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WO2025064402A1 - Méthode de traitement de la drépanocytose par ciblage des cellules sénescentes - Google Patents

Méthode de traitement de la drépanocytose par ciblage des cellules sénescentes Download PDF

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WO2025064402A1
WO2025064402A1 PCT/US2024/047044 US2024047044W WO2025064402A1 WO 2025064402 A1 WO2025064402 A1 WO 2025064402A1 US 2024047044 W US2024047044 W US 2024047044W WO 2025064402 A1 WO2025064402 A1 WO 2025064402A1
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scd
cells
subject
hscs
mice
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Shannon MCKINNEY-FREEMAN
Aditya BARVE
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St Jude Childrens Research Hospital
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St Jude Childrens Research Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate

Definitions

  • 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.
  • 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.
  • hematopoietic stem cells are administered or harvested from the subject, e.g., from peripheral blood or bone marrow of a healthy subject or subject with a sickle cell disease.
  • the hematopoietic stem cells are genetically modified and/or transplanted.
  • 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
  • percent short-term HSCs ST-HSCs; FIG. IB
  • FIGS. 2A-2D show that sickle cell disease induces hallmarks of senescence in HSCs from individuals with SCD.
  • 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.
  • N 9 for recipients of cells isolated from SCD mice treated with vehicle or ABT-263.
  • HSCs Hematopoietic stem cells
  • HSC transplantation therapies which can involve transplantation of bone marrow- or mobilized peripheral blood-derived stem cells.
  • HSC transplantation therapies can involve transplantation of bone marrow- or mobilized peripheral blood-derived stem cells.
  • HSC transplantation therapies can involve transplantation of bone marrow- or mobilized peripheral blood-derived stem cells.
  • HSCs 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.
  • HSCs in individuals and mice with sickle cell disease are enriched for cells with molecular programs and biomarkers of senescence.
  • 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.
  • 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.
  • 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.
  • sickle cell disease 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.
  • sickle cell disease 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • BH Bcl-2 homology
  • 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.
  • 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.
  • 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.
  • the methods of the invention provide for the administration of a Bcl-2 inhibitor.
  • 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.
  • the methods of the invention provide for the administration of a Bcl-xL inhibitor.
  • 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.
  • 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.
  • 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 AMG 232; CAS No. 1352066-68-2
  • Sulanemadlin AMG 232; CAS No. 1451199-98-6
  • Siremadlin NDP-HDM201; CAS No. 1448867-41-1
  • Alrizomadlin APG-115; CAS No. 1818393-16-6
  • Brigimadlin BI 907828; CAS No.
  • 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-
  • 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.
  • cardiac glycoside or aglycone JFD00244, Cyclosporine, Tyrphostin AG879, Cantharidin, Diphenyleneiodonium chloride, Rottierin, 2,3-Dimethoxy-1,4-n
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a subject may be identified using biomarkers of senescence to evaluate the quality of their bone marrow HSC pool.
  • 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.
  • 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.
  • the subject may be a pediatric patient or pediatric subject.
  • the pediatric patient is under 18 years old.
  • the pediatric patient may be an adolescent of between 12 and 18 years.
  • the pediatric patient may be a child of under 12 years.
  • the subject may be an adult patient.
  • the adult patient is 18 years old or older.
  • the adult patient is 21 years old or older.
  • the cardiovascular disorder is any cardiovascular disorder associated with sickle cell disease.
  • 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).
  • CAD coronary artery disease
  • IHD ischemic heart disease
  • mitral stenosis and regurgitation angina
  • hypertrophic cardiomyopathy diabetic cardiomyopathy
  • supraventricular and ventricular arrhythmias cardiac dysrhythmia
  • cardiac dysrhythmia cardiac dysrhythmia
  • atrial fibrillation (AF) new onset of atrial fibrillation
  • the organ damage is damage to one or more organs selected from spleen, brain, eyes, lungs, liver, heart, kidneys, penis, joints, bones, and skin.
  • organ damage may be any type known to be caused by sickle cell disease.
  • the organ damage is stroke (e.g., ischemic stroke or a hemorrhagic stroke).
  • 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.
  • At least one senolytic agent is administered.
  • 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.
  • two inhibitors of one or more anti-apoptotic proteins in the Bcl-2 family may be administered.
  • one inhibitor of one or more anti-apoptotic proteins in the Bcl-2 family may be administered.
  • 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.
  • the senolytic agent may be administered to a subject at a dose from about 0.1 mg/kg to about 500 mg/kg.
  • 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.
  • 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
  • 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.
  • 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.
  • the excipient may be a diluent.
  • the diluent may be compressible (i.e., plastically deformable) or abrasively brittle.
  • 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.
  • 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.
  • the excipient may be a filler.
  • Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone.
  • 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.
  • the excipient may be a buffering agent.
  • 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).
  • the excipient may be a pH modifier.
  • the pH modifying agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.
  • 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.
  • suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.
  • 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.
  • the excipient may be a preservative.
  • 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.
  • the excipient may be a lubricant.
  • suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate or stearic acid.
  • 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.
  • 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 .
  • 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).
  • 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,
  • 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.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-osseous, intra-articular, or intrasternal injection, or infusion techniques.
  • a composition may be a food supplement or a cosmetic.
  • Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets, and granules.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils.
  • the pharmaceutical composition is applied as a topical ointment or cream.
  • the active ingredient may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • 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.
  • 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.
  • a suitable vehicle is suitable for delivering a composition of the present invention.
  • 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.
  • 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.
  • 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.
  • 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).
  • PA phosphatidic acid
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • DPG diphosphatidylglycerol
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • 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 (lino
  • 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.
  • the phospholipids may come from any natural source, and, as such, may include a mixture of phospholipids.
  • 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'- tetramethylindocarb
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • microemulsions can and will have a multitude of different microscopic structures including sphere, rod, or disc shaped aggregates.
  • 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.
  • 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.
  • At least one senolytic agent may be delivered in a dendritic macromolecule, or a dendrimer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • stem cells e.g., blood-producing hematopoietic stem cells (HSCs)
  • HBBs blood-producing hematopoietic stem cells
  • 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).
  • the subject may additionally benefit from one or more of the treatment regimens described herein.
  • treatment described herein may be administered to the subjects prior to collection of their hematopoietic stem cells for genetic modification.
  • treatment described herein may be administered to the subjects before they are administered drug products for ex vivo or in vivo genetic modification.
  • the treatment described herein may be administered to allogeneic donors before they donate hematopoietic stem cells for transplantation into another individual.
  • 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., HIF
  • 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.
  • the subject may be further administered at least one mobilizing agent during or after treatment with the at least one senolytic agent.
  • 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.
  • G-CSF granulocyte colony-stimulating factor
  • 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.
  • the mobilizing agent is motixafortide with or without filgrastim.
  • 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.
  • 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.
  • HSCs are collected and isolated from the peripheral blood of the subject.
  • 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).
  • 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.
  • 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.
  • At least 1.0*10 6 , 5> ⁇ 10 6 , l.QxlO 7 , 5*10 7 , lxl0 e or 2*10 8 CD34 + cells are isolated using the method of this invention.
  • the amount of CD34+ cells isolated is sufficient to meet the threshold requirement for stem cell transplant (e.g., a target dose of 2> ⁇ 10 6 CD34+ cells/kg recipient weight).
  • the subject treated with the at least one senolytic 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.
  • HSCs from a healthy donor may or may not be genetically modified.
  • treatment of the donor with at least one senolytic agent may lead to better long-term outcomes for the recipients of the HSCs.
  • recipients of such HSCs may have a sickle cell disease.
  • the subject treated with the at least one senolytic agent has sickle cell disease.
  • 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.
  • 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.
  • 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.
  • 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.
  • a senolytic agent increases the total HSC yield from a subject with sickle cell disease leading to the collection of better quality 'product 1 for gene-editing and autologous HSC transplantation protocols used to treat the sickle cell disease and improve gene-editing treatment outcomes.
  • 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.
  • 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.
  • NCT04362293 institutional bone marrow transplant protocol
  • Bone marrow mononuclear cells 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 10 6 cells/vial and stored in liquid nitrogen.
  • PBS phosphate buffered saline
  • PBS fetal bovine serum
  • 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.
  • 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).
  • 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)
  • 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.
  • HSCs Long-CD45 + CD34+CD38-CD90 + CD45RA-
  • MPP Long” CD45+CD34+CD38-CD90-CD45RA
  • MLP Long”CD45 + CD34 + CD38”CD90” CD45RA+
  • 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).
  • 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
  • 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 ⁇ .
  • 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).
  • 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).
  • FACS fluorescence- activated cell sorting cell sorting
  • 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.
  • 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 IxlO 5 WBM competitor cells (CD45.1+).
  • 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.
  • 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).
  • 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.
  • CD45- BV711 HI30
  • Lineage-FITC UCHT1; HCD14; 3G8; HIB19; 2H7; HCD56
  • CD34-APC-Cy7 581
  • CD38-PE-Cy7 HIT2
  • 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).
  • 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.
  • 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.
  • ROS oxidative species
  • 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.
  • 4% paraformaldehyde Electrodehyde
  • RNA-Sequencing of Mouse LT-HSCs and Human HSPCs 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).
  • RNA-Seq 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).
  • 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.
  • 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.
  • 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.
  • CLR centered logratio
  • 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.
  • total bone marrow cellularity and multipotent progenitor frequencies were unaltered in young or middle-aged SCD mice.
  • LT-HSCs 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).
  • yH2AX phosphorylated histone H2AX
  • 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.
  • Principal component analysis 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.
  • 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. (2016) 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.
  • 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.
  • Bone marrow HSPCs Lineage-CD34 + CD38-
  • 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).
  • bone marrow HSPCs of individuals with SCD display elevated levels of cellular stress and hallmarks of senescence.
  • HSC/MPPs Lineage ⁇ Sca-l + c-kit + cells
  • gH2AX gH2AX
  • ABT-263 treatment restored the hematopoietic repopulating activity of SCD mice to that of control animals (FIGS. 3F-3G).
  • ABT-263 can also reduce systemic inflammation by clearing cells that have acquired the senescence-associated secretory phenotype (Grezella et al. (2016) 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).
  • ABT-263 may benefit HSPCs during SCD through both indirect and direct mechanisms.

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Abstract

L'invention concerne des méthodes de traitement, de prévention, de réduction ou d'élimination d'une manifestation ou d'une complication associée à la drépanocytose chez un sujet et d'amélioration de la préparation de cellules souches hématopoïétiques à partir d'un sujet à l'aide d'agents sénolytiques tels que des inhibiteurs de Bcl.
PCT/US2024/047044 2023-09-22 2024-09-17 Méthode de traitement de la drépanocytose par ciblage des cellules sénescentes Pending WO2025064402A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200361946A1 (en) * 2017-08-09 2020-11-19 Prelude Therapeutics, Incorporated Selective Inhibitors Of Protein Arginine Methytransterase 5 (PRMT5)
US20210260040A1 (en) * 2017-10-18 2021-08-26 Epizyme, Inc. Methods of using ehmt2 inhibitors in treating or preventing blood disorders
US20220339141A1 (en) * 2019-09-18 2022-10-27 Aprea Therapeutics Ab Combination treatment with a p53 reactivator and an inhibitor of an antiapoptotic bcl-2 family protein
US20220378955A1 (en) * 2019-09-17 2022-12-01 Actinium Pharmaceuticals, Inc. Radiolabeling of anti-cd45 immunoglobulin and methods of use thereof

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US20200361946A1 (en) * 2017-08-09 2020-11-19 Prelude Therapeutics, Incorporated Selective Inhibitors Of Protein Arginine Methytransterase 5 (PRMT5)
US20210260040A1 (en) * 2017-10-18 2021-08-26 Epizyme, Inc. Methods of using ehmt2 inhibitors in treating or preventing blood disorders
US20220378955A1 (en) * 2019-09-17 2022-12-01 Actinium Pharmaceuticals, Inc. Radiolabeling of anti-cd45 immunoglobulin and methods of use thereof
US20220339141A1 (en) * 2019-09-18 2022-10-27 Aprea Therapeutics Ab Combination treatment with a p53 reactivator and an inhibitor of an antiapoptotic bcl-2 family protein

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DRYSDALE CLAIRE M.; NASSEHI TINA; GAMER JACKSON; YAPUNDICH MORGAN; TISDALE JOHN F.; UCHIDA NAOYA: "Hematopoietic-Stem-Cell-Targeted Gene-Addition and Gene-Editing Strategies for β-hemoglobinopathies", CELL STEM CELL, ELSEVIER, CELL PRESS, AMSTERDAM, NL, vol. 28, no. 2, 4 February 2021 (2021-02-04), AMSTERDAM, NL , pages 191 - 208, XP086487316, ISSN: 1934-5909, DOI: 10.1016/j.stem.2021.01.001 *

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