p-value threshold was applied. Functional analysis for DE genes was performed using GOSeq to test for over-representation of pathways and gene sets assembled from KEGG, Gene Ontology, MSigDB, CellAge, SenNet, and SenMayo. Functional terms and pathways with FDR- adjusted enrichment p-values < 0.05 were considered significant. Gene Ontology term network representations were created in Cytoscape (version 3.10.2) with enrichment results from GOSeq.

[00100] Sample Preparation and Analysis for Mouse CITE-Seq. C-kit enriched non-SCD control and SCD age match bone marrow samples were processed for CITE-seq using Biolegend TotalSeq- A antibodies (Ly-6A, CD150, CD48, CD16/CD32, CD105, CD41, and CD71). Samples were incubated at 4°C for 30 minutes in 50 pL of Cell Staining buffer (Biolegend) containing TotalSeq-A antibodies (0.02 pg/mL per antibody), washed and resuspended in 2% FBS (1.5xl0⁶/mL) for downstream single cell RNA sequencing. Labeled cells were processed for single cell RNA sequencing using the Chromium Single Cell 3' Reagent Kit v3 (lOx Genomics) and sequenced on an Illumina NovaSeq 6000 platform. Raw reads were processed using Cell Ranger software (v5.0.1), and reads were aligned to the mouse reference genome GRCm38 using STAR (v2.7.0a). Doublets were detected and filtered using Scrublet (vO.2.3). Cells were further filtered by RNA to those containing 2000 to 40000 counts, 200 to 8000 RNA genes, and less than 35% mitochondrial gene counts. For counts from RNA, total cell counts were normalized to 10000 and natural log transformed using Scanpy functions (vl.9.3). Protein count normalization was performed using centered logratio (CLR) transformation.

Example 2 : Senolytics Restore Hematopoietic Stem Cell Function in Sickle Cell Disease

[00101] Flow cytometry, quantitative functional assays, and molecular profiling were employed to investigate HSPC phenotypes and function in mice and young individuals with SCD. Importantly, treatment of young SCD mice with the BCL2/BCLXL inhibitor, ABT-263, increased numbers and restored HSPC long-term repopulating ability post-transplant. These findings reveal that premature onset of aging phenotypes correlate with a loss of function in the HSPC pool of mice and individuals with SCD and that restoration of HSC function is of use in the treatment of SCD.

[00102] Altered Frequency, Diminished Quiescence, and Increased Cellular Stress in HSCs from SCD Mice. To gain a deeper understanding of how SCD pathophysiology affects bone marrow HSPCs over time, bone marrow was isolated from young (two-months-old) and middle-aged (six-month-old) mice with SCD and interrogated by flow cytometry for HSPC frequencies (i.e. long-term HSCs, LT-HSCs; short-term HSCs, ST-HSCs; multipotent progenitors 2, MPP2s; MPP3s; and MPP4s). Although LT-HSC frequency was modestly elevated in young SCD mice, it was diminished substantially in middle-aged SCD mice, relative to non-SCD control mice. ST-HSC frequency was similarly reduced in middle-aged SCD mice, but not in young SCD mice. Importantly, total bone marrow cellularity and multipotent progenitor frequencies were unaltered in young or middle-aged SCD mice. These findings were also confirmed in another transgenic SCD mouse model, which also displayed elevated frequencies of LT-HSC and ST-HSC in young SCD mice, with a substantial reduction in HSC frequency by middle-age. [00103] The cell cycle and apoptotic status of HSCs in SCD mice was subsequently analyzed using flow cytometry. No differences in apoptosis were observed in LT-HSCs or ST-HSCs relative to non-SCD mice. However, more LT-HSCs and ST-HSCs were in S-G2/M and fewer were in GO in middle-aged SCD mice, relative to controls. Consistently, LT-HSCs displayed elevated EdU uptake in vivo in middle-aged SCD mice relative to non-SCD mice. This increased cycling correlated with an accumulation of DNA damage and ROS, as assessed by phosphorylated histone H2AX (yH2AX) colocalized with the DNA damage repair protein 53BP1 and increased CellRox®-Green, respectively (FIGS. 1A-1F). These data are consistent with increased replicative and cellular stress resulting in unrepaired DNA damage, as previously reported in stressed and aged LT-HSCs (Rossi et al. (2007) Nature 447:725-729; Rube et al. (2011) PLoS One 6:el7487).

[00104] Young and Middle-Aged Mice with SCD Display a Severe Loss of Functional HSCs. Numbers of functional long-term blood repopulating cells (i.e., HSCs) were quantified in the bone marrow of young and middle-aged mice with SCD by limiting dilution transplantation (Sieburg et al. (2002) Exp. Hematol. 30:1436-1443). Here, 8-12-week-old lethally irradiated CD45 .1+CD45.2⁺ C57BL/6 mice were transplanted with 5000-100000 bone marrow cells isolated from young or middle-aged SCD mice or their non-SCD aged-matched controls (CD45.2+) along with 100000 competitor bone marrow cells from JaxBoy mice (CD45.1⁺). CD45.2⁺ reconstitution of peripheral blood (PB) was monitored for at least 16 weeks post-transplant. Engraftment was defined as ^2% total CD45.2⁺ PB and ^1% CD45.2+ cells in myeloid cells, B cells, and T cells. Total %CD45.2⁺ PB and numbers of engrafted mice was lower in recipients of bone marrow from young and middle-aged SCD mice across cell doses with no evidence of lineage skewing. Extreme limiting dilution analysis (Hu & Smyth (2009) J. Immunol. Methods 347:70-78) of engrafted recipients revealed about 6.4-fold (p=0.007) and 4.2-fold (p=0.002) fewer repopulating cells in the bone marrow of young and middle-aged SCD mice, relative to non-SCD mice (FIGS. 1H-1I). Thus, SCD mice display a profound and lasting loss of functional HSCs by two months of age.

[00105] HSCs from Middle-Aged Mice with SCD Display Evidence of Senescence-Associated Transcriptional and Functional Changes. To better understand the observed loss of HSCs in SCD mice, bulk RNA-sequencing was performed on LT-HSCs purified from the bone marrow of middle-aged SCD (n=4) and non-SCD (n=5) littermates. Principal component analysis (PCA) revealed separation between most SCD and non-SCD samples along PCI, explaining more than a quarter of the variance in the dataset. While correlations between biological replicates were >0.95 in all cases, substantially more variability was observed among SCD than non-SCD samples, as illustrated by both Pearson correlation and PCA across PCs 2 and 3. Differential expression analysis identified 122 and 84 significantly up or down-regulated genes, respectively, in SCD compared to non-SCD LT-HSCs (DESeq2 FDR p-value < 0.05, LFC magnitude £ 0.5). Pathway enrichment analysis of downregulated genes revealed two major molecular signatures: (1) downregulation of positive regulators of p53 and (2) impaired biogenesis of genes encoding ribosomal and histone proteins. Indeed, nearly 30 genes encoding ribosomal proteins and histone variants were downregulated in LT-HSCs of middle- aged SCD mice, relative to controls. Reduced protein levels of Rpl21 and H2A as representative ribosomal and histone proteins were confirmed by western blot, respectively.

[00106] As increased DNA damage, oxidative stress, and reduced ribosome and histone biogenesis are often seen during senescence, LT-HSCs were subsequently interrogated for molecular signatures of senescence (Tur et al. (2019) Aging (Albany NY) 11:2512-2540; Payea et al. (2021) Mol. Cell Biol. 41; Lessard et al. (2018) Nat. Cell Biol. 20:789-799; Nishimura et al. (2015) Cell Rep. 10:1310-1323; Funayama et al. (2006) J. Cell Biol. 175:869-880; Lopez et al. (2012) Aging (Albany NY) 4:823-842; Baker & Sedivy (2013) Nat. Struct. Mol. Biol. 17:1218-1225). Enhanced lysosomal p- galactosidase activity outside physiologic pH (i.e. senescence associated p-gal, SA-p-gal) and increased cell size are conserved and well-characterized hallmarks of senescence in many cellular contexts, including HSCs (Lengefeld et al. (2021) Sci. Adv. 7:eabk0271). A significant increase in SA-p-gal* cells was observed in LT-HSCs and ST- HSCs of middle-aged SCD mice, relative to non-SCD mice (FIG. 1G). SA-p-gal* LT-HSCs were also larger in size, compared to SA-p-gal- LT-HSCs, and these cells were enriched in HSCs from SCD mice, relative to controls. The senescence-associated genes P21 and Bcl2 were also upregulated by quantitative RT- PCR in LT-HSCs from middle-aged SCD mice. These data indicate a model in which some bone marrow HSCs are driven into senescence during aging in SCD mice.

[00107] Hematopoietic Stem and Progenitor Cells (HSPCs) Isolated from Young Individuals with SCD Display Loss of Function and Increased Senescence. Given the observed loss of function and enrichment for cells with molecular hallmarks of senescence in bone marrow HSCs of middle-aged mice with SCD, it was determined whether phenotypes observed in this preclinical model are also evident in the bone marrow of individuals with SCD. Flow cytometry was used to examine the frequencies of HSPCs in bone marrow samples obtained from children and young adults with SCD (n=15, ages 6 to 23 years) and without SCD (n=ll, ages 2 to 21 years). The frequency of immunophenotypic HSCs (Lineage~CD34*CD38~CD90⁺CD45RA~) was significantly elevated in individuals with SCD relative to non-SCD individuals. In contrast, MPP (Lineage“CD34⁺CD38~CD90~ CD45RA") and multi-lymphoid progenitors (MLPs, Lineage- CD34⁺CD38~CD90~CD45RA+) frequencies were unchanged.

[00108] Bone marrow HSPCs (Lineage-CD34⁺CD38-) from individuals with SCD ■ and non-SCD individuals were subsequently examined for signatures of cellular stress and senescence, including DMA damage, SA-p-gal activity, and expression of canonical senescence mediators (Gonzalez-Gualda et al. (2021) FEBS J. 288:56-80). The frequency of HSPCs with elevated DNA damage and SA-p-gal activity was significantly increased in individuals with SCD relative to controls (FIGS. 2A-2B). HSPCs from individuals with SCD also displayed elevated levels of the cell cycle inhibitors, p!6 and p21 FIGS. 2C-2D). Thus, bone marrow HSPCs of individuals with SCD display elevated levels of cellular stress and hallmarks of senescence.

[00109] To assess function in the HSPC compartment of individuals with SCD, Lineage^_CD45⁺CD34⁺ HSPCs were isolated from the bone marrow of young individuals with SCD and age- matched controls and plated directly into semi-solid media supplemented with hematopoiesis-promoting cytokines (Petzer et al. (1996) Proc. Natl. Acad. Sci. USA 93:1470-1474) (i.e. colony forming unit (CFU) potential). HSPCs from individuals with SCD displayed highly variable CFU potential, relative to controls, with some displaying very little CFU output.

[00110] Analysis of HSCs from individuals with SCD. To better understand dysfunction in the HSPC pool in individuals with SCD, bulk RNA-sequencing was performed on Lineage’CD34⁺CD38~ HSPCs isolated from the bone marrow of young individuals with SCD and age-matched controls (n=7 and n=4, respectively). Each sequenced sample had at least 12,500 reliably expressed protein coding genes (counts^lO). Similarly to our observations in mice with SCD, substantially more heterogeneity in gene expression was observed in HSPCs isolated from individuals with SCD than in controls, as reflected by PCA and correlations between samples. Consequently, relatively few differential expressed genes were detected when all SCD samples were compared with non- SCD controls (29 upregulated and 4 downregulated in SCD relative to controls when a moderate threshold was applied). Encouragingly, gamma globin (HBG1/HBG2) was consistently upregulated in SCD individuals; HBG expression was increased in individuals responsive to hydroxyurea therapy. Upregulation of IL4R and IL18BP was also observed, indicating a compensatory response to chronic inflammation, as well as TREML4, a mediator of pro-inflammatory signaling downstream of Toll-like receptors.

[00111] Senolytic Treatment of SCD Mice Restores Function to Bone Marrow HSC Pool. Clearance of senescent mouse HSPCs using the BCL2/BCLXL inhibitor, ABT-263 (Navitoclax), can reverse transplantation defects associated with exposure to senescence-inducing stresses, such as aging and total body irradiation (Chang et al. (2016) Nat. Med. 22:78-83). Given the observed increase in HSPCs displaying molecular and functional hallmarks of senescence in the bone marrow of mice and individuals with SCD, it was determined whether ABT-263 could restore HSC function and numbers to mice with SCD. As HSC dysfunction manifests in SCD mice as young as 2 months, two-month-old SCD and non-SCD mice were treated with two cycles of ABT-263 (50 mg/kg by oral gavage daily for two weeks followed by two weeks off) or vehicle control (FIG. 3A). Two weeks after the final administration of ABT-263, mice were euthanized, and their bone marrow examined for HSPC frequency and function via transplantation. A significant increase in LT-HSCs and a trend toward increased c-Kit⁺ HSPCs was observed in SCD mice treated with ABT-263 compared to vehicle-treated mice, with no significant difference in ST-HSCs (FIGS. 3B- 3D) . Further, fewer HSC/MPPs (Lineage~Sca-l⁺c-kit⁺ cells) in treated SCD mice displayed high levels of DNA damage, as measured by gH2AX, relative to controls (FIG. 3E). Importantly, ABT-263 treatment restored the hematopoietic repopulating activity of SCD mice to that of control animals (FIGS. 3F-3G). These data indicate that targeting of senescent cells can restore function to the bone marrow during SCD. In addition, the benefits of ABT-263 treatment on HSPCs likely extend beyond culling of senescent HSPCs. ABT-263 can also reduce systemic inflammation by clearing cells that have acquired the senescence-associated secretory phenotype (Grezella et al. (2018) Stem Cell Res. Ther. 9:108; Yang et al. (2020) Aging (Albany NY) 12:12750-12770) or via senolytic-independent mechanisms (Stenger et al. (2019) Blood 134:2249-2260). Thus, ABT-263 may benefit HSPCs during SCD through both indirect and direct mechanisms.

Claims

What is claimed is:

1. A method for treating, preventing, reducing, or eliminating a manifestation or complication associated with sickle cell disease in a subject, comprising administering to the subject an effective amount of at least one senolytic agent thereby treating, preventing, reducing, or eliminating a manifestation or complication associated with sickle cell disease sickle in the subject.

2. The method of claim 1, wherein the at least one senolytic agent is a B-cell lymphoma (Bel) inhibitor, protein kinase B (Akt) inhibitor, and/or MDM2 inhibitor.

3. The method of claim 2, wherein the Bel inhibitor inhibits Bcl-2, Bcl-xL, Bcl-xS and/or Mell.

4. The method of claim 3, wherein the Bel inhibitor inhibits Bcl-2 and is selected from the group consisting of Navitoclax, Venetoclax, ABT-737, Obatoclax, oblimersen, Pelcitoclax, Lisaftoclax, LP-118, LP-108, HA14-1, TW-37, and pharmaceutically acceptable salts thereof.

5. A method for improving the quality of hematopoietic stem cells of a subject comprising administering at least one senolytic agent to the subject thereby improving the quality of the subject's hematopoietic stem cells.

6. The method of claim 5, further comprising administering a mobilizing agent to the subject.

7. The method of claim 5 or 6, further comprising collecting or harvesting the hematopoietic stem cells from the subject.

8. The method of claim 7, wherein the hematopoietic stem cells are collected or harvested from peripheral blood or bone marrow.

9. The method of claim 5, further comprising genetically modifying the subject's hematopoietic stem cells.

10. The method of claim 7, further comprising genetically modifying and/or transplanting the collected or harvested hematopoietic stem cells.

11. The method of claim 5, wherein the subject is healthy or has sickle cell disease.

12. The method of claim 5, wherein the at least one senolytic agent is a B-cell lymphoma (Bel) inhibitor, protein kinase B (Akt) inhibitor, and/or MDM2 inhibitor.

13. The method of claim 12, wherein the Bel inhibitor inhibits Bcl-2, Bcl-xL, Bcl-xS and/or Mell.

14. The method of claim 13, wherein the Bel inhibitor inhibits Bcl-2 and is selected from the group consisting of Navitoclax, Venetoclax, ABT-737, Obatoclax, oblimersen, Pelcitoclax, Lisaftoclax, LP-118, LP-108, HA14-1, TW-37, and pharmaceutically acceptable salts thereof.