Treatment of TLR-4 mediated diseases and conditions with aptamers targeting TLR-4

Abstract

To provide broad spectrum molecules with the capability of binding specifically to and inhibiting TLR-4 and that are useful as therapeutic agents against a broad range of diseases and conditions related to the overexpression or overactivation of TLR-4.SOLUTION: Provided is an aptamer for use in ameliorating or improving at least a symptom or sequelae of acute cardiac infarction, the aptamer having a length between 40 and 100 nucleotides and being selected from a specific sequence group, and (i) the aptamer specifically binding to an epitope on the extracellular domain of TLR-4, (ii) binding of the aptamer to the epitope reducing and / or inhibiting TLR-4 activation, and the aptamer being administered during, prior, or immediately after the acute cardiac infarction.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure provides methods for the treatment of TLR-4-mediated diseases and conditions, comprising administering nucleic acid aptamers that specifically target the extracellular domain of TLR-4. [Background technology]

[0002] Toll-like receptors (TLRs) are a family of pattern recognition receptors first identified for their role in innate immune activation and can also regulate the activation of adaptive immune responses. TLR-4 was the first TLR characterized in mammals. The most important endogenous TLR-4 ligands are molecules released in response to tissue or cellular injury. Therefore, TLR-4 is involved in many very common pathologies associated with tissue cellular injury, such as stroke.

[0003] The involvement of TLRs in innate immunity, and in particular in multiple pathologies, has led to increased interest in developing agonists and antagonists of these receptors as pharmacological targets. However, there are few drugs that can regulate TLR-4, and moreover, the drugs that can regulate TLR-4 and are currently being developed for TLR-4-mediated treatment or prevention are generally suitable for the treatment of certain conditions or a limited number of conditions. Therefore, there is a need in the art for broad-spectrum molecules that can specifically bind to and inhibit TLR-4 and are useful as therapeutic agents for a wide range of diseases and conditions associated with TLR-4 overexpression or overactivation. Summary of the Invention

[0004] The present disclosure provides aptamers for use in ameliorating or ameliorating at least some symptoms or sequelae of acute myocardial infarction, (a) the aptamer has a length of 40 to 100 nucleotides and is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4 (or any aptamer sequence in Table 1 or a combination thereof); (i) the aptamer specifically binds to an epitope on the extracellular domain of TLR-4; (ii) binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation, or (b) the aptamer is a functionally equivalent variant of the aptamer of (a) having at least 85% sequence identity to SEQ ID NO: 1, 2, 3, or 4 (or any aptamer sequence in Table 1 or a combination thereof), wherein the functionally equivalent variant is SEQ ID NO: 1, 2, 3, or 4 (or any aptamer sequence in Table 1 or a combination thereof) and retains the ability to specifically bind to and reduce and / or inhibit activation of TLR-4; The aptamer is administered during, before, or immediately after an acute myocardial infarction.

[0005] In embodiments, administration of the aptamer causes a reduction in the infarct area, in particular a reduction of the infarct area by at least 25% compared to control conditions.

[0006] In another embodiment, administration of the aptamer causes a reduction in fibrosis and / or necrosis caused by acute myocardial infarction.

[0007] In another embodiment, administration of the aptamer comprises: (i) improved cardiac function; (ii) reduced extracellular matrix degradation; (iii) improvement of cardiac remodeling; (iv) preservation of ventricular anatomy; (v) reduction in infarct progression; or (vi) resulting in any combination thereof.

[0008] The present disclosure also provides an aptamer as defined above for use in ameliorating or ameliorating at least some symptoms or sequelae of a neuromuscular or neurodegenerative disease or condition, wherein the aptamer is administered during, before, or after the onset of the neuromuscular or neurodegenerative disease or condition.

[0009] In one embodiment, administration of the aptamer comprises: (i) reduced demyelination, (ii) reduction of axonal damage, or (iii) causing their combination.

[0010] In another embodiment, administration of the aptamer causes an inhibition of demyelination by at least 20-80% compared to a control condition (eg, administration of a placebo).

[0011] In another embodiment, administration of the aptamer causes a reduction in (ie, protection against) axonal damage by at least 10-30% compared to a control condition (eg, administration of a placebo).

[0012] In some embodiments, the neuromuscular or neurodegenerative disease or condition is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, Alzheimer's disease, and vascular dementia.

[0013] In some embodiments, the aptamer used for treatment is ApTOLL. In other embodiments, the aptamer is administered at a dose range of about 0.5 mg / dose to about 14 mg / dose. In some embodiments, the aptamer is administered at a dose range of about 0.007 mg / kg per dose to about 0.2 mg / kg per dose. In some embodiments, the aptamer is formulated in PBS (sodium chloride, potassium chloride, disodium hydrogen phosphate dihydrate, and potassium dihydrogen phosphate) pH 7.4 containing magnesium chloride hexahydrate, and optionally containing A-trehalose dihydrate. In embodiments, the aptamer is administered intravenously by infusion.

[0014] The present disclosure also provides methods of treating attacks of TLR-4-mediated diseases and conditions (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or multiple sclerosis) in a subject in need thereof, comprising administering to the subject at least one dose of a nucleic acid aptamer 40 to 80 nucleobases in length, which binds to an epitope on the extracellular domain of TLR-4, and binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation. In some embodiments, binding of the aptamer to the epitope reduces TLR-4 activation. In some embodiments, binding of the aptamer to the epitope inhibits TLR-4 activation.

[0015] In some embodiments, the method further comprises administering an additional therapy or a combination thereof. In some embodiments, the additional therapy is a second TLR-4 antagonist. In some embodiments, the additional therapy is surgical intervention. In some embodiments, the additional therapy comprises administering an anti-inflammatory agent, a nucleic acid, a peptide, or a combination thereof. In some embodiments, the peptide comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the nucleic acid comprises an antisense oligonucleotide, an antimir, an siRNA, or an shRNA.

[0016] In some embodiments, the nucleic acid aptamer comprises a sequence at least 70% identical to SEQ ID NO: 1, 2, 3, or 4 (or any aptamer sequence or combination thereof in Table 1), or a combination thereof. In some embodiments, the nucleic acid aptamer further comprises a biologically active molecule covalently or non-covalently attached to the aptamer. In some embodiments, the nucleic acid aptamer cross-competes with or binds to the same TLR-4 epitope as SEQ ID NO: 1, 2, 3, or 4 (or any aptamer sequence or combination thereof in Table 1). In some embodiments, the nucleic acid aptamer cross-competes with or binds to an epitope that overlaps with the TLR-4 epitope recognized by the nucleic acid aptamer of SEQ ID NO: 1, 2, 3, or 4 (or any aptamer sequence or combination thereof in Table 1).

[0017] In some embodiments, the nucleic acid aptamer is administered in a dosage regimen comprising multiple doses. In some embodiments, the multiple doses are administered simultaneously, sequentially, or a combination thereof. In some embodiments, the multiple doses comprise 2, 3, 4, or 5 doses. In some embodiments, each dose comprises 0.007 to 0.2 mg / kg of the nucleic acid aptamer.

[0018] In some embodiments, the nucleic acid aptamer is administered intravenously, intraarterially, or intraperitoneally. In some embodiments, the TLR-4 mediated disease or condition is an ischemic disease or condition. In some embodiments, the ischemic condition is myocardial infarction or ischemic stroke. In some embodiments, the TLR-4 mediated disease or condition is a hemorrhagic condition. In some embodiments, the hemorrhagic condition is hemorrhagic stroke or hemorrhagic transformation. In some embodiments, the TLR-4 mediated disease or condition is a neuromuscular disease or condition. In some embodiments, the neuromuscular disease or condition is a neurodegenerative disease or condition. In some embodiments, the neurodegenerative disease or condition is multiple sclerosis. [Brief explanation of the drawings]

[0019] [Figure 1] 1 shows the primary, secondary, and tertiary sequences of the aptamer of the present disclosure (ApTOLL, SEQ ID NO: 1). [Figure 2] In vitro antagonism of aptamers ApTLR#1R and ApTLR#4F is shown. HEK-blue cells expressing hTLR-4 coupled to the activation reporter system SEAP were incubated with the selective TLR-4 agonist LPS (200 ng / ml) for 1 hour before adding aptamers (0.2–200 nM) to the incubation medium. hTLR-4 activation was quantified, demonstrating concentration-dependent antagonism of both aptamers in the presence of LPS. [Figure 3] Schematic diagram showing the sequence optimization of aptamers ApTLR#1R and ApTLR#4F. Parts of the sequences of aptamers ApTLR#1R and ApTLR#4F that are not involved in obtaining the 3D structure were deleted, leading to the corresponding truncated forms ApTLR#1RT and ApTLR#4FT. [Figure 4] Figure 1 shows confirmation of maintenance of hTLR-4 binding ability of truncated aptamers ApTLR#1RT and ApTLR#4FT. A) Flow cytometry chart showing quantification of ApTLR#1RT (red line) and ApTLR#4FT (blue line) for hTLR-4 expressed in 293-hTLRA cells compared to control HEK293 cells. B) Flow cytometry chart showing slight changes in truncated aptamer binding to hTLR-4 after stimulation of 293-hTLRA cells with LPS. [Figure 5] Confirmation of the antagonistic effect of truncated aptamers ApTLR#1RT and ApTLR#4FT in HEK-blue cells expressing hTLR-4. A) hTLR-4 activation quantified by the reporter system SEAP is shown compared to the parent aptamers ApTLR#1R and ApTLR#4F. B) Time window of h-TLR-4 activation quantified by SEAP. [Figure 6]Figure 1 shows the inhibition of hTLR-4 activated by endogenous ligands (DAMPs). hTLR-4 activity assay showing the inhibitory effects of ApTLR#1R, ApTLR#4F and the corresponding truncated forms (0.2-200 nM) on activation mediated by endogenous TLR-4 agonists. [Figure 7] Figure 1 shows the inhibitory effect of ApTOLL on downstream TLR-4 cellular effectors. A) Schematic showing the chemistry underlying the Griess assay for NOx detection. B) Quantification of NOx concentrations in the culture medium of peritoneal mouse macrophages activated with the TLR-4 agonist LPS and incubated with ApTOLL (20 and 200 nM) after 1 hour. [Figure 8] Figure 1 shows the in vitro binding affinity of ApTOLL for TLR-4. Quantification of the % of receptor saturation after administration of different concentrations of ApTOLL to cynomolgus monkey monocytes (A) and human monocytes (B). [Figure 9] Figure 1 shows the agonistic effect of ApTOLL on other TLRs. TLR activity assay in cell lines expressing human TLRs 2, 3, 4, 5, 7, 8, and 9. No agonistic effect was detected after incubation with ApTOLL (20 and 200 nM). [Figure 10] Figure 1 shows hTLR2 and hTLR5 activity assays in HEK-blue-hTLR2 and HEK-blue-hTLR5 cells activated with the hTLR2 and hTLR5 agonists Pam3 and FLAT-ST, respectively. Incubation with ApTOLL (20 and 200 nM) showed no inhibitory effect on activation of hTLR2 and hTLR5 cells previously activated by the appropriate agonists. [Figure 11]Protective effects of ApTOLL immediately following experimental stroke in mice. A) Quantification of infarct volume in an ApTOLL dose-response study showing protection at 0.45 mg / kg and 0.9 mg / kg administered intraperitoneally (ip) 10 minutes after pMCAO. B) Quantification of infarct volume in TLR4 knockout mice showing no effect of ApTOLL. C) Quantification of infarct volume in wild-type animals when ApTOLL was administered intravenously. (*) One-way ANOVA p<0.05 vs. vehicle. [Figure 12] Figure 1 shows ApTOLL-mediated protection in a mouse model of permanent middle cerebral artery occlusion using electrocoagulation. Quantification of infarct volume 24 hours after ischemia when 0.91 mg / kg of ApTOLL was administered 10 minutes after occlusion. (*) t-Student p<0.05 vs. vehicle. [Figure 13] Administration of two and three doses of ApTOLL (10 minutes, 2 hours, and 6 hours post-ischemia) in rats after permanent middle cerebral artery occlusion by electrocoagulation is shown. A) Quantification of infarct volume in the ApTOLL multiple dose study showing protection when 0.45 mg / kg of aptamer was administered at 10 minutes, 10 minutes, and 2 hours, and 10 minutes, 2 hours, and 6 hours post-ischemia. (*) One-way ANOVA p<0.05 vs. vehicle. [Figure 14] Figure 1 shows ApTOLL-mediated protection after ischemia-reperfusion in rats. A) Quantification showing reduction in infarct size 24 hours after tMCAO in Wistar rats. B) Quantification of infarct volume after ApTOLL or vehicle treatment in SD rats. (*) t-Student p<0.05 vs. vehicle. [Figure 15] Figure 1 shows a scheme for the design of the therapeutic time window of protection for ApTOLL after stroke in mice. Quantification of infarct size 24 hours after permanent ischemia in mice administered ApTOLL 10 minutes, 2 hours, or 6 hours after pMCAO shows a similar degree of protection at all times tested. (*) One-way ANOVA p<0.05 vs. vehicle. [Figure 16]Figure 1 shows post-ischemic cytokine measurements in animals treated with ApTOLL / vehicle. Quantification of cytokine levels in plasma 24 hours after pMCAO. Results show a significant decrease in several pro-inflammatory cytokines in plasma from animals treated with ApTOLL. (*) t-Student p<0.05 vs. vehicle. [Figure 17] Long-term anatomical and functional protection induced by acute ApTOLL administration (10 min after occlusion) in mice. A) Quantification of brain edema by T2W-MRI at 24, 48, and 72 hours after stroke, demonstrating sustained protection in ApTOLL-treated mice; B) Quantification of infarct size at 21 days after stroke on Nissl-stained sections, demonstrating long-term protection mediated by acute ApTOLL administration; C-D) Quantification of stride length at 21 days after stroke, demonstrating no neurological deficits in ApTOLL-treated animals; E) Photographs showing the stained path in the footprint test and the different distances that may have changed as a result of stroke. (*) Student's t-test p<0.05 vs. vehicle (A, B) or two-way ANOVA p<0.05 vs. sham (C, D). (#) Two-way ANOVA p<0.05 vs. MCAO vehicle. [Figure 18] Figure 1 shows long-term motor protection induced by acute administration of ApTOLL (10 min after occlusion) in rats. Assessment of neurological function by the motor score test up to 21 days after pMCAO showed significant protection at 2 and 7 days after ApTOLL-induced stroke (n=8). Data represent mean ± SEM. Two-way ANOVA followed by Bonferroni test (*p<0.05 vs. vehicle). [Figure 19] Figure 1 shows the antiendotoxin effect of ApTOLL (0.91 mg / kg, 10 minutes after LPS injection) in a mouse model of sepsis. A) % weight loss in mice at 8 and 24 hours after intraperitoneal LPS injection (20 mg / kg); B) % body temperature decrease in mice at 8 and 24 hours after intraperitoneal injection (20 mg / kg); C) cumulative sepsis score in mice at 24 hours (showing a significant reduction in animals injected with ApTOLL); D) Survival curve up to 72 hours after LPS injection (20 mg / kg) (showing increased survival in animals injected with ApTOLL). [Figure 20] A flowchart of the manufacturing process for IMP ApTOLL drug product is shown. IMP was manufactured under full GMP conditions. [Figure 21]

[0033] Figure 1 shows the effect of intravenous administration of ApTOLL on physiological parameters. When compared to intravenous administration of vehicle, no relevant effect of administration of the aptamer on a range of physiological parameters measured in the blood was observed. [Figure 22] Compound-treated human mixed cortical neurons, cortical glutamatergic neurons, and cortical GABAergic neurons are shown. A) Cell viability (note that 0.01 μM is actually the untreated control condition (0 μM) and is used only for logarithmic graphing purposes). B) Photomicrographs from cultures after 10 days of treatment. [Figure 23] Effect of a single intravenous dose of ApTOLL on respiratory function in rats. A) Respiratory rate. B) Tidal volume. C) Minute ventilation. [Figure 24] Figure 1 shows aptamer binding to plasma proteins. Elution plots showing fluorescent ApTOLL in the bound and unbound fractions of human (A), rat (B), and NHP (C) plasma proteins. The gray-shaded area corresponds to the peak of unbound aptamer. Plots show data from three independent samples separately. [Figure 25] Detection of ApTOLL in peripheral and central cells is shown. A) Flow cytometry peripheral analysis of Alexa Fluor 488-labeled ApTOLL (4FT-488, 0.91 mg / kg) in WT and TLR4-KO mice. B) Alexa Fluor 488-labeled ApTOLL in the granulocyte region 5 minutes after aptamer administration in WT mice. C) Distribution of Alexa Fluor 488-labeled ApTOLL within the cerebral infarct region 24 hours after intravenous injection. The distribution pattern of the aptamer (green) within the ischemic core was confirmed by probing with Cy3-conjugated anti-Alexa-488 antibody (c, red). D) Unconjugated ApTOLL was used as a negative control. [Figure 26]Resistance of ApTOLL to degradation by λ-exonuclease A), DNAse I B), and in rat, monkey, and human plasma C) at 37° C. Representative gels from three experiments are shown. [Figure 27] ApTOLL histograms are shown. Incubation with ApTOLL (20 nM) showed no inhibitory effect on the activation of any selected targets, neither GPCRs, ion channels, kinases, nuclear receptors, transporters, nor other non-kinase enzymes. A) Uptake results. B) Binding assay. [Figure 28] In vitro absorption is shown. Incubation with ApTOLL (20 nM) did not show any inhibitory effect on the selected transporters. [Figure 29] Figure 1 shows a histogram of ApTOLL. % inhibition of control values ​​after administration of ApTOLL (20 nM). The results show that there is no significant effect on the inhibition of any of the CYP enzymes evaluated. [Figure 30] Figure 1 shows the induction of CYP enzymes. The fold induction of vehicle activity after administration of ApTOLL (2-20-200 nM). The cutoff value was previously determined using 10 known CYP derivatives and 5 known CYP non-derivatives. The results show no significant effect on the induction of any of the CYP enzymes evaluated. [Figure 31] In vitro cytotoxicity assays for ApTOLL are shown. Cell viability assays (A) MTT activity and B) LDH measurements quantify the effect of 24 and 48 hours of incubation of HEPG2 and HL60 cell lines with ApTOLL (2-2000 nM), demonstrating the absence of cytotoxic effects at biologically active concentrations (2-20 nM). (*) Student's t-test p<0.05 vs. control cells. [Figure 32] The design of the groups participating in the GJ96ND study (a pharmacokinetic study in Sprague Dawley rats) is shown. [Figure 33] A summary of the tmax, Cmax and AUCt values ​​obtained in the MC47KC study (cynomolgus monkey toxicity study) is shown. [Figure 34]Figure 1 shows the in vitro bacterial cytotoxicity assay ApTOLL. Cytotoxicity results are expressed as percent of control growth (OD650). [Figure 35] The in vitro bacterial cytotoxicity assay ApTOLL is shown in addition to that present in Figure 34. Cytotoxicity results are expressed as percent of control growth (OD650). [Figure 36] In vitro Ames test for ApTOLL is shown. A weak positive is denoted as "+" if p<0.05. A strong positive is denoted as "++" if p<0.01. A very strong positive is denoted as "+++" if p<0.001. When possible, compounds with scores significantly below background are flagged. This may indicate low levels of cytotoxicity not detectable in the growth assay. Compounds are flagged as follows: if p<0.05, flagged as "<", if p<0.01, flagged as "<<", and if p<0.001, flagged as "<<<". A hyphen (-) indicates a negative result. [Figure 37] In addition to those shown in Figure 36, in vitro Ames test results for ApTOLL are shown. [Figure 38] Figure 1 shows the in vitro micronucleus assay of ApTOLL. % of micronucleated cells after treatment with ApTOLL at different concentrations. "+" p<0.05 (by t-test) and % of micronucleated cells is at least 3-fold higher than background level. "+ / -" p<0.05 (by t-test) and % of micronucleated cells is at least 2-fold higher than background level. "-" p>0.05 (by t-test) and % of micronucleated cells is less than 2-fold higher than background level. CYTO: High cytotoxicity, insufficient number of scorable cells (>80% cytotoxicity). [Figure 39]A) Schematic of the time window study design in rats. Quantification of infarct volume (B) and edema (C) 72 hours after transient cerebral ischemia in rats when ApTOLL was administered 30 minutes before reperfusion (BR) and 10 minutes, 2 hours, 6 hours, 9 hours, 12 hours, or 24 hours after reperfusion. Protection in tMCAO rats, extending the therapeutic window up to 12 hours, and protection when ApTOLL was administered before reperfusion are confirmed. [Figure 40] Figure 1 shows the effect of ApTOLL on cardiac muscle contractility. Left ventricular echocardiographic parameters A) ejection fraction (%) and B) fractional shortening (%) were recorded from each rat before (basal) and 72 hours after ischemia-reperfusion myocardial infarction (IR). Treatment was administered by intravenous injection of vehicle (PBS with MgCl2, n=7) or a single dose (0.45 mg / Kg, n=11) of ApTOLL 10 minutes after reperfusion. (*) Data shown represent mean ± SEM. t-Student **p<0.01 vs. vehicle. [Figure 41] Figure 1 shows the results of a preclinical study of multiple sclerosis. Clinical scores in an EAE model: disease progression in mice intravenously injected with vehicle (n=7) or 0.91 mg / Kg ApTOLL (n=12) at the onset of symptoms. (*) Data shown represent the mean ± SEM. t-Student *p<0.05, **p<0.01, ***p<0.001 vs. vehicle. [Figure 42] The effect of ApTOLL on OPCs from 7-day-old rats is shown. A) Cell viability determined by MTT assay was expressed as % of control (n=3). H2O2 was used as a mortality control. B) Proliferation was quantified by immunocytochemistry and expressed as % of cells BrdU+ / Olig2+ relative to Olig2+ (n=6). C) Differentiation was quantified by immunocytochemistry and expressed as % of cells MBP+ / Olig2+ relative to Olig2+ (n=5). T3 (thyroid hormone) was used as a differentiation control. (*) Data shown represent mean ± SEM. Student's t-test *p<0.05 vs. vehicle. [Figure 43]Quantification of infarct volume in a multiple-dose ApTOLL study in rats after permanent middle cerebral artery occlusion by electrocoagulation is shown. One (10 min), two (10 min and 2 h), three (10 min, 2 h, and 6 h), four (10 min, 2 h, 6 h, and 24 h), or five (10 min, 2 h, 6 h, 24 h, and 48 h) doses of 0.45 mg / kg aptamer were administered after cerebral ischemia. Protection was observed at all doses tested. All groups were compared to their respective vehicle groups (doses 1, 2, 3, and 4 were compared to the vehicle group euthanized at 48 h, and group 5 and its vehicle control group were euthanized at 72 h). (*) Student's t-test, p<0.05 vs. vehicle. [Figure 44] 1 shows the clinical scores in an experimental autoimmune encephalomyelitis (EAE) mouse model administered ApTOLL (i.e., 0.91 mg / kg) 24 hours after the onset of symptoms. [Figure 45] Figure 1 shows the results of ApTOLL in a mouse EAE model of MS. Clinical score evolution after intravenous administration of different doses of ApTOLL in independent assays. The number of animals used for each dose was as follows: 0.45 mg / kg dose: 6 EAE-ApTOLL, 15 EAE-VEH, and 5 shams; 0.91 mg / kg dose: 13 EAE-ApTOLL, 6 EAE-VEH, and 20 shams; 1.82 mg / kg dose: 8 EAE-ApTOLL, 7 EAE-VEH, and 8 shams; 3.6 mg / kg dose: 5 EAE-ApTOLL, 15 EAE-VEH, and 5 shams. EAE-ApTOLL = EAE model mice treated with ApTOLL. EAE-VEH = EAE model mice treated with vehicle. [Figure 46] Figure 1 shows a comparison of four doses of ApTOLL (intravenous, 0.45 mg / kg, 0.91 mg / kg, 1.82 mg / kg, and 3.6 mg / kg) studied in the EAE model of MS. Data show a follow-up of the clinical course of animals treated with each dose of ApTOLL compared to the vehicle group. [Figure 47]Figure 1 shows the results of a study of myelin loss measured by eriochrome-cyanin staining in spinal cord sections from animals treated with different doses of ApTOLL or vehicle. Quantification of the percentage of demyelination relative to the area of ​​white matter in each experimental group is shown. ApTOLL induces a decrease in the area of ​​demyelination at all doses studied. [Figure 48] Figure 1 shows the results of histological studies of remyelination, axonal damage, and inflammation in EAE-ApTOLL (0.91 and 1.82 mg / kg) compared to EAE-VEH mice. Graphical representation of myelin area (quantification of MBP marker). Graphical representation of axonal damage area (quantification of NFH marker). Quantification of the percentage of microglia relative to total cells (Iba1 marker). [Figure 49] Quantification of oligodendroglial lineage Olig2+ cells, mature cells (CC1+), and oligodendrocyte precursor cells (PDGFRα) after EAE-ApTOLL (0.91 and 1.82 mg / kg) and EAE-VEH administration is shown. [Figure 50] Figure 1 shows plasma cardiac troponin I (cTnI) levels at 8 and 24 hours after reperfusion in a porcine model of ischemia / reperfusion myocardial infarction. Values ​​are expressed as mean ± standard deviation. *p<0.002 ApTOLL 24 hours (n=10) vs. vehicle 24 hours (n=10). [Figure 51] Figure 1 shows porcine cardiac function at 7 days after reperfusion, expressed as EF (ejection fraction) and FS (fractional shortening). N=9 ApTOLL (aptamer, intravenous, 0.078 mg / kg) / 8 placebo (control). Data are expressed as mean ± standard deviation. EF: *p<0.0006 aptamer vs. control. FS: *p<0.003 aptamer vs. control. [Figure 52]Figure 1 shows the reduction in infarct area after double catheterization performed 7 days after treatment with ApTOLL (intravenous, 0.078 mg / kg) or vehicle. A) TTC / Evans blue double staining was performed on 0.5 cm cardiac sections, showing healthy areas (marked H), areas at risk (R), and necrotic (infarcted) areas (white). B) Quantification of infarct area is expressed as a percentage of the area at risk. Values ​​are expressed as mean ± standard deviation. *p<0.002 placebo (vehicle) vs. ApTOLL. [Figure 53] (A) Center panel: Brightfield micrograph (20x magnification) of 0.5 μm heart sections 7 days after reperfusion and H&E staining. Outer panels: Magnification (60x) of the center panel. N=5 ApTOLL / 4 placebo. (B) Brightfield micrograph of 0.5 μm heart sections stained with Masson's trichrome 7 days after reperfusion. N=5 ApTOLL / 4 placebo. [Figure 54] Confocal microscopy detection of matrix metallopeptidase 9 (MMP-9) in cardiac sections from pigs treated with ApTOLL or placebo MMP-9 after 7 days of reperfusion. Nuclei were stained with the fluorescent probe 4',6-diamidino-2-phenylindole (DAPI). N=5 ApTOLL / 4 placebo. Values ​​are expressed as mean ± standard deviation. *p<0.001 placebo vs. ApTOLL. [Figure 55] Tissue distribution of ApTOLL measured by qPCR. (A) Quantification of ApTOLL in the heart, lung, kidney, spleen, liver, small intestine, pancreas, thymus, and ependymal fat. (B) Quantification of ApTOLL in the spleen, kidney, and liver. (C) Distribution of ApTOLL in the ischemic (ipsilateral and contralateral hemispheres) and naive rat brain. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure is directed to methods for treating TLR-4-mediated diseases and conditions (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis) comprising administering to a patient in need thereof at least one therapeutically effective dose of at least one nucleic acid of the present disclosure (e.g., ApTOLL), alone or in combination with at least another therapy commonly used to treat the disease or condition, e.g., pharmacological and / or mechanical thrombolysis (e.g., thrombectomy) in myocardial infarction. Also provided are nucleic acid aptamers, chemically modified nucleic acid aptamers, pharmaceutical compositions and formulations comprising the aptamers, dosages and administration regimens for practicing the methods of the disclosure, kits and articles of manufacture, and methods of manufacture and formulation.

[0021] The diseases and conditions disclosed herein represent a broad sample of TLR-4-mediated diseases and conditions that affect different tissues and organs, have different causes, and have a wide variety of symptoms and sequelae, demonstrating that the nucleic acid aptamers of the present disclosure are broad-spectrum agents that can be successfully applied to a variety of diseases, conditions, and their symptoms and sequelae through modulation of TLR-4-mediated cell signaling.

[0022] TLR-4-mediated diseases and conditions include acute diseases and conditions such as enterocolitis, influenza, ischemic stroke, sepsis, renal ischemia-reperfusion, liver ischemia-reperfusion, intracerebral hemorrhage, or myocardial ischemia; subacute diseases and conditions such as multiple sclerosis, addiction withdrawal, adenomyosis, keratitis, or pulmonary inflammation; and chronic diseases and conditions such as rheumatoid arthritis, atherosclerosis, asthma, lupus, osteoporosis, transplant rejection, dermatitis, psoriasis, obesity, type II diabetes, neuropathic pain, hypertension, RLA, aortic aneurysm, colon cancer, diffuse axonal injury, or chronic pain. TLR-4-mediated diseases and conditions also include, for example, breast cancer, lung cancer, pancreatic cancer, skin cancer, gastrointestinal cancer, liver cancer, bladder cancer, head and neck cancer, esophageal cancer, stomach cancer, colorectal cancer, ovarian cancer, cervical cancer, or prostate cancer. See, for example, Mai et al. (2013) OncoTargets and Therapy 6:1573-87, which is incorporated herein by reference in its entirety. Inhibiting TLR-4 can reduce cell migration and invasion in cancer, and thus inhibiting TLR-4 can reduce cancer metastasis. TLR-4 inhibition can also reduce hepatic steatosis. Thus, the methods and compositions disclosed herein can be applied to the treatment of any of the TLR-4-mediated diseases and conditions disclosed herein, alone or in combination with therapeutic interventions (e.g., pharmacological and / or surgical) commonly used to treat such TLR-4-mediated diseases and conditions. Furthermore, the methods and compositions disclosed herein can be used to treat art-known symptoms and / or sequelae associated with any of the TLR-4-mediated diseases and conditions disclosed herein, as well as other TLR-4-mediated diseases and conditions known in the art. For example, with respect to the use of the methods disclosed herein to treat conditions such as cancer, the disclosed methods and compositions can, for example, reduce or prevent tumor growth, slow progression, inhibit or reduce angiogenesis, inhibit or reduce tumor invasion, inhibit or reduce metastasis, increase survival rates, increase quality of life, improve prognosis, and the like.

[0023] Overexpression of TLR-4 may contribute to chemotherapy resistance, for example, the resistance to paclitaxel in ovarian cancer and the resistance to siRNA therapy in prostate cancer.TLR-4 signal transduction is also associated with the resistance to chemotherapy in liver cancer.Therefore, the methods and compositions disclosed herein can be used to reduce, prevent or reverse the resistance to chemotherapy in cancer patients.

[0024] TLR-4 signaling in immune and inflammatory cells in the tumor microenvironment can lead to the production of inflammatory cytokines, which can further polarize tumor-associated macrophages, convert fibroblasts into tumor-promoting cancer-associated fibroblasts, convert dendritic cells into tumor-associated DCs, and activate the pro-tumorigenic function of immature myeloid cells. Thus, in some embodiments, the methods and compositions of the present disclosure can (i) inhibit or reduce the production of inflammatory cytokines, (ii) reduce or inhibit the polarization of tumor-associated macrophages, (iii) reduce or inhibit the conversion of fibroblasts into tumor-promoting cancer-associated fibroblasts, (iv) reduce or inhibit the conversion of dendritic cells into tumor-associated DCs, (v) reduce or inhibit the activation of the pro-tumorigenic function of immature myeloid cells, or (vi) any combination thereof.

[0025] Increased TLR-4 activation is associated with insulin resistance. Thus, in the context of obesity or diabetes, the methods and compositions disclosed herein can be used to reduce or prevent insulin resistance.

[0026] TLR-4 activation during intrauterine infection leads to contraction of uterine smooth muscle. Thus, the methods and compositions disclosed herein can be used to prevent or inhibit uterine smooth muscle contraction.

[0027] Activation of TLR-4 is also associated with several autoimmune inflammatory diseases, such as human systemic sclerosis (SSc), rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, psoriasis, multiple sclerosis, or autoimmune diabetes, and in particular, inhibition of TLR-4 has been observed to reduce fibrosis, such as skin or lung fibrosis. Therefore, the methods and compositions disclosed herein can be used to treat or ameliorate the symptoms of autoimmune inflammatory diseases associated with increased expression and / or activation of TLR-4, such as human systemic sclerosis (SSc), rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, psoriasis, multiple sclerosis, or autoimmune diabetes. In some embodiments, the methods and compositions disclosed herein can be used to inhibit or reduce fibrosis in autoimmune inflammatory diseases associated with increased expression and / or activation of TLR-4, such as human systemic sclerosis (SSc), rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, psoriasis, multiple sclerosis, or autoimmune diabetes.

[0028] In some embodiments, the methods and compositions disclosed herein can be used to treat, prevent (e.g., suppress, inhibit, or slow), or ameliorate any of the symptoms and sequelae of central nervous system diseases, including amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, Alzheimer's disease, and vascular dementia diseases.

[0029] Before the present disclosure is described in more detail, it should be understood that the present disclosure is not limited to the particular compositions or process steps described, and as such may, of course, vary. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has distinct components and features that may be readily separated from or combined with the features of any of the other aspects without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0030] The headings provided herein are not limitations on the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0031] Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0032] I. Definition In order that this description may be more readily understood, certain terms are defined first. Additional definitions are set forth throughout the detailed description.

[0033] It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, a "nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should further be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as an antecedent basis for using exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of a negative limitation.

[0034] Furthermore, "and / or" as used herein is to be interpreted as a specific disclosure of each of two specific features or components, regardless of the presence or absence of the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0035] Whenever an embodiment is described herein with the word "comprising," it is understood that similar embodiments that are otherwise described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0037] Units, prefixes, and symbols are indicated in the format accepted by the Systeme International de Unites (SI). Numerical ranges are inclusive of the numbers defining the range. When a range of values ​​is described, it is to be understood that each intervening integer value, and each fraction thereof, between the stated upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range may independently be included in or excluded from the range, and each range including either or both extremes, or excluding neither limit, is also encompassed by the present disclosure. Thus, ranges described herein are understood to be abbreviations for all values ​​within the range, inclusive of the stated endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0038] When a value is explicitly stated, it should be understood that values ​​that are approximately the same quantity or amount as the stated value are also within the scope of the present disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, where different elements or groups of elements are individually disclosed, their combinations are also disclosed. Where any element of the disclosure is disclosed as having multiple alternatives, examples of that disclosure in which each alternative is excluded, alone or in any combination with other alternatives, are also disclosed herein. Multiple elements of the disclosure may have such exclusions, and all combinations of elements with such exclusions are disclosed herein.

[0039] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise noted, nucleotide sequences are written left to right in the 5' to 3' direction. Nucleotides are referred to herein by their commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, "a" represents adenine, "c" represents cytosine, "g" represents guanine, "t" represents thymine, and "u" represents uracil. .

[0040] Amino acid sequences are written left to right in amino to carboxy direction. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0041] About: The term "about" is used herein to mean approximately, roughly, around, or within the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" can modify numerical values ​​above and below the stated value by, for example, a variance of up to or below 10 percent (higher or lower). As used herein, when applied to a series of values ​​or ranges, the terms "about" or "at least about" apply equally to all members of the list. Thus, "at least about 1, 2, 3, 4..." are interchangeable with "at least about 1, at least about 2, at least about 3, at least about 4...."

[0042] Administration: The terms "administration," "administering," and grammatical variations thereof refer to the introduction of a composition, such as an aptamer of the present disclosure (e.g., ApTOLL), into a subject via a pharmaceutically acceptable route. Introduction of a composition, such as an aptamer of the present disclosure, into a subject can be by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, periocular, or topical. Administration includes self-administration and administration by another.

[0043] A suitable route of administration allows the composition or aptamer (e.g., ApTOLL) to perform its intended function. For example, if the suitable route is intravenous or intraarterial, the composition is administered by introducing the composition or agent into a vein or artery of the subject.

[0044] Antagonist: As used herein, the term "antagonist" refers to a molecule that blocks or attenuates agonist-mediated responses when bound to a receptor, rather than inducing a biological response itself. Many antagonists achieve their efficacy by competing with endogenous ligands or substrates at structurally defined binding sites on receptors. Antagonists can be competitive, non-competitive, or uncompetitive antagonists. In some embodiments of the present disclosure, the antagonist is a TLR-4 antagonist, for example, an aptamer of the present disclosure, such as ApTOLL.

[0045] Antibody: As used herein, the term "antibody" includes natural or partially or wholly synthetic immunoglobulins and fragments thereof. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" further includes polypeptides comprising a framework region from an immunoglobulin gene or a fragment thereof that specifically binds and recognizes an antigen. The use of the term antibody is meant to include whole, polyclonal, monoclonal, and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric, mouse-human, mouse-primate, and primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides. Antibodies include bispecific and multispecific antibodies, so long as they exhibit the desired biological activity or function. In some embodiments of the present disclosure, the biologically active molecule is a molecule comprising an antibody or an antigen-binding fragment thereof.

[0046] Approximately: As used herein, the term "approximately," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately," unless otherwise specified or clear from the context, refers to a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value (except when such a value exceeds 100% of possible values).

[0047] Aptamer: As used herein, the term "aptamer" refers to a single-stranded nucleic acid chain that adopts a specific tertiary structure that enables it to bind to a molecular target with high specificity and affinity comparable to that of a monoclonal antibody through interactions other than traditional Watson-Crick base pairing. Generally, aptamers are selected from combinatorial libraries by the systematic evolution of ligands by exponential enrichment (SELEX) technique. SELEX is used to identify DNA and RNA aptamers that recognize and selectively bind extracellular and intracellular target molecules with high specificity and nanomolar affinity. When folded under physiological conditions, aptamers acquire a unique three-dimensional structure based on their nucleotide sequence, and it is this tertiary structure of the aptamer that confers selectivity and affinity for their target.

[0048] Aptamer binding site: The term "aptamer binding site" refers to a region within the extracellular region of TLR-4 that contains a continuous or discontinuous site (i.e., an epitope) to which a complementary aptamer specifically binds. Thus, an aptamer binding site can extend beyond the epitope and include additional regions within the TLR-4 sequence that can determine properties such as binding affinity and / or stability, or influence properties such as antigen-enzymatic activity or dimerization. Thus, even if two aptamers bind to the same epitope within the extracellular region of TLR-4, if the aptamers establish distinct intermolecular contacts with amino acids outside the epitope, such aptamers are considered to bind to distinct aptamer binding sites.

[0049] Aptamer of the present disclosure: The term "aptamer of the present disclosure" and grammatical variations thereof refer to an aptamer that can bind to an epitope located in the extracellular domain of TLR-4 and modulate TLR-4-mediated signal transduction, e.g., act as a TLR-4 antagonist. In some embodiments, the aptamer of the present disclosure prevents or reduces activation of the NF-κB intracellular signaling pathway and / or inflammatory cytokine production. In some embodiments, the aptamer of the present disclosure blocks the inflammatory response released after the onset of a disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or multiple sclerosis). In some embodiments, the aptamers of the present disclosure are aptamers of SEQ ID NOs: 1-4, or variants thereof (e.g., aptamers having a certain percentage of sequence identity to the aptamers of SEQ ID NOs: 1-4) or derivatives thereof (e.g., aptamers of SEQ ID NOs: 1-4 or variants thereof comprising at least one biologically active molecule covalently or non-covalently attached to the aptamer).

[0050] In other embodiments, the aptamer of the present disclosure is an aptamer that competes with the aptamers of SEQ ID NOs: 1 to 4 for binding to the TLR-4 extracellular domain. In yet other embodiments, the aptamer of the present disclosure is an aptamer that binds to a TLR-4 extracellular domain epitope that partially or completely overlaps with the epitope bound by the aptamer of SEQ ID NOs: 1 to 4. In other embodiments, the aptamer of the present disclosure is an aptamer disclosed in Table 1 or a variant or derivative thereof.

[0051] Binding: The term "binding" refers to the physical interaction between at least two entities, for example, an aptamer and its target epitope, an aptamer and a target protein, or an aptamer and a target cell.

[0052] Binding affinity: "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an aptamer of the present disclosure) and its binding partner (e.g., TLR-4). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an aptamer and TLR-4). The affinity of molecule X for partner Y is generally determined by its K a (association constant) or its dissociation constant (K d ) (which is the reciprocal of the association constant). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity binding molecules, e.g., low-affinity aptamers, generally bind slowly to target epitopes and tend to dissociate easily, while high-affinity molecules, e.g., high-affinity aptamers, generally bind faster to target epitopes and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure.

[0053] The ability of an aptamer of the present disclosure (e.g., ApTOLL) to specifically bind to TLR-4 can be determined, for example, by in vitro binding assays such as enzyme-linked oligonucleotide assay (ELONA), enzyme-linked aptamer adsorbent assay (ELASA), precipitation and quantitative PCR (qPCR), or by fluorescent techniques such as aptahistochemistry, aptacytochemistry, fluorescence microscopy, or flow cytometry. Similarly, both the ability to specifically bind to TLR-4 and the affinity of the aptamer for TLR-4 can be determined by techniques well known to those skilled in the art, such as gel mobility shift assays, surface plasmon resonance (SPR), dynamic capillary electrophoresis, and fluorescent binding assays. Briefly, the fluorescent binding assay consists of the incubation of TLR-4-coated magnetic balls with different concentrations (e.g., 0-100 nM) of a labeled aptamer of the invention (e.g., carboxyfluorescein, FAM), and the subsequent elution and detection of bound aptamer; the dissociation constant (Kd) is calculated by nonlinear fit analysis.

[0054] Binding specificity: The term "specificity" or "binding specificity" refers to the ability of a binding molecule, e.g., an aptamer of the present disclosure, to preferentially bind to one epitope over another, and does not necessarily imply high affinity. The terms "binding specificity" and "specificity" are used interchangeably and can refer to both (i) a specific portion of a binding molecule (e.g., an aptamer) and (ii) the ability of a binding molecule to specifically bind to a particular epitope. A binding molecule, e.g., an aptamer, "specifically binds" when there is a specific interaction between the aptamer and its target epitope. The term "specifically binds" means that the aptamer is generated to bind to its target epitope. The term "non-specific binding" means that the aptamer is not generated to specifically bind to the target epitope, but binds to the epitope through some non-specific means.

[0055] Biologically active molecule: As used herein, the term "biologically active molecule" refers to any molecule that can be covalently or non-covalently attached to an aptamer of the present disclosure (e.g., ApTOLL), which can have a therapeutic or prophylactic effect in a subject in need thereof, or can be used for diagnostic purposes. Thus, by way of example, the term biologically active molecule includes proteins (e.g., antibodies, proteins, polypeptides, and their derivatives, fragments, and variants), lipids and their derivatives, carbohydrates (e.g., glycan moieties in glycoproteins), or small molecules. In some embodiments, the biologically active molecule is a radioisotope. In some embodiments, the biologically active molecule is a detectable moiety, such as a radionuclide, a fluorescent molecule, or an imaging agent. In some embodiments, the biologically active molecule can be covalently attached to an aptamer of the present disclosure. In some embodiments, the biologically active molecule is directly attached to the aptamer. In other embodiments, the biologically active molecule is attached to the aptamer via a linker.

[0056] Conserved: As used herein, the term "conserved" refers to nucleotides or amino acid residues of a polynucleotide or polypeptide sequence, respectively, that occur unchanged in the same position in two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are more conserved between related sequences than nucleotides or amino acids that appear elsewhere in the sequences.

[0057] In some embodiments, two or more sequences are said to be "fully conserved" or "identical" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to portions, regions, or features thereof.

[0058] Cross-competition: As used herein with respect to binding molecules, e.g., aptamers of the present disclosure, the terms "compete" or "cross-compete" mean that a first binding molecule, e.g., a first aptamer, binds to an epitope in a manner sufficiently similar to the binding of a second binding molecule, e.g., a second aptamer, such that the result of binding of the first binding molecule to its cognate epitope is detectably reduced in the presence of the second binding molecule compared to binding of the first binding molecule in the absence of the second binding molecule.

[0059] Alternatively, the binding of a second binding molecule to its epitope can, but need not, also be detectably reduced in the presence of the first binding molecule. That is, a first binding molecule can inhibit the binding of a second binding molecule to its epitope without that second molecule inhibiting the binding of the first binding molecule to its respective epitope. However, if each binding molecule detectably inhibits the binding of the other binding molecule to its cognate epitope (or epitope, in the case of bispecific binding molecules), whether to the same extent, a greater extent, or a lesser extent, the binding molecules are said to "cross-compete" with each other for binding of their respective epitopes. Both competing and cross-competing binding molecules are encompassed by the present disclosure.

[0060] Aptamers are said to "bind to the same epitope" or "contain the same binding site" or have "essentially the same binding" properties if they cross-compete so that only one aptamer can bind to an epitope at a given time, i.e., one binding molecule prevents the binding or modulatory effect of the other.

[0061] Competition herein means at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or greater than about 100% relative inhibition, as determined, for example, by competition ELONA or ELASA analysis or any suitable method known in the art. It may be desirable to set a higher threshold of relative inhibition as a measure of what is an appropriate level of competition in a particular situation. Thus, for example, a competitive binding criterion can be set such that a relative inhibition of at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or about 95%, or even about 100% is detected before an aptamer is considered sufficiently competitive.

[0062] Derived from: As used herein, the terms "derived from" or "derivative" (e.g., "nucleic acid derivative" or "aptamer derivative"), or any grammatical variations thereof, refer to a component isolated from or created using a particular molecule (e.g., a nucleic acid aptamer of the present disclosure). For example, a nucleic acid sequence (e.g., an aptamer) derived from a first nucleic acid sequence (e.g., a parent aptamer) may contain a nucleotide sequence identical to or substantially similar to the nucleotide sequence of the first nucleic acid sequence. In the case of nucleotides, the derived species can be obtained, for example, by naturally occurring mutagenesis, artificially directed mutagenesis, or artificially random mutagenesis. The mutagenesis used to derive the nucleotide can be intentionally directed, intentionally random, or a mixture of each. Mutagenesis of a nucleotide to create a different nucleotide derived from the original one can be a random event (e.g., caused by polymerase infidelity), and the identification of the derived nucleotide can be performed by an appropriate screening method.

[0063] In some embodiments, derived nucleotide sequences of the present disclosure can be generated, for example, using combinatorial chemistry, by chemically modifying nucleotide units at specific positions, by substituting nucleotide units at specific positions with nucleotide analogs, by modifying the chemical bonds in the backbone, by fusing or conjugating the nucleotide sequence with a biologically active molecule, or any combination thereof.

[0064] In some embodiments, the derived nucleic acid sequence is, for example, (i) conjugation to another therapeutic agent (e.g., another TLR antagonist); (ii) conjugation to a moiety that facilitates targeting (e.g., a ligand, binding moiety, or moiety that directs the aptamer to a particular cell or tissue); (iii) conjugation to a moiety that modulates, i.e., increases or decreases, plasma half-life (e.g., by modulating resistance to nucleases or altering renal or hepatic clearance); (iv) conjugation to a delivery moiety (e.g., PEG or a biopolymer such as a lipid, peptide, or carbohydrate that will facilitate transport across the blood-brain barrier); Iwa (v) It may be produced by any combination thereof.

[0065] In some embodiments, a nucleotide sequence (e.g., an aptamer) derived from a first nucleotide sequence (e.g., a parent aptamer) is at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity, and the first nucleotide sequence retains the biological activity of the second nucleotide sequence (e.g., in the case of an aptamer of the present disclosure, the ability to specifically bind to its TLR-4 epitope and inhibit TLR-4).

[0066] Complementary: The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each containing a nucleic acid sequence) related to each other by Watson-Crick base pairing rules, or between an oligomer and a target gene. For example, the nucleic acid sequence "TGA(5'→3')" is complementary to the nucleic acid sequence "ACT(3'→5')." Complementarity can be "partial," in which not all nucleobases of a first nucleic acid sequence match other nucleobases of a second nucleic acid sequence according to the base pairing rules. For example, in some embodiments, the complementarity between a given nucleic acid sequence and another nucleic acid sequence can be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Continuing with this example, there can be "complete" or "perfect" (100%) complementarity between a given nucleic acid sequence and another nucleic acid sequence. The degree of complementarity between nucleic acid sequences significantly affects the efficiency and strength of hybridization between the sequences.

[0067] Effective amount: As used herein, the term "effective amount" of an agent, e.g., an aptamer of the present disclosure (e.g., ApTOLL), refers to an amount sufficient to produce a beneficial or desired result, e.g., a clinical result; therefore, the "effective amount" depends on the context in which it is being applied. For example, in the context of administering an agent to treat a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or multiple sclerosis, or to ameliorate or prevent (e.g., suppress, inhibit, or delay) sequelae and / or symptoms associated with a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or multiple sclerosis, an effective amount of the agent, e.g., an aptamer of the present disclosure, is an amount sufficient to reduce or diminish, for example, tissue damage, tissue inflammation, physiological, physical, or behavioral symptoms or sequelae, or any combination thereof, e.g., compared to the response obtained without administration of the agent.

[0068] The term "effective amount" can be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose." In certain embodiments, this term refers to an amount of an aptamer of the present disclosure (e.g., ApTOLL) that can treat, prevent, reduce, or ameliorate, for example, a symptom or sequela of a TLR-4-mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or multiple sclerosis.

[0069] In certain embodiments, the term refers to the amount of an aptamer of the present disclosure (e.g., ApTOLL) required to achieve (i) a reduction in damaged tissue, (ii) a reduction in inflammation, (iii) an improved neurological outcome, (iv) a decrease in the level of an inflammatory biomarker (e.g., interferon-γ, interleukin-12p70, TNFα, IL-6, or any combination thereof), (iv) an improvement in motor and / or behavioral scores (e.g., improved mobility or response to stimuli), (v) an increase in survival rate, (vi) an improvement in quality of life, (vii) a reduction in pain or discomfort, or (viii) any combination thereof in a subject in need thereof, compared to a reference value obtained from an untreated subject or a population of untreated subjects.

[0070] Epitope: As used herein, the term "epitope" refers to a protein determinant (e.g., an amino acid subsequence of TLR-4) that can bind to a binding molecule, e.g., an aptamer of the present disclosure, such as ApTOLL. Epitopes typically consist of surface groupings of chemically active molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural and charge characteristics. The portion of an aptamer that recognizes an epitope is called a paratope. Epitopes are classified into two categories: structural epitopes and linear epitopes, based on their structure and interaction with the paratope. Structural epitopes are composed of discontinuous sections of the target protein (e.g., TLR-4) amino acid sequence. These epitopes interact with the aptamer paratope based on the 3D surface features and shape or tertiary structure of the target protein (e.g., TLR-4). In contrast, linear epitopes interact with the paratope based on their primary structure. A linear epitope is formed by a contiguous sequence of amino acids from a target protein (eg, TLR-4).

[0071] Excipient: The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound, e.g., a nucleic acid aptamer of the present disclosure (e.g., ApTOLL).

[0072] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules). Generally, the term "homology" refers to the evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestry. In the context of this disclosure, the term homology encompasses both identity and similarity.

[0073] In some embodiments, polymer molecules are considered to be "homologous" to one another if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the monomers within the molecules are identical (exactly the same monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (e.g., polynucleotide sequences).

[0074] Identity: As used herein, the term "identity" refers to the overall monomer conservation between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules). The term "identical" without any additional qualifiers, e.g., nucleic acid A is identical to nucleic acid B, meaning that the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., "70% identical" is equivalent to describing them as having, e.g., "70% sequence identity."

[0075] The percent identity calculation of two polymer molecules, for example, polynucleotide sequences, can be carried out by, for example, aligning the two sequences for optimal comparison purposes (for example, for optimal alignment, gaps can be introduced into one or both of the first and second polynucleotide sequences, and non-identical sequences can be ignored for comparison purposes).In certain embodiments, the length of the sequence aligned for comparison purposes is at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the length of the reference sequence.Then, in the case of polynucleotides, the bases at corresponding base positions are compared.

[0076] If a position in the first sequence is occupied by the same base as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm.

[0077] Suitable software programs are available from various sources for aligning both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq performs comparisons between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs include, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) (www.ebi.ac.uk / Tools / psa).

[0078] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (Clustal W, Clustal X, or Clustal Omega), and MUSCLE.

[0079] Different regions in a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own sequence identity percentage.Please note that sequence identity percentage values ​​are rounded to the nearest tenth.For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2.Please also note that length values ​​are always integers.

[0080] In certain embodiments, the percentage identity (%ID) between a first amino acid or nucleic acid sequence and a second amino acid or nucleic acid sequence is calculated as %ID=100×(Y / Z), where Y is the number of amino acid residues or nucleic acid bases scored as identical matches in the alignment of the first and second sequences (e.g., as aligned by visual inspection or a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0081] Those skilled in the art will understand that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence-sequence comparisons driven exclusively by primary sequence data. It will also be understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, available at www.tcoffee.org, or available, for example, from EBI. It will also be understood that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.

[0082] Inhibits TLR-4: The terms "inhibits TLR-4," "inhibition of TLR-4," "TLR-4 inhibition," and grammatical variations thereof, refer to the activation and / or activity of TLR-4, e.g., blocking and / or reducing the transmission of signals through TLR-4. In the context of the present disclosure, TLR-4 is considered to be inhibited by an aptamer (e.g., ApTOLL) of the present disclosure if the signaling activity of TLR-4 is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% compared to the activity of TLR-4 in the presence of a natural agonist, e.g., lipopolysaccharide (LPS). Lipopolysaccharide, also known as endotoxin, is the major glycolipid component of the outer cell wall of Gram-negative bacteria. LPS molecules typically consist of strain-specific distal polysaccharide side chains known as O antigens, a hydrophilic core oligosaccharide, and a hydrophobic domain called lipid A.

[0083] In some embodiments, the term inhibiting TLR-4 refers to, for example, (i) blocking or complete inhibition of TLR-4 activation, (ii) reducing or partially inhibiting TLR-4 activation, (iii) blocking or complete inhibition of TLR-4 signaling activity, (iv) reducing or partially inhibiting TLR-4 signaling activity, or (v) any combination thereof by an aptamer of the present disclosure.

[0084] The ability of the disclosed aptamers (e.g., ApTOLL) to inhibit TLR-4 can be determined by a series of assays available in the art. In some embodiments, the ability of the disclosed aptamers to inhibit TLR-4 is determined by an in vitro assay using cells expressing recombinant TLR-4 and a reporter gene, the expression of which is associated with the activation of the recombinant TLR-4. Those skilled in the art will recognize that there are multiple variations of this method depending on the cells and recombinant gene used. An example of this assay is contained, for example, in U.S. Pat. No. 10,196,642, which is incorporated herein by reference in its entirety. Other available techniques include measuring the levels of inflammatory cytokines, such as IL-1, IL-8, TNF-α, and IL-12, released by cells expressing TLR-4.

[0085] Isolated: As used herein, the terms "isolated," "purified," and "extracted," and grammatical variations thereof, are used interchangeably and refer to the state of a preparation of a desired composition of the present disclosure (e.g., an aptamer of the present disclosure) that has undergone one or more processes of purification. In some embodiments, isolation or purification, as used herein, is a process of removing or partially removing a composition of the present disclosure (e.g., a fraction) from a sample containing contaminants. In some embodiments, an isolated composition has no detectable undesired activity, or the level or amount of undesired activity is below an acceptable level or amount. In other embodiments, an isolated composition has an amount and / or concentration of a desired composition of the present disclosure at or above an acceptable amount and / or concentration and / or activity. In other embodiments, an isolated composition is concentrated compared to the starting material from which the composition is obtained. The enrichment can be at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% relative to the starting material.

[0086] In some embodiments, the isolated preparation is substantially free of residual biological material. In some embodiments, the isolated preparation is 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any contaminating biological material. Residual biological material can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.

[0087] Linked: As used herein, the term "linked" refers to a first amino acid sequence or polynucleotide sequence (e.g., an aptamer of the present disclosure) that is covalently or non-covalently joined or attached to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence (e.g., an aptamer of the present disclosure) can be directly joined or juxtaposed to the second amino acid or polynucleotide sequence, or an intervening sequence can covalently join the first sequence to the second sequence. The term "linked" not only refers to the fusion of the first polynucleotide sequence to the second polynucleotide sequence at the 5' or 3' end, but also includes the insertion of the entire first polynucleotide sequence (or second polynucleotide sequence) into any two nucleotides in the second polynucleotide sequence (or first polynucleotide sequence, respectively). The first polynucleotide sequence can be linked to the second polynucleotide sequence by a phosphodiester bond or a linker. The linker can be, for example, a polynucleotide.

[0088] Mismatch: The term "mismatch" or "mismatches" refers to one or more nucleic acid bases (consecutive or separate) in a first nucleic acid sequence (e.g., an aptamer of the present disclosure) that do not match a second nucleic acid sequence (e.g., a variant or derivative of an aptamer of the present disclosure) according to base-pairing rules. While perfect complementarity is often desired, some embodiments can include one or more, but preferably 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, mismatches between aptamer variants relative to the parent aptamer. Variation anywhere within the aptamer is included. In certain embodiments, the aptamers of the present disclosure comprise terminal, internal nucleobase sequence variants, if present, typically within about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the 5' and / or 3' terminal subunits. In certain embodiments, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleobases can be removed and still provide on-target binding.

[0089] Modulate: As used herein, the terms "modulate," "modulate," and grammatical variations thereof, when applied to a particular concentration, level, expression, function, or behavior, generally refer to the ability to change by increasing or decreasing, e.g., by directly or indirectly promoting / stimulating / upregulating or interfering / inhibiting / downregulating, a particular concentration, level, expression, function, or behavior, to act as an antagonist or agonist. In some cases, a modulator may increase and / or decrease a particular concentration, level, activity, or function compared to a control, or compared to an average level of activity that would generally be expected, or compared to a control level of activity.

[0090] Nucleic Acid: "Nucleic Acid," "Nucleic Acid Molecule," "Nucleic Acid Sequence," "Polynucleotide," and grammatical variations thereof, are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single-stranded form or a double-stranded helix.

[0091] A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and is not limited to any particular tertiary form. Thus, the term includes, among others, linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and double-stranded DNA found in chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to normal translation, which provides only the 5' to 3' sequence along the non-transcribed strand of DNA (i.e., the strand with sequence homology to mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. The "nucleic acid composition" of the present disclosure can contain one or more nucleic acids (e.g., nucleic acid aptamers) as described herein.

[0092] The term nucleic acid also encompasses variants such as peptide nucleic acids (PNAs), locked nucleic acids (LNAs), and combinations thereof, modifications thereof, including modified nucleotides, etc. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc.

[0093] Parenteral administration: As used herein, the phrases "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In some embodiments, parenteral administration is intravenous or intraarterial. In some embodiments, intravenous or intraarterial administration is by bolus administration, for example, by slow bolus administration of a pharmaceutical composition comprising an aptamer of the present disclosure (e.g., ApTOLL).

[0094] Pharmaceutically acceptable carrier: The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," and grammatical variations thereof, include any of the agents approved by a regulatory agency of the U.S. Federal government or listed in the U.S. Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause the production of undesirable physiological effects to an extent that would prohibit administration of the composition to a subject and does not abolish the biological activity and properties of the administered compound. Included are generally safe, non-toxic, and desirable excipients and carriers that are useful in preparing pharmaceutical compositions.

[0095] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to one or more of the compounds described herein, e.g., an aptamer of the present disclosure, such as ApTOLL, mixed, intermixed, or suspended with one or more other chemical components, such as pharmaceutically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate administration of a preparation of an aptamer to a subject.

[0096] Polynucleotide: The term "polynucleotide" is used interchangeably with "nucleic acid" and refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. In some embodiments, the term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). It also includes modified forms of polynucleotides, for example, by alkylation and / or by capping, as well as unmodified forms.

[0097] In some embodiments, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), e.g., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), or double-stranded RNA (dsRNA), any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, whether spliced ​​or unspliced, and other polymers containing a normonucleotide backbone, e.g., polyamides (e.g., peptide nucleic acids "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that allows for base pairing and base stacking as found in DNA and RNA.

[0098] In some embodiments, the polynucleotide may be, for example, a nucleic acid aptamer (e.g., ApTOLL) of the present disclosure. In some embodiments, the polynucleotide is DNA. In some embodiments, the DNA is synthetic DNA, for example, synthetic ssDNA. In some embodiments, the synthetic DNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class are replaced with unnatural nucleobases (e.g., all uridines in the polynucleotides disclosed herein can be replaced with unnatural nucleobases, for example, 5-methoxyuridine).

[0099] Polypeptide: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. Polymers can contain modified amino acids. These terms also encompass amino acid polymers modified, naturally or by intervention, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. As used herein, the term "polypeptide" refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs thereof. A polypeptide can be a single polypeptide or a multimolecular complex, such as a dimer, trimer, or tetramer. They can also include single-chain or multi-chain polypeptides. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids. In some embodiments, a "peptide" can be 50 amino acids or less in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, the polypeptide can be covalently or non-covalently attached to the aptamer of the present disclosure.

[0100] Prevent: As used herein, the terms "prevent," "inhibit," "suppress," and variations thereof, as applied to a disease or condition disclosed herein, or a symptom or sequela thereof, refer, for example: (i) partially or completely delaying the onset of a disease, disorder, and / or condition, e.g., any TLR-4-mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke); (ii) partially or completely delaying the onset of one or more symptoms, characteristics, or clinical signs of a particular disease, disorder, and / or condition, e.g., any TLR-4-mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke); (iii) partially or completely delaying the onset of one or more symptoms, characteristics, or signs of a particular disease, disorder, and / or condition, e.g., any TLR-4-mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke); (iv) partially or completely slowing the progression of a particular disease, disorder, and / or condition, e.g., any TLR-4-mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia, or ischemic stroke); and / or (v) reducing the risk of developing pathology associated with a disease, disorder, and / or condition, e.g., any TLR-4-mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia, or ischemic stroke).

[0101] In some embodiments, prevention, inhibition, or suppression of outcomes is achieved through prophylactic treatment, for example, by administering an aptamer of the present disclosure.

[0102] Prophylactic: As used herein, "prophylactic" refers to a therapeutic agent or course of action used to prevent, inhibit, suppress the onset of a disease or condition, e.g., any TLR-4 mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke), or to prevent, inhibit, suppress, or delay the symptoms associated with a disease or condition, e.g., any TLR-4 mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke).

[0103] In some embodiments, a preventive effect can be achieved by administering an aptamer of the present disclosure, e.g., ApTOLL, to a subject at risk for any TLR-4-mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke), or to a subject at risk for certain symptoms or sequelae after the onset of any TLR-4-mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke).

[0104] Prevention: As used herein, "prevention" refers to measures taken to maintain health, prevent, inhibit, suppress, or delay the onset of a TLR-4 mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke), or to prevent, inhibit, suppress, or delay symptoms associated with the occurrence of a TLR-4 mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke). In some embodiments, the aptamers of the present disclosure can be used to prevent a TLR-4 mediated disease or condition disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke).

[0105] Similarity: As used herein, the term "similarity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of percent similarity between polymer molecules can be performed in the same manner as calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as understood in the art. It is understood that the percentage of similarity is subject to the comparison scale used, i.e., whether amino acids are compared according to, for example, their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or a combination thereof.

[0106] Subject: The terms "subject," "patient," "individual," and "host," and variations thereof, are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired, including, but not limited to, humans, domestic animals (e.g., dogs, cats, etc.), livestock (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.). The methods described herein are applicable to both human therapy and veterinary applications.

[0107] References to "TLR-4" throughout this disclosure refer to human TLR-4 in relation to a human subject and to the respective orthologs when the subject is not a human subject, i.e., for example, veterinary applications of the methods disclosed herein to equine, feline, or canine subjects require the inhibition of equine, feline, or canine TLR-4 by an aptamer of the present disclosure capable of specifically binding to the extracellular domain of equine, feline, or canine TLR-4.

[0108] Subject in need thereof: As used herein, the phrase "subject in need thereof" includes a subject, such as a mammalian subject, who would benefit from administration of an aptamer of the present disclosure, e.g., ApTOLL, to improve hemostasis.

[0109] Susceptible: A subject who is "susceptible" or "at risk" for a disease, disorder, and / or condition, or its symptoms or sequelae, has not been diagnosed with and / or does not exhibit symptoms of the disease, disorder, and / or condition, but has a tendency to develop the disease or its symptoms.

[0110] In some embodiments, a subject susceptible to or at risk for a disease, disorder, and / or condition (e.g., ischemic stroke) may be characterized by one or more of the following: (1) a genetic mutation associated with the onset of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with the onset of the disease, disorder, and / or condition; (3) an increase and / or decrease in the expression and / or activity of a protein and / or nucleic acid associated with the onset of the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with the onset of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection with a microorganism associated with the onset of the disease, disorder, and / or condition.

[0111] In some embodiments, a subject susceptible to or at risk for a disease, disorder, and / or condition will develop the disease, disorder, and / or condition, hi some embodiments, a subject susceptible to or at risk for a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0112] Systemic administration: As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to the administration of a compound, drug, or other substance other than by direct administration into the central nervous system, where it enters the patient's system and is therefore subject to metabolism and other similar processes, e.g., intravenous or intra-arterial administration.

[0113] Target cell: As used herein, the term "target cell" refers to specific cells that express TLR-4, including, inter alia, myeloid cells such as monocytes, macrophages, microglial cells, granulocytes, and immature dendritic cells, as well as cells of other lineages such as neurons. In certain embodiments, the target cell is a monocyte or macrophage. In some embodiments, the target cell is a microglia cell. In some embodiments, the target cell is a granulocyte. In some embodiments, the target cell is an immature dendritic cell. In some embodiments, the target cell is a neuron. In some embodiments, the aptamers of the present disclosure bind to TLR-4 expressed on the surface of the target cells disclosed herein.

[0114] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a composition comprising an aptamer of the present disclosure (e.g., ApTOLL) sufficient to produce a desired therapeutic, pharmacological, and / or physiological effect in a subject in need thereof. Because prevention can be considered a treatment, a therapeutically effective amount can be a "prophylactically effective amount."

[0115] The term "therapeutically effective amount" also refers to the amount of a composition comprising an aptamer of the present disclosure (e.g., ApTOLL) that is delivered, sufficient to: (i) treating a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (ii) ameliorating the symptoms of a TLR-4 mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (iii) ameliorating the sequelae of a TLR-4-mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (iv) preventing, inhibiting, suppressing, or delaying a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (v) delaying a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (vi) delaying the sequelae of a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (vii) preventing, inhibiting, suppressing, or delaying the onset of a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (viii) preventing, inhibiting, suppressing, or delaying the recurrence of a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (ix) any combination thereof (or any of the acts disclosed below in the definition of the term "treatment") when administered to a subject (a) suffers from a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (b) susceptible to or at risk for a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (c) susceptible to or at risk of recurrence or worsening of a TLR-4-mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia, or ischemic stroke; or (d) at risk for a TLR-4-mediated disease or condition, e.g., due to an underlying infection, disease, disorder, condition, or lifestyle, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia, or ischemic stroke; or (e) any combination thereof.

[0116] Therapeutically Effective Outcome: As used herein, the term "therapeutically effective outcome" means (i) suffers from a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (ii) susceptible to or at risk for a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (iii) susceptible to or at risk of recurrence or worsening of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (iv) at risk for a TLR-4-mediated disease or condition, e.g., due to an underlying infection, disease, disorder, condition, or lifestyle, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia, or ischemic stroke; or (v) In any combination thereof, In particular, effectively (a) treating a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (b) ameliorating the symptoms of a TLR-4 mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (c) ameliorating the sequelae of a TLR-4-mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia, or ischemic stroke; (d) preventing, inhibiting, suppressing, or delaying a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (e) delaying a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (f) delaying the sequelae of a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (g) preventing, inhibiting, suppressing, or delaying the onset of a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke; (h) preventing, inhibiting, suppressing, or delaying the recurrence of a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia, or ischemic stroke; or (i) means an outcome of treatment (e.g., administration of at least one dose of an aptamer of the present disclosure, e.g., ApTOLL) sufficient for any combination thereof (or any of the effects disclosed below in the definition of the term "treatment").

[0117] TLR-4: As used herein, the term "TLR-4" refers to the membrane receptor Toll-like receptor 4. Activation of TLR-4 produces a signaling cascade that leads to the release of inflammatory cytokines, such as IL-1, IL-8, TNF-α, IL-6, and IL-12, causing inflammation and cell damage. The TLR-4 receptor is also sometimes referred to as ARMD10, CD284, TLR-4, or hTOLL. In humans, the TLR-4 receptor was registered in GenBank on May 27, 2014, under accession number 000206.2 and is encoded by the TLR4 gene. TLR-4 has several isoforms. The amino acid numbering used to describe the location of different structural domains of TLR-4 refers to the 839 amino acid-long isoform (isoform 1, Uniprot: O00206-1). Amino acid residues 1-23 constitute the signal sequence, residues 24-631 constitute the extracellular domain, residues 632-652 constitute the transmembrane domain, and residues 653-839 constitute the cytoplasmic domain. TLR-4 isoform 2 (Uniprot: O00206-2) lacks amino acids 1-40 of the canonical isoform 1 sequence. Thus, the extracellular domain of isoform 2 contains amino acids 41-631 of isoform 1. TLR-4 isoform 3 (Uniprot: O00206-3) lacks amino acids 1-200 of the canonical isoform 1 sequence. Thus, the extracellular domain of isoform 3 contains amino acids 201-631 of isoform 1.

[0118] The term TLR-4 also encompasses polymorphisms and natural variants, such as the allele TLR-4*B (Gly-299, Ile-399), which is associated with a blunted response to inhaled LPS, or natural variants with one or more of the following naturally occurring substitutions: T175A, Q188R, C246S, E287D, D299G, C306W, V310G, N329S, F342Y, L385F, T399I, S400N, F443L, E474K, Q510H, K694R, R763H, or Q834H.

[0119] In certain embodiments, the aptamers of the present disclosure specifically bind to an epitope located in the extracellular domain of TLR-4 isoform 1 (ie, amino acids 24-631 of TLR-4 isoform 1).

[0120] In non-human subjects, the term TLR-4 refers to each TLR-4, isoforms, polymorphs, and naturally occurring variants.

[0121] Treatment: As used herein, the terms "treat," "treatment," and "therapy" refer to reducing the severity of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, or ischemic stroke; ameliorating or eliminating one or more symptoms or sequelae associated with the disease or condition; or providing a beneficial effect to a subject with a disease or condition, without necessarily curing the disease or condition. The term also includes prophylaxis or prevention (e.g., suppressing, inhibiting, or delaying) of a disease or condition or its symptoms or sequelae.

[0122] In some embodiments, the term refers to preventing (e.g., suppressing or inhibiting) a disease or condition; curing a disease or condition; delaying the onset of a disease or condition; reducing the severity of a disease or condition; ameliorating one or more symptoms; ameliorating one or more sequelae; preventing (e.g., suppressing, inhibiting, or delaying) one or more symptoms; preventing (e.g., suppressing, inhibiting, or delaying) one or more sequelae; delaying one or more symptoms; delaying one or more sequelae; ameliorating one or more symptoms; ameliorating one or more sequelae; shortening the duration of one or more symptoms; shortening the duration of one or more sequelae; reducing the frequency of one or more symptoms; reducing the frequency of one or more sequelae; reducing the severity of one or more symptoms; reducing the severity of one or more sequelae; improving quality of life; increasing survival; preventing (e.g., suppressing, inhibiting, or delaying) the recurrence of a disease or condition; clinical intervention to delay the recurrence of a disease or condition; or any combination thereof, e.g., with respect to that expected in the absence of treatment with at least one aptamer of the present disclosure. In some embodiments, the disease or condition is a pathology characterized by increased expression of TLR-4 and / or increased TLR-4 activation.

[0123] II. Treatment of TLR-4-mediated diseases with TLR-4-binding aptamers The present disclosure provides a method of treating a TLR-4-mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, or ischemic stroke, in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of a nucleic acid aptamer (e.g., ApTOLL) about 40 to about 100 nucleobases in length, e.g., about 40 to about 80 nucleobases in length, or a variant or derivative thereof, wherein the aptamer, variant, or derivative binds to an epitope on the extracellular domain of TLR-4, and binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation. In one embodiment, the TLR-4-mediated disease or condition is ischemic stroke, or a symptom or sequela thereof. In another embodiment, the TLR-4-mediated disease or condition is myocardial infarction, or a symptom or sequela thereof. In yet another embodiment, the TLR-4-mediated disease or condition is hemorrhagic stroke, or a symptom or sequela thereof. In some embodiments, the TLR-4-mediated disease or condition is hemorrhagic transformation, or a symptom or sequela thereof. In other embodiments, the TLR-4-mediated disease or condition is multiple sclerosis, or a symptom or sequela thereof.

[0124] It is understood that all of the methods disclosed herein can alternatively be formulated as nucleic acid aptamers (e.g., ApTOLL) having a length of about 40 to about 100 nucleobases, e.g., about 40 to about 80 nucleobases, or variants or derivatives thereof, as described above, for use in treating TLR-4-mediated diseases or conditions, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, or ischemic stroke. Alternatively, use of the nucleic acid aptamer for preparing a medicament for treating such TLR-4-mediated diseases or conditions is also provided.

[0125] Also provided is a method of preventing (e.g., suppressing, inhibiting, or delaying) at least one symptom or sequela of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, or ischemic stroke, in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of a nucleic acid aptamer (e.g., ApTOLL) about 40 to about 100 nucleobases in length, e.g., about 40 to about 80 nucleobases in length, or a variant or derivative thereof, wherein the aptamer, variant, or derivative binds to an epitope on the extracellular domain of TLR-4, and wherein binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation.

[0126] The present disclosure also provides a method of ameliorating at least one symptom of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, or ischemic stroke, in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of a nucleic acid aptamer (e.g., ApTOLL) about 40 to about 100 nucleobases in length, e.g., about 40 to about 80 nucleobases in length, or a variant or derivative thereof, wherein the aptamer, variant, or derivative binds to an epitope on the extracellular domain of TLR-4, and wherein binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation.

[0127] As used herein, the term "ischemic stroke" refers to a type of stroke (also known as cerebrovascular disease, cerebral infarction, or apoplexy) characterized by neurological deficits caused by an abnormally sudden and significant reduction in cerebral blood flow. In ischemic stroke, blood perfusion is lost due to a sudden and immediate interruption of blood flow due to occlusion of one of the arteries that irrigate the cerebral mass, producing the appearance of an infarcted area. Arterial occlusion generally results from atherosclerosis or embolism (cerebral embolism) originating elsewhere, typically the heart or another artery. Ischemic stroke is a pathology characterized by increased TLR-4 expression and / or increased TLR-4 activation. Given that TLR-4 activation produces a signaling cascade that leads to the release of inflammatory cytokines such as IL-1, IL-8, TNF-α, IL-6, and IL-12, causing inflammation and cellular damage, pathologies characterized by increased TLR-4 expression and / or increased TLR-4 activation may further be characterized as having an inflammatory component.

[0128] In some embodiments, ischemic stroke may be caused by thrombosis, emboli, or hypoperfusion.In some embodiments, ischemic stroke may be caused by, for example, atherosclerosis, vasculitis, vertebral and carotid artery dissection, polythrombocythemia, hypercoagulable state, infection, valve vegetation, mural thrombus, arterio-arterial embolism from proximal source, fat embolism, septic embolism, systemic hypotension, sickle cell anemia, compressed blood vessel, ventricular tachycardia, thrombus, cardiac respiratory arrest, stroke, or heart failure resulting in congenital heart defect.Therefore, the present disclosure provides a method for treating any of these diseases or conditions in a subject who needs treatment (e.g., a subject who has suffered from ischemic stroke, is at risk of ischemic stroke, or is at risk of recurrence of ischemic stroke), comprising administering at least one therapeutically effective dose of at least one aptamer (e.g., ApTOLL) of the present disclosure to the subject.

[0129] Symptoms and sequelae of ischemic stroke include, for example, loss of consciousness, blindness, tonic eye deviation, global aphasia, dysgraphia, dyslexia, dyscalculia, disorientation, spatial neglect, visual neglect, facial sensory and / or motor symptoms and deficits, sensory and / or motor symptoms of the extremities (upper, lower, or both), urinary incontinence, akinetic mutism, transcortical motor aphasia, confusion, motor hemispatial neglect, hemiplegia, facial paralysis, sensory loss, dysarthria, inattention, ipsilateral hemianopsia, CN deficit, dizziness, spatial disorientation, inability to volitional movements, diplopia, dysphagia, transient ALOC, drop attacks, lightheadedness, quadriplegia, coma, locked-in syndrome, death, Millard-Gabler syndrome, sparing of vertical eye movements, one and a half half syndrome, medial inferior pontine syndrome, nystagmus, ataxia, decreased proprioception, medial middle pontine syndrome, contralateral paralysis, pharyngeal / vocal cord / facial myoclonus, lateral superior pontine syndrome, Horner's syndrome, gaze palsy, facial / limb / trunk pain or loss of temperature, unilateral headache, visual field defect, visual ataxia, lateral midbrain syndrome, contralateral hemiataxia, tremor, hyperkinesia, medial midbrain syndrome, lateral inferior pontine syndrome, facial paralysis, loss of corneal reflex, hearing loss, limb and gait ataxia, Wallenberg's syndrome, hoarseness, hand clumsiness syndrome, medial bulbar syndrome, tongue deviation, or anterior spinal artery syndrome.

[0130] Accordingly, the present disclosure also provides a method of treating, preventing (e.g., suppressing, inhibiting or delaying), or ameliorating any of the symptoms and sequelae of ischemic stroke disclosed herein, or any combination thereof, in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of at least one aptamer (e.g., ApTOLL) of the present disclosure.

[0131] As used herein, the term "hemorrhagic stroke" refers to a condition in which a blood vessel in the brain ruptures, depriving an area of ​​the brain dependent on that artery of blood. Furthermore, the spilled blood compresses brain structures, including other blood vessels, increasing the area affected by ischemia secondary to intracerebral hemorrhage. Symptoms of hemorrhagic stroke may include complete or limited loss of consciousness, nausea, vomiting, sudden, severe headache, weakness or numbness in the face, legs, or arms on one side of the body, seizures, dizziness, loss of balance, speech or swallowing problems, confusion, or disorientation. The most common cause is an aneurysm. A rare cause is an arteriovenous malformation (AVM). There are two types of hemorrhagic stroke: intracerebral hemorrhage and subarachnoid hemorrhage. In general, the ischemic events caused by hemorrhagic stroke can lead to the sequelae described above for ischemic stroke.

[0132] Accordingly, the present disclosure also provides a method of treating, preventing (e.g., suppressing, inhibiting or delaying), or ameliorating any of the symptoms and sequelae of hemorrhagic stroke (either intracerebral hemorrhage or subarachnoid hemorrhage) disclosed herein, or any combination thereof, in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of at least one aptamer (e.g., ApTOLL) of the present disclosure.

[0133] As used herein, the term "hemorrhagic transformation" refers to the transformation of, for example, the quiescent infarction resulting from ischemic stroke into hemorrhagic infarction.Therefore, this term refers to the hemorrhage that occurs in dead or dying tissue, for example, brain tissue that has been deprived of its normal blood supply by ischemic stroke.The spectrum of hemorrhagic transformation ranges from mild punctate hemorrhage (hemorrhagic infarction) to severe massive hemorrhage (parenchymal hematoma).In general, for example, hemorrhagic transformation resulting from ischemic stroke can cause the sequelae described above for ischemic stroke.

[0134] The present disclosure also provides a method of treating, preventing (e.g., suppressing, inhibiting, or delaying), or ameliorating any of the symptoms and sequelae of hemorrhagic transformation (e.g., hemorrhagic infarction or parenchymal hematoma) disclosed herein, or any combination thereof, in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of at least one aptamer (e.g., ApTOLL) of the present disclosure.

[0135] As used herein, the term "myocardial infarction" (also known as "infarction" or "heart attack") refers to a pathology characterized by insufficient blood supply to the heart accompanied by tissue damage due to a blockage in one of the coronary arteries. Ischemia or insufficient oxygen supply to the myocardium resulting from such a blockage leads to angina, which, if recirculated quickly enough, does not cause cardiac tissue death. However, if this anoxia persists, the myocardium is damaged and eventually necrosis, i.e., infarction, occurs. The cause of myocardial infarction is often atherosclerosis. Another possible cause is spasm of the coronary arteries. Myocardial infarction can lead to heart failure, arrhythmia, cardiogenic shock, or cardiac arrest. Risk factors include, among others, hypertension, smoking, diabetes, physical inactivity, obesity, high blood cholesterol, poor diet, and excessive alcohol intake. Impaired blood flow to the myocardium can trigger an ischemic cascade. Myocardial infarction can cause tissue damage (primarily necrosis) and the formation of a collagen scar. Tissue death and myocardial scarring alter the heart's normal conduction pathways and weaken the affected area. Thus, myocardial infarction can lead to sequelae such as abnormal heart rhythms (arrhythmias), heart block, ventricular aneurysms, cardiac inflammation, or cardiac rupture.

[0136] Accordingly, the present disclosure also provides a method of treating, preventing (e.g., suppressing, inhibiting or delaying), or ameliorating any of the symptoms and sequelae of myocardial infarction disclosed herein, or any combination thereof, in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of at least one aptamer (e.g., ApTOLL) of the present disclosure.

[0137] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after a myocardial infarction results in an improvement in cardiac function of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to cardiac function (e.g., as determined by measuring ejection fraction and / or fractional shortening) observed in an untreated subject or a population of untreated subjects.

[0138] As used herein, the term "fractional shortening" refers to a measure of the pumping function of the heart. It is the ratio of the diameter of the left ventricle when relaxed to the diameter of the left ventricle when contracted.

[0139] As used herein, the term "ejection fraction" refers to the volume fraction (or portion of the total) of fluid (usually blood) that is ejected from a chamber (usually the heart) with each contraction (or heartbeat). Ejection fraction is widely used as a measure of the heart's pumping efficiency and is used to classify types of heart failure. It is also used as an indicator of the severity of heart failure.

[0140] The term "multiple sclerosis" as used herein refers to a pathology characterized by the development of demyelinating, neurodegenerative, and chronic lesions in the central nervous system. The cause is currently unknown, although the involvement of various autoimmune mechanisms has been demonstrated. In patients with multiple sclerosis, lymphocytes cross the blood-brain barrier and affect myelin, resulting in an inflammatory process assisted by macrophages and glial cells.

[0141] Demyelination destroys the ability of parts of the nervous system to communicate, resulting in a variety of signs and symptoms, including physical, mental, and sometimes psychiatric problems. Specific symptoms may include double vision, blindness in one eye, muscle weakness, sensory problems, or coordination problems. Multiple sclerosis comes in several forms, and new symptoms may occur in isolated attacks (relapsing forms) or accumulate over time (progressive forms). Between attacks, symptoms may disappear completely, but persistent neurological problems often remain, especially as the disease progresses.

[0142] Multiple sclerosis can cause a variety of symptoms, including changes in sensation (hypoesthesia), muscle weakness, abnormal muscle spasms, difficulty moving; coordination and balance disorders; problems with speaking (dysarthria) or swallowing (dysphagia); vision problems (nystagmus, optic neuritis, phosphenes, or diplopia); ataxia, tremors, pain, spasms, sexual dysfunction, spasticity, fatigue, and acute or chronic pain syndromes, bladder and bowel difficulties, cognitive impairment, or emotional symptomatology (mainly major depression). The primary clinical measure of disability progression and symptom severity is the Expanded Disability Status Scale (EDDS). Some of the most common cognitive impairments affect recent memory, attention, processing speed, visuospatial abilities, and executive function.

[0143] The main pathophysiological feature of MS, as in other primary demyelinating diseases, is the loss of myelin in the central nervous system, both in white and gray matter. On the other hand, the autoimmune component underlying the pathology of multiple sclerosis is a promoter of the processes of inflammation, demyelination, and axonal network damage, in which TLR-4 and its activation via inflammatory signaling play a key role.

[0144] Among the pathophysiological processes underlying the disease, axonal demyelination in the central and peripheral nervous systems plays a key role and is the basis for the symptoms presented by individuals affected by the disease. Myelin is a cellular differentiation that allows the correct transmission of nerve impulses and is physiologically synthesized by oligodendrocytes (in the central nervous system) and neuroblastoma cells (in the peripheral nervous system).

[0145] Accordingly, the present disclosure also provides a method of treating, preventing (e.g., suppressing, inhibiting or delaying), or ameliorating any of the symptoms and sequelae of multiple sclerosis disclosed herein, or any combination thereof, in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of at least one aptamer (e.g., ApTOLL) of the present disclosure.

[0146] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject with multiple sclerosis results in a decrease in clinical score, with a higher clinical score associated with a higher degree of disability and severity of symptoms, and the observed clinical score is less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, or less than about 30% of the clinical score value observed in an untreated subject or a population of untreated subjects.

[0147] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject with multiple sclerosis results in an increase in mobility that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% greater than the increase in mobility observed when the subject is treated with fingolimod (GYLENYA®) or methylprednisolone (URBASON®).

[0148] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) can result in an increase in proliferation of oligodendrocyte progenitor cells that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% greater than the level of proliferation observed in oligodendrocyte progenitor cells growing in the absence of an aptamer of the present disclosure (e.g., ApTOLL).

[0149] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) can result in an increase in differentiation of oligodendrocyte progenitor cells that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% greater than the level of differentiation observed in oligodendrocyte progenitor cells grown in the absence of an aptamer of the present disclosure (e.g., ApTOLL).

[0150] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject in need thereof can result in remyelination of neuronal tissue damaged, for example, as a result of an acute (e.g., ischemic stroke, intracerebral hemorrhage, hemorrhagic stroke, or hemorrhagic transformation), subacute (e.g., multiple sclerosis), or chronic (e.g., diffuse axonal injury) TLR-4-mediated disease or condition. In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject in need thereof can result in neuronal proliferation and / or neuronal differentiation of neuronal tissue damaged, for example, as a result of an acute (e.g., ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage), subacute (e.g., multiple sclerosis), or chronic (e.g., diffuse axonal injury) TLR-4-mediated disease or condition. Accordingly, the present disclosure provides methods for remyelination of neuronal tissue damaged as a result of an acute (e.g., ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage), subacute (e.g., multiple sclerosis), or chronic (e.g., diffuse axonal injury) TLR-4-mediated disease or condition, comprising administering to a subject at least one therapeutically effective dose of a nucleic acid aptamer 40 to 80 nucleobases in length (e.g., ApTOLL) or a variant or derivative thereof, wherein the aptamer, variant, or derivative binds to an epitope on the extracellular domain of TLR-4, and wherein binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation.

[0151] In some embodiments, the aptamer of the disclosure (e.g., ApTOLL) is administered within 16 hours of the onset of a TLR-4 mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke. In some embodiments, the aptamer of the disclosure (e.g., ApTOLL) is administered within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 95, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 400, 410, 415, 420, 425, 430, 435, 440, 445, 80, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440 , 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 670, 675, 680, 685, 690, 695, 700, 705, 710, 7 administered in less than 15, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, or 960 minutes.

[0152] In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) is administered less than about 1 hour, less than about 2 hours, less than about 3 hours, less than about 4 hours, less than about 5 hours, less than about 6 hours, less than about 7 hours, less than about 8 hours, less than about 9 hours, less than about 10 hours, less than about 11 hours, less than about 12 hours, less than about 13 hours, less than about 14 hours, less than about 15 hours, less than about 16 hours, less than about 17 hours, less than about 18 hours, less than about 19 hours, less than about 20 hours, less than about 21 hours, less than about 22 hours, less than about 23 hours, or less than about 24 hours after the onset of a TLR-4 mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke.

[0153] In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) is administered about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke.

[0154] In some embodiments, an aptamer of the present disclosure (eg, ApTOLL) is administered shortly after the onset of a TLR-4 mediated disease or condition, eg, myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke.

[0155] In some embodiments, additional doses of an aptamer of the present disclosure (e.g., ApTOLL) are then administered after the initial dose. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional doses are administered after the initial dose. In some embodiments, several doses are administered on the same day. In some embodiments, one or more booster doses are followed by one or more maintenance doses. In some embodiments, all doses contain the same amount of an aptamer of the present disclosure (e.g., ApTOLL).

[0156] In some embodiments, additional doses of an aptamer of the present disclosure (e.g., ApTOLL) are administered about 2 hours and about 6 hours after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke. In other embodiments, additional doses of an aptamer of the present disclosure (e.g., ApTOLL) are further administered about 2 hours, about 6 hours, about 12 hours, and about 24 hours after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke.

[0157] In some embodiments, particularly in the case of acute TLR-4-mediated diseases or conditions, the aptamers (e.g., ApTOLL) of the present disclosure are generally administered within minutes (e.g., 10-60 minutes), hours (e.g., 1-48 hours), or days after the acute event. In other embodiments, for example, in subacute (e.g., multiple sclerosis) or chronic (e.g., rheumatoid arthritis) TLR-4-mediated diseases or conditions, the aptamers (e.g., ApTOLL) of the present disclosure can be administered for weeks, months, or years.

[0158] In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of about 0.5 mg / day to about 80 mg / day. In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of at least about 0.5 mg / day, at least about 1 mg / day, at least about 2 mg / day, at least about 5 mg / day, at least about 10 mg / day, at least about 15 mg / day, at least about 20 mg / day, at least about 25 mg / day, or at least about 30 mg / day. In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of 0.5 mg / day. In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of 1 mg / day. In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of 2 mg / day. In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of 5 mg / day. In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of 10 mg / day. In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of 15 mg / day. In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of 20 mg / day. In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of 25 mg / day. In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of 30 mg / day.

[0159] In some embodiments, a dose of approximately 14 mg / kg / day of the aptamer of the present disclosure (e.g., ApTOLL) is considered a no-observed-adverse-effect level (NOAEL) when the aptamer is administered twice a day (e.g., 6 hours apart) by intravenous or intra-arterial route (bolus) for 14 days. In some embodiments, the maximum recommended starting dose (MRSD) to be administered to a healthy subject is approximately 31.5 mg for a subject weighing 70 kg. In some embodiments, the maximum recommended starting dose (MRSD) to be administered to a healthy subject is approximately 0.5 mg for a subject weighing 70 kg.

[0160] In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) is administered at a dose of about 0.007 mg / kg (i.e., approximately 0.5 mg / day for a 70 kg subject). In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) is administered at a dose of at least about 0.1 mg / kg, at least about 0.2 mg / kg, at least about 0.3 mg / kg, at least about 0.4 mg / kg, at least about 0.5 mg / kg, at least about 0.6 mg / kg, at least about 0.7 mg / kg, at least about 0.8 mg / kg, at least about 0.9 mg / kg, at least about 1 mg / kg, at least about 1.1 mg / kg, at least about 1.2 mg / kg, or at least about 1.3 mg / kg. g, at least about 1.4 mg / kg, at least about 1.5 mg / kg, at least about 1.6 mg / kg, at least about 1.7 mg / kg, at least about 1.8 mg / kg, at least about 1.9 mg / kg, at least about 2 mg / kg, at least about 2.1 mg / kg, at least about 2.2 mg / kg, at least about 2.3 mg / kg, at least about 2.4 mg / kg, at least about 2.5 mg / kg, at least about 2.6 mg / kg, at least about 2.7 mg / kg, at least about 2.8 mg / kg, at least about 2.9 mg / kg, at least about 3 mg / kg, at least about 3.1 mg / kg, at least about 3.2 mg / kg, at least about 3.3 mg / kg, at least about 3.4 mg / kg, at least about 3.5 mg / kg, at least about 3.6 mg / kg, at least about 3.7 mg / kg, at least about 3.8 mg / kg, at least about 3.9 mg / kg, at least about 4 mg / kg, at least about 4.1 mg / kg, at least about 4.2 mg / kg, at least about 4.3 mg / kg, at least about 4.4mg / kg, at least about 4.5mg / kg, at least about 4.6mg / kg, at least about 4.7mg / kg, at least about 4.8mg / kg, at least about 4.9mg / kg, at least about 5mg / kg, at least about 5.1mg / kg, at least about 5.2mg / kg, at least about 5.3mg / kg, at least about 5.4mg / kg, at least about 5.5mg / kg, at least about 5.6mg / kg, at least about 5.7mg / kg, at least about 5.8mg / kg, at least about 5.9mg / kg, at least about 6mg / kg, at least about 6.1mg / kg, at least about 6.2mg / kg, at least about 6.3mg / kg, at least about 6.4mg / kg, at least about 6.5mg / kg, at least about 6.6mg / kg, at least about 6.7mg / kg, at least about 6.8mg / kg, at least about 6.9mg / kg, at least about 7mg / kg, at least about 7.1mg / kg, at least about 7.2mg / kg, at least about 7.3mg / kg, at least about 7.4mg / kg g, at least about 7.5 mg / kg, at least about 7.6 mg / kg, at least about 7.7 mg / kg, at least about 7.8 mg / kg, at least about 7.9 mg / kg, at least about 8 mg / kg, at least about 8.1 mg / kg, at least about 8.2 mg / kg, at least about 8.3 mg / kg, at least about 8.4 mg / kg, at least about 8.5 mg / kg, at least about 8.6 mg / kg, at least about 8.7 mg / kg, at least about 8.8 mg / kg, at least about 8.9 mg / kg, at least about 9 mg / kg, at least about 9.1 mg / kg, at least about 9.2 mg / kg, at least about 9.3 mg / kg, at least about 9.4 mg / kg, at least about 9.5 mg / kg, at least about 9.6 mg / kg, at least about 9.7 mg / kg, at least about 9.8 mg / kg, at least about 9.9 mg / kg, at least about 10 mg / kg, at least about 11 mg / kg, at least about 12 mg / kg, at least about 13 mg / kg, at least about 14 mg / kg, at least about 15 mg / kg, at least about 16 mg / kg, at least about 17 mg / kg, at least about 18 mg / kg, at least about 19 mg / kg, at least about 20 mg / kg, at least about 21 mg / kg, at least about 22 mg / kg, at least about 23 mg / kg, at least about 24 mg / kg, at least about 25 mg / kg, at least about 26 mg / kg, at least about 27 mg / kg, at least about 28 mg / kg, at least about 29 mg / kg, or at least about 30 mg / kg.

[0161] In some embodiments, an aptamer of the disclosure (e.g., ApTOLL) has a therapeutic effect of at least about 0.001 mg / kg / day, at least about 0.002 mg / kg / day, at least about 0.003 mg / kg / day, at least about 0.004 mg / kg / day, at least about 0.005 mg / kg / day, at least about 0.006 mg / kg / day, at least about 0.007 mg / kg / day, at least about 0.008 mg / kg / day, at least about 0.009 mg / kg / day, at least about 0.010 mg / kg / day, at least about 0.015 mg / kg / day, at least about 0.020 mg / kg / day, at least about 0.025 mg / kg / day, at least about 0.030 mg / kg / day, at least about 0.035 mg / kg / day, at least about 0.040 mg / kg / day, at least about 0.045 mg / kg / day, at least about 0.046 mg / kg / day, at least about 0.047 mg / kg / day, at least about 0.048 mg / kg / day, at least about 0.049 mg / kg / day, at least about 0.050 mg / kg / day, at least about 0.051 mg / kg / day, at least about 0.052 mg / kg / day, at least about 0.053 mg / kg / day, at least about 0.054 mg / kg / day, at least about 0.055 mg / kg / day, at least about 0.056 mg / kg / day, at least about 0.057 mg / kg / day, at least about 0.058 mg / kg / day, at least about 0.059 mg / kg / day, at least about 0.060 mg / kg / day, at least about mg / kg / day, at least about 0.045 mg / kg / day, at least about 0.050 mg / kg / day, at least about 0.055 mg / kg / day, at least about 0.060 mg / kg / day, at least about 0.065 mg / kg / day, at least about 0.070 mg / kg / day, at least about 0.075 mg / kg / day, at least about 0.080 mg / kg / day, at least about 0.085 mg / kg / day, at least about 0.090 mg / kg / day, at least about 0.095 mg / kg / day, at least about 0.1 mg / kg / day, about 0.11 mg / kg / day, at least about 0.12 mg / kg / day, at least about 0.13 mg / kg / day, at least about 0.14 mg / kg / day, or at least about 0.15 mg / kg / day.

[0162] In some embodiments, the aptamer of the disclosure (e.g., ApTOLL) provides a dose of at least about 1 μg / kg / day, at least about 1.1 μg / kg / day, at least about 1.2 μg / kg / day, at least about 1.3 μg / kg / day, at least about 1.4 μg / kg / day, at least about 1.5 μg / kg / day, at least about 1.6 μg / kg / day, at least about 1.7 μg / kg / day, at least about 1.8 μg / kg / day, at least about 1.9 μg / kg / day, at least about 2 μg / kg / day, at least about 2.1 μg / kg / day, at least about 2.2 μg / kg / day day, at least about 2.3 μg / kg / day, at least about 2.4 μg / kg / day, at least about 2.5 μg / kg / day, at least about 2.6 μg / kg / day, at least about 2.7 μg / kg / day, at least about 2.8 μg / kg / day, at least about 2.9 μg / kg / day, at least about 3 μg / kg / day, at least about 3.1 μg / kg / day, at least about 3.2 μg / kg / day, at least about 3.3 μg / kg / day, at least about 3.4 μg / kg / day, at least about 3.5 μg / kg / day, at least about 3.6 μg / kg / day, at least about 3.7 μg / kg / day kg / day, at least about 3.8 μg / kg / day, at least about 3.9 μg / kg / day, at least about 4 μg / kg / day, at least about 4.1 μg / kg / day, at least about 4.2 μg / kg / day, at least about 4.3 μg / kg / day, at least about 4.4 μg / kg / day, at least about 4.5 μg / kg / day, at least about 4.6 μg / kg / day, at least about 4.7 μg / kg / day, at least about 4.8 μg / kg / day, at least about 4.9 μg / kg / day, at least about 5 μg / kg / day, at least about 5.1 μg / kg / day, at least about 5.2 μg / kg / day, at least about 5.3 μg / kg / day, at least about 5.4 μg / kg / day, at least about 5.5 μg / kg / day, at least about 5.6 μg / kg / day, at least about 5.7 μg / kg / day, at least about 5.8 μg / kg / day, at least about 5.9 μg / kg / day, at least about 6 μg / kg / day, at least about 6.1 μg / kg / day, at least about 6.2 μg / kg / day, at least about 6.3 μg / kg / day, at least about 6.4 μg / kg / day, at least about 6.5 μg / kg / day, at least about 6.6 μg / kg / day, at least about 6.7 μg / kg / day, at least about 6.8 μg / kg / day, at least about 6.9 μg / kg / day, at least about 7 μg / kg / day, at least about 7.1 μg / kg / day, at least about 7.2 μg / kg / day, at least about 7.3 μg / kg / day, at least about 7.4 μg / kg / day, at least about 7.5 μg / kg / day, at least about 7.6 μg / kg / day, at least about 7.7 μg / kg / day, at least about 7.8 μg / kg / day, at least about 7.9 μg / kg / day, at least about 8 μg / kg / day, at least about 8.1 μg / kg / day, at least about 8 0.2 μg / kg / day, at least about 8.3 μg / kg / day, at least about 8.4 μg / kg / day, at least about 8.5 μg / kg / day, at least about 8.6 μg / kg / day, at least about 8.7 μg / kg / day, at least about 8.8 μg / kg / day, at least about 8.9 μg / kg / day, at least about 9 μg / kg / day, at least about 9.1 μg / kg / day, at least about 9.2 μg / kg / day, at least about 9.3 μg / kg / day, at least about 9.4 μg / kg / day, at least about 9.5 μg / kg / day, at least about 9.6 μg / kg / day, at least Also about 9.7 μg / kg / day, at least about 9.8 μg / kg / day, at least about 9.9 μg / kg / day, at least about 10 μg / kg / day, at least about 10.1 μg / kg / day, at least about 10.2 μg / kg / day, at least about 10.3 μg / kg / day, at least about 10.4 μg / kg / day, at least about 10.5 μg / kg / day, at least about 10.6 μg / kg / day, at least about 10.7 μg / kg / day, at least about 10.8 μg / kg / day, at least about 10.9 μg / kg / day, at least about 11 μg / kg / day, and at least about 11. 1 μg / kg / day, at least about 11.2 μg / kg / day, at least about 11.3 μg / kg / day, at least about 11.4 μg / kg / day, at least about 11.5 μg / kg / day, at least about 11.6 μg / kg / day, at least about 11.7 μg / kg / day, at least about 11.8 μg / kg / day, at least about 11.9 μg / kg / day, at least about 12 μg / kg / day, at least about 12.1 μg / kg / day, at least about 12.2 μg / kg / day, at least about 12.3 μg / kg / day, at least about 12.4 μg / kg / day, at least about 12.5 μg / kg / day, at least about 12.6 μg / kg / day, at least about 12.7 μg / kg / day, at least about 12.8 μg / kg / day, at least about 12.9 μg / kg / day, at least about 13 μg / kg / day, at least about 13.1 μg / kg / day, at least about 13.2 μg / kg / day, at least about 13.3 μg / kg / day, at least about 13.4 μg / kg / day, at least about 13.5 μg / kg / day, at least about 13.6 μg / kg / day, at least about 13.7 μg / kg / day, at least about 13.8 The compound may be administered at a dose of at least about 13.9 μg / kg / day, at least about 14 μg / kg / day, at least about 14.1 μg / kg / day, at least about 14.2 μg / kg / day, at least about 14.3 μg / kg / day, at least about 14.4 μg / kg / day, at least about 14.5 μg / kg / day, at least about 14.6 μg / kg / day, at least about 14.7 μg / kg / day, at least about 14.8 μg / kg / day, at least about 14.9 μg / kg / day, or at least about 15 μg / kg / day.

[0163] In some embodiments, the aptamer of the disclosure (e.g., ApTOLL) is administered at a dose of at least about 1 μg / kg / day to at least about 2 μg / kg / day, at least about 2 μg / kg / day to at least about 3 μg / kg / day, at least about 3 μg / kg / day to at least about 4 μg / kg / day, at least about 4 μg / kg / day to at least about 5 μg / kg / day, at least about 5 μg / kg / day to at least about 6 μg / kg / day, at least about 6 μg / kg / day to at least about 7 μg / kg / day, at least about 7 μg / kg / day to at least about 8 μg / kg / day / kg / day, at least about 8 μg / kg / day to at least about 9 μg / kg / day, at least about 9 μg / kg / day to at least about 10 μg / kg / day, at least about 10 μg / kg / day to at least about 11 μg / kg / day, at least about 11 μg / kg / day to at least about 12 μg / kg / day, at least about 12 μg / kg / day to at least about 13 μg / kg / day, at least about 13 μg / kg / day to at least about 14 μg / kg / day, or at least about 14 μg / kg / day to at least about 15 μg / kg / day.

[0164] In some embodiments, an aptamer of the disclosure (e.g., ApTOLL) is administered at a dosage of at least about 1 μg / kg / day to at least about 3 μg / kg / day, at least about 3 μg / kg / day to at least about 6 μg / kg / day, at least about 6 μg / kg / day to at least about 9 μg / kg / day, at least about 9 μg / kg / day to at least about 12 μg / kg / day, or at least about 12 μg / kg / day to at least about 15 μg / kg / day.

[0165] In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dose of at least about 1 μg / kg / day to at least about 4 μg / kg / day, at least about 4 μg / kg / day to at least about 8 μg / kg / day, at least about 8 μg / kg / day to at least about 12 μg / kg / day, or at least about 11 μg / kg / day to at least about 15 μg / kg / day.

[0166] In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered at a dosage of at least about 1 μg / kg / day to at least about 5 μg / kg / day, at least about 5 μg / kg / day to at least about 10 μg / kg / day, or at least about 10 μg / kg / day to at least about 15 μg / kg / day.

[0167] In some embodiments, the aptamer of the disclosure (e.g., ApTOLL) is administered at a dose of at least about 6.5 μg / kg / day to at least about 7.5 μg / kg / day, at least about 6 μg / kg / day to at least about 8 μg / kg / day, at least about 5.5 μg / kg / day to at least about 8.5 μg / kg / day, at least about 5 μg / kg / day to at least about 9 μg / kg / day, at least about 4.5 μg / kg / day to at least about 9.5 μg / kg / day, at least about 4 μg / kg / day to at least about 10 μg / kg / day, at least about 3.5 μg / kg / day to at least about 10.5 μg / kg / day, at least about 3 μg / kg / day to at least The compound is administered at a dose of about 11 μg / kg / day, at least about 2.5 μg / kg / day to at least about 11.5 μg / kg / day, at least about 2 μg / kg / day to at least about 12 μg / kg / day, at least about 1.5 μg / kg / day to at least about 12.5 μg / kg / day, at least about 1 μg / kg / day to at least about 13 μg / kg / day, at least about 1 μg / kg / day to at least about 13.5 μg / kg / day, at least about 1 μg / kg / day to at least about 14 μg / kg / day, at least about 1 μg / kg / day to at least about 14.5 μg / kg / day, or at least about 1 μg / kg / day to at least about 15 μg / kg / day.

[0168] The dosages disclosed above can be administered as a single dose or multiple doses throughout the day. Thus, a total daily dose of 0.6 mg can be administered, for example, as two 0.3 mg doses, or three 0.2 mg doses, or five 0.1 mg doses.

[0169] In some embodiments, an aptamer of the disclosure (e.g., ApTOLL) has a T of about 0.5 hours, about 0.6 hours, about 0.7 hours, about 0.8 hours, about 0.9 hours, about 1 hour, about 1.1 hours, about 1.2 hours, about 1.3 hours, about 1.4 hours, about 1.5 hours, about 1.6 hours, about 1.7 hours, about 1.8 hours, about 1.9 hours, about 2 hours, about 2.1 hours, about 2.2 hours, about 2.3 hours, about 2.4 hours, about 2.5 hours, about 2.6 hours, about 2.7 hours, about 2.8 hours, about 2.9 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, or about 8 hours.1 / 2 (plasma half-life). In one particular embodiment, the T of the aptamer (e.g., ApTOLL) 1 / 2 is about 0.8 hours and 1.4 hours. In one particular embodiment, the T of an aptamer (e.g., ApTOLL) 1 / 2 In one particular embodiment, the T of ApTOLL in human plasma is about 1.4 hours. 1 / 2 is about 8 hours.

[0170] In some embodiments, the aptamer (e.g., ApTOLL) of the present disclosure is administered in multiple doses. In one embodiment, the aptamer is administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. In some embodiments, the aptamer is administered in three doses. In some embodiments, the three doses are administered on the same day. In some embodiments, the first dose is administered less than 1 hour after the onset of a TLR-4-mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke, for example, 10 minutes after the onset of a TLR-4-mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke. In some embodiments, the second dose is administered less than 3 hours after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke, e.g., about 2 hours after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke. In some embodiments, the third dose is administered less than 8 hours after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke, e.g., about 6 hours after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke.

[0171] In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) is administered intravenously or intraarterially. In certain embodiments, the aptamer of the present disclosure is administered as a bolus. In some embodiments, the bolus is a slow bolus.

[0172] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject with a TLR-4 mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, multiple sclerosis, (i) reduction of damaged tissue; (ii) reduced inflammation; (iii) improved prognosis and outcomes; (iv) a decrease in the level of an inflammatory biomarker (e.g., interferon-γ, interleukin-12p70, TNFα, IL-6, or any combination thereof); (v) improving quality of life; (vi) improvement in functional scores, e.g., motor scores (e.g., improved mobility); (vii) increased survival; or (v) resulting in any combination thereof.

[0173] The above effects relate to a control subject or control subject population that has or has had a TLR-4 mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, multiple sclerosis, but that has not been administered an aptamer of the present disclosure, e.g., ApTOLL.

[0174] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4-mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, results in a 20% to 75% reduction in tissue damage (e.g., brain or heart tissue) relative to untreated subjects or a reference value obtained from a control population of untreated subjects. In one particular embodiment, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4-mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, results in an approximately 65% ​​reduction in tissue damage (e.g., brain or heart tissue) relative to untreated subjects or a reference value obtained from a control population of untreated subjects.

[0175] In some embodiments, administration of an aptamer of the present disclosure to a subject following the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, results in a reduction in tissue damage (e.g., brain tissue or heart tissue) of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% relative to a reference value obtained from an untreated subject or a control population of untreated subjects.

[0176] In certain embodiments, administration of the aptamer of the present disclosure (e.g., ApTOLL) to a subject in need thereof reduces the size of the damaged or diseased area (e.g., infarct area after an ischemic event), which is significantly smaller when administered in a multiple-dose regimen. For example, in certain embodiments, administration of three doses of the aptamer (e.g., 10 minutes, 2 hours, and 6 hours after infarction) reduces the size of the damaged or diseased area (e.g., infarct area after an ischemic event) by at least 24%, compared to the approximately 19% reduction observed when a single dose is administered 10 minutes after infarction.

[0177] In some embodiments, administration of a multiple dose regimen of an aptamer of the present disclosure (e.g., ApTOLL) to a subject in need thereof reduces the size of the damaged or diseased area (e.g., infarct area after an ischemic event) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or at least about 50% compared to the size of the damaged or diseased area (e.g., infarct area after an ischemic event) observed after administration of a corresponding single dose regimen. , at least about 45%, at least 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 135%, at least about 140%, at least about 145%, at least about 150% , at least about 155%, at least about 160%, at least about 165%, at least about 170%, at least about 175%, at least about 180%, at least about 185%, at least about 190%, at least about 195%, at least about 200%, at least about 205%, at least about 210%, at least about 215%, at least about 220%, at least about 225%, at least about 230%, at least about 235%, at least about 240%, at least about 245%, at least about 250%, at least about 255%. 5%, at least about 260%, at least about 265%, at least about 270%, at least about 275%, at least about 280%, at least about 285%, at least about 290%, at least about 295%, at least about 300%, at least about 305%, at least about 310%, at least about 315%, at least about 320%, at least about 325%, at least about 330%, at least about 335%, at least about 340%, at least about 345%, or at least about 350% reduction.

[0178] In some embodiments, the treatment of the TLR-4-mediated diseases or conditions disclosed herein, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, by administering at least one aptamer (e.g., ApTOLL) of the present disclosure can be combined with other therapeutic and / or prophylactic treatments. For example, the aptamers of the present disclosure can be administered together with biologically active molecules such as anticoagulants, anti-inflammatory agents, or blood pressure regulators.

[0179] In some embodiments, administration of the aptamer of the present disclosure (e.g., ApTOLL) can be combined with, for example, surgical intervention, such as thrombectomy, in subjects suffering from myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke. In some embodiments, administration of the aptamer of the present disclosure can be combined with catheterization, such as balloon catheterization, or stent insertion. In some embodiments, arterial recanalization can be induced pharmacologically (e.g., thrombolysis), mechanically (e.g., endovascular thrombectomy), or a combination thereof.

[0180] In some embodiments, administration of the aptamer of the present disclosure (e.g., ApTOLL) is performed before, during, or after surgery (e.g., thrombectomy), or a combination thereof. In some embodiments, administration of the aptamer of the present disclosure (e.g., ApTOLL) is performed before, during, or after thrombolysis, such as pharmacological thrombolysis, pharmacodynamic thrombolysis, mechanical thrombectomy, or a combination thereof. In some embodiments, the thrombectomy is stent retrieval thrombectomy, balloon embolectomy, direct aspiration thrombectomy, surgical embolectomy, or any combination thereof.

[0181] In some embodiments, the methods of treating ischemic stroke disclosed herein include thrombolysis (e.g., pharmacodynamic thrombolysis) and / or thrombectomy (e.g., mechanical thrombectomy) in combination with administration of an aptamer of the present disclosure (e.g., ApTOLL), and the combined treatment achieves an improvement in tissue damage (e.g., infarct area) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, or more than about 10% compared to the effectiveness in reducing tissue damage (e.g., infarct area) observed after administration of an aptamer of the present disclosure (e.g., ApTOLL) in the absence of thrombolysis. at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 105%, at least about 110%, at least about 115%, at least about 120%, at least about 125%, at least about 130%, at least about 135%, at least about 140%, at least about 145%, at least about at least about 150%, at least about 155%, at least about 160%, at least about 165%, at least about 170%, at least about 175%, at least about 180%, at least about 185%, at least about 190%, at least about 195%, at least about 200%, at least about 205%, at least about 210%, at least about 215%, at least about 220%, at least about 225%, at least about 230%, at least about 235%, at least about 240%, at least about 245%, at least about 250%, at least about 255%, or at least about 350%, or at least about 260%, at least about 265%, at least about 270%, at least about 275%, at least about 280%, at least about 285%, at least about 290%, at least about 295%, at least about 300%, at least about 305%, at least about 310%, at least about 315%, about 320%, at least about 325%, at least about 330%, at least about 335%, at least about 340%, at least about 345%, or at least about 350% increase in effectiveness in reducing tissue damage (e.g., reduction in infarct area).

[0182] In some embodiments of the present disclosure, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4-mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, provides a protective effect.

[0183] Thus, in some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4 mediated disease or condition, e.g., ischemic stroke, results in at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 1 9, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours, resulting in a sustained reduction in the incidence of a particular complication, e.g., cerebral infarction.

[0184] In some embodiments, administration of an aptamer of the disclosure (e.g., ApTOLL) to a subject following the onset of a TLR-4 mediated disease or condition, e.g., ischemic stroke, results in at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 96 hours, resulting in a sustained reduction in the incidence of a particular complication, e.g., cerebral infarction.

[0185] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4 mediated disease or condition, such as a myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke, event, results in a reduction in the risk of death of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours, resulting in a sustained protective effect (e.g., reduced recurrence, reduced tissue damage, reduced inflammation, reduced symptoms and / or sequelae, or any combination thereof).

[0186] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4 mediated disease or condition, such as myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke, results in a decrease in the incidence of TLR-4-mediated disease or condition after administration of an aptamer of the present disclosure (alone or in combination with pharmacological intervention (e.g., thrombolysis) and / or mechanical intervention (e.g., endovascular thrombectomy)) of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, or 96 hours.

[0187] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke, results in a persistent protective effect (e.g., reduced recurrence, reduced tissue damage, reduced inflammation, reduced symptoms and / or sequelae, or any combination thereof) for at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, or at least about 28 days after the onset of the TLR-4-mediated disease or condition.

[0188] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4 mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, or ischemic stroke, results in a decrease in TLR-4 activity of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 14, at least about 16, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, or at least about 28 days.

[0189] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, results in a reduction in the volume of damaged tissue (e.g., infarct volume) of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% relative to the volume of damaged tissue observed in a control subject or control population in the absence of treatment with an aptamer of the present disclosure.

[0190] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, results in a reduction in the volume of damaged tissue (e.g., infarct volume) of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% relative to the volume of damaged tissue observed in a control subject or control population in the absence of treatment with an aptamer of the present disclosure.

[0191] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4-mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, results in a reduction in tissue damage (e.g., cortical or myocardial damage) of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, about 35%, or at least about 40% relative to tissue damage (e.g., cortical or myocardial damage) observed in a control subject or control population in the absence of treatment with an aptamer of the present disclosure.

[0192] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4 mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, results in an improvement in neurological recovery of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% relative to the neurological recovery observed in a control subject or control population in the absence of treatment with an aptamer of the present disclosure.

[0193] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4 mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, results in an improvement in motor function of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% relative to the motor score observed in a control subject or control population in the absence of treatment with an aptamer of the present disclosure.

[0194] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject after the onset of a TLR-4 mediated disease or condition, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, ischemic stroke, or multiple sclerosis, results in a reduction in plasma protein levels of a pro-inflammatory biomarker by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% relative to the plasma protein levels of the pro-inflammatory biomarker observed in a control subject or control population in the absence of treatment with an aptamer of the present disclosure.

[0195] In some embodiments, the aptamer (e.g., ApTOLL) of the present disclosure can be administered via intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In certain embodiments, the aptamer (e.g., ApTOLL) of the present disclosure is administered intravenously or intraarterially, for example, via infusion or bolus. In some embodiments, administration is via a slow bolus, i.e., the dose is administered via an injection lasting about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, or about 15 minutes.

[0196] In some embodiments, the aptamers of the present disclosure (eg, ApTOLL) can be used in conjunction with other agents or treatments suitable for treating ischemic conditions and / or thrombi, such as thrombolysis as described above.

[0197] In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) used in the methods disclosed herein can be combined with one or more therapies (pharmacological and / or surgical) for the treatment of ischemic stroke known in the art. In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) used in the methods disclosed herein can be combined with one or more therapies (pharmacological and / or surgical) for the treatment of myocardial infarction known in the art. In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) used in the methods disclosed herein can be combined with one or more therapies (pharmacological and / or surgical) for the treatment of myocardial infarction known in the art. In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) used in the methods disclosed herein can be combined with one or more therapies (pharmacological and / or surgical) for the treatment of hemorrhagic stroke known in the art. In some embodiments, the aptamer (e.g., ApTOLL) of the present disclosure used in the methods disclosed herein can be combined with one or more therapies (pharmacological and / or surgical) known in the art for the treatment of hemorrhagic transformation. In some embodiments, the aptamer (e.g., ApTOLL) of the present disclosure used in the methods disclosed herein can be combined with one or more therapies (pharmacological and / or surgical) known in the art for the treatment of multiple sclerosis.

[0198] In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) can be administered in combination with, for example, a TLR-4 antagonist, an anti-inflammatory agent, a nucleic acid, a peptide or protein, or a combination thereof. In some embodiments, the methods disclosed herein can also be combined with surgical procedures such as carotid endarterectomy and / or carotid stenting.

[0199] In some embodiments, the methods disclosed herein comprise administration of at least one aptamer of the present disclosure (e.g., ApTOLL), alone or in combination with pharmacological or mechanical thrombolysis, and optionally in combination with ibudilast, TAK242, NI-0101, eritoran, edaravone, uric acid, fingolimod, natalizumab, minocycline, anakinra, nerinetide, or any combination thereof.

[0200] In some embodiments, the methods disclosed herein include co-administration of at least one of the disclosed aptamers (e.g., ApTOLL) as a combination therapy comprising administration of: (i) a TLR-4 antagonist selected from the group consisting of naloxone, (+)-naloxone, naltrexone, (+)-naltrexone, lipopolysaccharide (LPS), ibudilast, propentofylline, amitriptyline, ketotifen, cyclobenzaprine, mianserin, imipramine, lipid A analogues (e.g., eritoran or E5531), pinocembrin, palmitoylethanolamide, tapentadol, polypropyletherimine dendrimer glucosamine (DG), aminoalkyl glucosaminide 4-phosphate (e.g., CRX-526), ​​IAXO-102, Rs-LPS, TLR-IN-C34, TAK-242, E5564, or any combination thereof; (ii) antiplatelet drugs, e.g., aspirin or clopidogrel; (iii) anticoagulants, e.g., heparin, acenocoumarol, warfarin, dabigatran, or rivaroxaban; (iv) antioxidants, such as edaravone; (v) tissue plasminogen activator, or (vi) any combination thereof.

[0201] In some embodiments, the methods disclosed herein include co-administration of at least one of the disclosed aptamers (e.g., ApTOLL) as a combination therapy, and administration of nucleic acids capable of silencing expression of genes involved in pathologies characterized by increased expression of TLR-4 and / or increased activation of TLR-4, e.g., antisense oligonucleotides (e.g., antisense RNA, antisense DNA, or antisense RNA / DNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), anti-microRNA (antimir); peptides such as signaling peptides and target-binding peptides (e.g., antibodies or antigen-binding fragments thereof, or compounds comprising antibodies or antigen-binding fragments thereof, such as antigen-drug conjugates or immunotoxins).

[0202] In some embodiments, the methods disclosed herein comprise the administration of at least one aptamer of the present disclosure, e.g., ApTOLL or any of the aptamers disclosed below, particularly any of the aptamers disclosed in Table 1, or a variant or derivative thereof.

[0203] In some embodiments, the methods disclosed herein can be practiced using nucleic acids other than aptamers that, instead of reducing and / or inhibiting TLR-4 action by binding to the TLR-4 protein, directly or indirectly reduce and / or inhibit (e.g., deplete or abolish) TLR-4 expression by interacting with the TLR-4 gene or a transcription product of the TLR4 gene, such as the messenger RNA (mRNA) encoding TLR-4, or with a nucleic acid (e.g., miRNA) that regulates TLR-4 expression, such as an antisense oligonucleotide, siRNA, shRNA, or antimir. The methods disclosed herein also are contemplated for practice using gene therapy approaches that employ agents that transiently or permanently alter TLR-4 expression, such as CRISPR / Cas, TALEN, or ZFN. Also contemplated is the practice of the methods disclosed herein using agents that post-transcriptionally modify the activity of TLR-4 or alter the integration of TLR-4 into the plasma membrane, alter TLR-4 function (e.g., antibodies or small molecule drugs), alter TLR-4 trafficking and / or recycling, or alter TLR-4 signaling by pharmacological or gene therapy intervention upstream and / or downstream of the TLR-4 signaling pathway.

[0204] In some embodiments, the present disclosure provides nucleic acid aptamers for use in ameliorating or ameliorating at least some symptoms or sequelae of a disease or condition in a subject in need thereof, (a) The aptamer has a length of, for example, about 40 to about 100 nucleotides and is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4; (i) the aptamer specifically binds to an epitope on the extracellular domain of TLR-4; (ii) binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation, or (b) The aptamer is a functionally equivalent variant of the aptamer of (a) having, for example, at least 85% sequence identity to SEQ ID NO: 1, 2, 3, or 4, wherein the functionally equivalent variant is derived from SEQ ID NO: 1, 2, 3, or 4 and retains the ability to specifically bind to TLR-4 and reduce and / or inhibit its activation.

[0205] In some embodiments, the present disclosure provides a method of treating a disease or condition disclosed herein, comprising administering to a subject in need of treatment a nucleic acid, (a) The aptamer has a length of, for example, about 40 to about 100 nucleotides and is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4; (i) the aptamer specifically binds to an epitope on the extracellular domain of TLR-4; (ii) binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation, or (b) The aptamer is a functionally equivalent variant of the aptamer of (a) having, for example, at least 85% sequence identity to SEQ ID NO: 1, 2, 3, or 4, wherein the functionally equivalent variant is derived from SEQ ID NO: 1, 2, 3, or 4 and retains the ability to specifically bind to TLR-4 and reduce and / or inhibit its activation.

[0206] In some embodiments, the present disclosure provides a method for ameliorating, improving, inhibiting, or reducing at least some symptoms or sequelae of a disease or condition disclosed herein in a subject in need thereof, comprising administering to the subject a nucleic acid; (a) The aptamer has a length of, for example, about 40 to about 100 nucleotides and is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4; (i) the aptamer specifically binds to an epitope on the extracellular domain of TLR-4; (ii) binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation, or (b) The aptamer is a functionally equivalent variant of the aptamer of (a) having, for example, at least 85% sequence identity to SEQ ID NO: 1, 2, 3, or 4, wherein the functionally equivalent variant is derived from SEQ ID NO: 1, 2, 3, or 4 and retains the ability to specifically bind to TLR-4 and reduce and / or inhibit its activation.

[0207] In some embodiments, the methods disclosed herein can be performed using any of the aptamers disclosed in Table 1, or a combination thereof. Thus, in some embodiments, for example, an aptamer having a length of about 40 to about 100 nucleotides is selected from the group consisting of SEQ ID NOs: 1-16.

[0208] In some embodiments, for example, an aptamer having a length of about 40 to about 100 nucleotides is a functionally equivalent variant having at least 85% sequence identity to the aptamer of SEQ ID NO: 1-16, where the functionally equivalent variant is derived from SEQ ID NO: 1-16 and maintains the ability to specifically bind to TLR-4 and reduce and / or inhibit its activation.

[0209] In some embodiments, the aptamer has a length of about 45, about 59, about 68, about 76, or about 78 nucleotides. In some embodiments, the aptamer has a length of about 45 to about 78 nucleotides. In some embodiments, the aptamer has a length of about 59 to about 78 nucleotides. In some embodiments, the aptamer has a length of about 68 to about 78 nucleotides. In some embodiments, the aptamer has a length of about 45 to about 76 nucleotides. In some embodiments, the aptamer has a length of about 45 to about 68 nucleotides. In some embodiments, the aptamer has a length of about 45 to about 59 nucleotides. In some embodiments, the aptamer has a length of about 59 to about 76 nucleotides. In some embodiments, the aptamer has a length of about 59 to about 68 nucleotides. In some embodiments, the aptamer has a length of about 68 to about 76 nucleotides.

[0210] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) or a combination thereof to a subject having an ischemic condition and / or a thrombus can reduce infarct volume. In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) or a combination thereof to a subject having an ischemic condition and / or a thrombus can reduce infarct volume after administration of multiple doses (e.g., 1, 2, 3, 4, or 5 doses) of an aptamer of the present disclosure (e.g., ApTOLL) or a combination thereof.

[0211] In some embodiments, administration of multiple doses of an aptamer of the present disclosure (e.g., ApTOLL), or a combination thereof, can begin, for example, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or about 48 hours after occlusion. In some embodiments, a single dose is administered, for example, about 10 minutes after occlusion, and administration of the aptamer induces a reduction in infarct volume compared to a control condition, for example, compared to the infarct volume of a subject not treated with the aptamer.

[0212] In some embodiments, two doses are administered, for example, about 10 minutes and about 2 hours after occlusion. In some embodiments, three doses are administered, for example, about 10 minutes, about 2 hours, and about 6 hours after occlusion. In some embodiments, four doses are administered, for example, about 10 minutes, about 2 hours, about 6 hours, and about 24 hours after occlusion. In some embodiments, five doses are administered, for example, about 10 minutes, about 2 hours, about 6 hours, about 24 hours, and about 48 hours after occlusion. In some embodiments, such a dose regimen induces a reduction in infarct volume compared to a control condition, for example, compared to the infarct volume in a subject not treated with an aptamer.

[0213] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) or a combination thereof induces a reduction in infarct volume of at least about 10%, at least 15%, at least about 20%, or at least about 25% compared to a control condition, e.g., compared to the infarct volume of a subject not treated with the aptamer.

[0214] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject with an ischemic condition and / or thrombus reduces infarct volume when administered shortly after the ischemic event, e.g., about 5, 10, 15, 20, or 30 minutes after the ischemic event. In some embodiments, the reduction in infarct volume is about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% compared to the infarct volume observed under control conditions, e.g., compared to the infarct volume in a subject not treated with an aptamer of the present disclosure (e.g., ApTOLL).

[0215] In some embodiments, intravenous administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject with an ischemic condition and / or thrombus reduces infarct volume by about 65% when administered about 10 minutes after the ischemic event.

[0216] The present disclosure also provides a method for selecting a subject having an ischemic condition and / or thrombus for treatment with an aptamer of the present disclosure (e.g., ApTOLL), wherein the subject is selected from treatment if the subject has, for example, a vascular occlusion suitable for mechanical thrombectomy, as determined or confirmed, for example, by computed tomography angiography (CTA). In some embodiments, the criterion used for selection is a large vessel occlusion suitable for mechanical thrombectomy, as determined or confirmed by neuroimaging criteria (CT or MRI), such as: (i) magnetic resonance imaging (MRI) criteria: for example, a diffusion-weighted imaging (DWI) restriction volume of ≥ about 5 mL to ≤ about 70 mL as determined by RAPID® software, and / or (ii) Computed tomography (CT) criteria: Alberta Stroke Program Early CT Score (ASPECTS) of about 6 to about 10, and admission cerebral blood flow (CBF) < 30% and ≥ about 5 mL to ≤ about 70 mL, as determined, for example, by RAPID® software.

[0217] In some embodiments, the standard used for selecting an object is the time from the onset of symptoms.Therefore, in some embodiments, when the object is less than 6 hours, for example, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours or less than 1 hour from the onset of ischemic state and / or thrombus, the object is selected for treatment with the aptamer (for example, ApTOLL) of the present disclosure.

[0218] In some embodiments, the criteria used to select a subject for treatment with an aptamer of the present disclosure (e.g., ApTOLL) is whether the subject is a candidate for EVT treatment, e.g., thrombectomy.

[0219] In some embodiments, the subject is a human subject and the aptamer of the present disclosure (e.g., ApTOLL) is administered at a dose of about 0.007 mg / kg to about 0.2 mg / kg. Thus, in some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) is administered at a dose of about 0.007 mg / kg per dose, about 0.008 mg / kg per dose, about 0.009 mg / kg per dose, about 0.010 mg / kg per dose, about 0.011 mg / kg per dose, about 0.016 mg / kg per dose, about 0.018 mg / kg per dose, about 0.019 mg / kg per dose, about 0.020 mg / kg per dose, about 0.021 mg / kg per dose, about 0.022 mg / kg per dose, about 0.023 mg / kg per dose, about 0.024 mg / kg per dose, about 0.025 mg / kg per dose, about 0.026 mg / kg per dose, about 0.027 mg / kg per dose, about 0.028 mg / kg per dose, about 0.029 mg / kg per dose, about 0.030 mg / kg per dose, about 0.031 mg / kg per dose, about 0.032 mg / kg per dose, about 0.033 mg / kg per dose, about 0.034 mg / kg per dose, about 0.035 mg / kg per dose, about 0.036 mg / kg per dose, about 0.037 mg / kg per dose, about 0.038 mg / kg per dose, about 0.039 mg / kg per dose, about 0.040 mg / kg per dose, about 0.041 mg / kg per dose, about 0.042 mg / kg per dose, about 0.043 mg / kg per dose, about 0.04 0.012mg / kg, about 0.013mg / kg per dose, about 0.014mg / kg per dose, about 0.015mg / kg per dose, about 0.016mg / kg per dose, about 0.017mg / kg per dose, about 0.018mg / kg per dose, about 0.019mg / kg per dose, about 0.020mg / kg per dose, about 0.021mg / kg per dose, about 0.022mg / kg per dose, about 0.023mg / kg per dose, about 0.024mg / kg per dose, about 0.025mg / kg per dose g / kg, about 0.030 mg / kg per dose, about 0.035 mg / kg per dose, about 0.040 mg / kg per dose, about 0.045 mg / kg per dose, about 0.050 mg / kg per dose, about 0.055 mg / kg per dose, about 0.060 mg / kg per dose, about 0.065 mg / kg per dose, about 0.070 mg / kg per dose, about 0.075 mg / kg per dose, about 0.080 mg / kg per dose, about 0.085 mg / kg per dose, about 0.090 mg / kg per dose, about 0.095mg / kg per dose, about 0.100mg / kg per dose, about 0.105mg / kg per dose, about 0.110mg / kg per dose, about 0.115mg / kg per dose, about 0.120mg / kg per dose, about 0.125mg / kg per dose, about 0.130mg / kg per dose, about 0.135mg / kg per dose, about 0.140mg / kg per dose, about 0.145mg / kg per dose, about 0.150mg / kg per dose, about 0.155mg / kg per dose, about 0.It is administered to a human subject at a dose of about 160 mg / kg, about 0.165 mg / kg per dose, about 0.170 mg / kg per dose, about 0.175 mg / kg per dose, about 0.180 mg / kg per dose, about 0.185 mg / kg per dose, about 0.190 mg / kg per dose, or about 0.2 mg / kg per dose.

[0220] According to the above disclosure, considering a dose range of about 0.007 mg / kg to about 0.20 mg / kg, and considering a standard body weight of about 70 kg for a human subject, the amount of a standard single dose is about 0.5 mg / dose to about 10 mg / dose. Thus, in some embodiments, the aptamer (e.g., ApTOLL) of the present disclosure is administered at about 0.5 mg / dose, about 0.6 mg / dose, about 0.7 mg / dose, about 0.8 mg / dose, about 0.9 mg / dose, about 1 mg / dose, about 1.1 mg / dose, about 1.2 mg / dose, about 1.3 mg / dose, about 1.4 mg / dose, about 1.5 mg / dose, about 1.6 mg / dose, about 1.7 mg / dose, or about 1.8 mg / dose to treat any of the diseases or conditions disclosed herein, or to prevent, inhibit, or reduce any of the symptoms and / or sequelae associated with such diseases or conditions. The compound is administered to a human subject in an amount of about 1.9 mg / dose, about 2 mg / dose, about 2.5 mg / dose, about 3 mg / dose, about 3.5 mg / dose, about 4 mg / dose, about 4.5 mg / dose, about 5 mg / dose, about 5.5 mg / dose, about 6 mg / dose, about 6.5 mg / dose, about 7 mg / dose, about 7.5 mg / dose, about 8 mg / dose, about 8.5 mg / dose, about 9 mg / dose, about 9.5 mg / dose, about 10 mg / dose, about 11 mg / dose, about 12 mg / dose, about 13 mg / dose, about 14 mg / dose, about 15 mg / dose, about 16 mg / dose, about 17 mg / dose, about 18 mg / dose, about 19 mg / dose, or about 20 mg / dose.

[0221] In some embodiments, the present disclosure provides prophylactic methods for preventing the development of an inflammatory response in a subject who has suffered an acute myocardial infarction, comprising administration of an aptamer of the present disclosure (eg, ApTOLL).

[0222] In some embodiments, the present disclosure provides methods for selecting a subject who has suffered an acute myocardial infarction for treatment with an aptamer of the present disclosure (e.g., ApTOLL), the methods comprising, for example, (i) measuring, assessing, or quantifying the infarct area, (ii) assessing cardiac function, (iii) measuring biomarkers associated with tissue damage or tissue remodeling, or (iv) a combination thereof.

[0223] The present disclosure also provides a method for promoting or inducing recovery of cardiac function in a subject suffering from acute myocardial infarction, the method comprising administering an aptamer of the present disclosure (e.g., ApTOLL) to the subject. Also provided is a method for reducing necrosis (e.g., left ventricular necrosis) and / or fibrosis in a subject suffering from acute myocardial infarction, the method comprising administering an aptamer of the present disclosure (e.g., ApTOLL) to the subject. In some embodiments, recovery of cardiac function, reduction in infarct area relative to a control, reduction in necrosis (e.g., left ventricular necrosis) relative to a control, reduction in fibrosis relative to a control, or any combination thereof, can be observed at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after administering an aptamer of the present disclosure (e.g., ApTOLL) to the subject. In some embodiments, the troponin I level in a subject who has suffered an acute myocardial infarction and who has been administered an aptamer of the present disclosure (e.g., ApTOLL) is lower than the troponin I level in a subject who has not been administered the aptamer. In some embodiments, the lower troponin I level is detectable, for example, at about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, or about 48 hours after administering an aptamer of the present disclosure (e.g., ApTOLL) to the subject.

[0224] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject suffering from acute myocardial infarction can reduce the infarct area by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% relative to a control condition, e.g., relative to a subject not administered an aptamer of the present disclosure (e.g., ApTOLL).

[0225] In some embodiments, administration of an aptamer of the present disclosure (e.g., ApTOLL) to a subject suffering from acute myocardial infarction results in: (i) reducing the infarct area (e.g., the volume of the infarct area) relative to a control; (ii) maintaining cardiac tissue integrity; (iii) reducing or inhibiting fibrosis; (iv) inhibiting the expression of markers of extracellular matrix degradation; (v) reducing, reducing the risk of, or inhibiting erroneous cardiac remodeling; (vi) induce cardioprotection; (vii) reducing or inhibiting the degradation of the extracellular matrix; (viii) improving or promoting cardiac remodeling; (ix) preserving ventricular anatomy; (x) maintaining cardiac function; (xi) reducing infarction progression; (xii) improve myocardial repair; (xiii) increasing or restoring ventricular contractility; or (xiv) Any combination thereof is possible.

[0226] Thus, in some embodiments, the present disclosure provides a method for treating acute myocardial infarction in a subject: (i) reducing the infarct area (e.g., the volume of the infarct area) relative to a control; (ii) maintaining cardiac tissue integrity; (iii) reducing or inhibiting fibrosis; (iv) inhibiting the expression of markers of extracellular matrix degradation; (v) reducing, reducing the risk of, or inhibiting erroneous cardiac remodeling; (vi) induce cardioprotection; (vii) reducing or inhibiting the degradation of the extracellular matrix; (viii) improving or promoting cardiac remodeling; (ix) preserving ventricular anatomy; (x) maintaining cardiac function; (xi) reducing infarction progression; (xii) improve myocardial repair; (xiii) increasing or restoring ventricular contractility; or (xiv) A method for any combination thereof, Methods are provided that include administering to a subject an aptamer of the disclosure (e.g., ApTOLL).

[0227] In some embodiments, the expression of MMP-9 in a subject who has suffered an acute myocardial infarction, comprising administering to the subject an aptamer of the present disclosure (e.g., ApTOLL), is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% relative to expression in a subject who has not been administered an aptamer of the present disclosure (e.g., ApTOLL).

[0228] In some embodiments, the present disclosure provides a method for selecting a subject who has suffered an acute myocardial infarction for administration of an aptamer of the present disclosure (e.g., ApTOLL), the method comprising measuring the expression level of MMP-9 in the subject (e.g., protein expression level, mRNA expression level, or a combination thereof), and administering an aptamer of the present disclosure (e.g., ApTOLL) if MMP-9 is elevated relative to a control value, e.g., a value observed in a subject not treated with an aptamer of the present disclosure (e.g., ApTOLL) or a standard normal expression value.

[0229] In some embodiments, an aptamer of the present disclosure (eg, ApTOLL) is administered to a human subject suffering from acute myocardial infarction at a dose of about 0.007 mg / kg to about 0.20 mg / kg.Thus, in some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered to a human subject suffering from acute myocardial infarction at a dose of about 0.007 mg / kg per dose, about 0.008 mg / kg per dose, about 0.009 mg / kg per dose, about 0.010 mg / kg per dose, about 0.011 mg / kg per dose, about 0.012 mg / kg per dose, about 0.013 mg / kg per dose, about 0.014 mg / kg per dose, about 0.015 mg / kg per dose, about 0.016 mg / kg per dose, about 0.017 mg / kg per dose. g, about 0.018 mg / kg per dose, about 0.019 mg / kg per dose, about 0.020 mg / kg per dose, about 0.021 mg / kg per dose, about 0.022 mg / kg per dose, about 0.023 mg / kg per dose, about 0.024 mg / kg per dose, about 0.025 mg / kg per dose, about 0.030 mg / kg per dose, about 0.035 mg / kg per dose, about 0.040 mg / kg per dose, about 0.045 mg / kg per dose, about 0.050 mg / kg per dose, about 0.055 mg / kg per dose, about 0.060mg / kg per dose, about 0.065mg / kg per dose, about 0.070mg / kg per dose, about 0.075mg / kg per dose, about 0.080mg / kg per dose, about 0.085mg / kg per dose, about 0.090mg / kg per dose, about 0.095mg / kg per dose, about 0.100mg / kg per dose, about 0.105mg / kg per dose, about 0.110mg / kg per dose, about 0.115mg / kg per dose, about 0.120mg / kg per dose, about 0.125mg / kg per dose about 0.130 mg / kg, about 0.135 mg / kg per dose, about 0.140 mg / kg per dose, about 0.145 mg / kg per dose, about 0.150 mg / kg per dose, about 0.155 mg / kg per dose, about 0.160 mg / kg per dose, about 0.165 mg / kg per dose, about 0.170 mg / kg per dose, about 0.175 mg / kg per dose, about 0.180 mg / kg per dose, about 0.185 mg / kg per dose, about 0.190 mg / kg per dose, or about 0.2 mg / kg per dose.

[0230] According to the above disclosure, considering a dose range of about 0.007 mg / kg to about 0.20 mg / kg, and considering the standard body weight of a human subject suffering from acute myocardial infarction of about 70 kg, the amount of a standard single dose is about 0.5 mg / dose to about 10 mg / dose. Thus, in some embodiments, the aptamer (e.g., ApTOLL) of the present disclosure is administered to a human subject suffering from acute myocardial infarction at a dose of about 0.5 mg / dose, about 0.6 mg / dose, about 0.7 mg / dose, about 0.8 mg / dose, about 0.9 mg / dose, about 1 mg / dose, about 1.1 mg / dose, about 1.2 mg / dose, about 1.3 mg / dose, about 1.4 mg / dose, about 1.5 mg / dose, about 1.6 mg / dose, about 1.7 mg / dose, about 1.8 mg / dose, about 1.9 mg / dose, about 2 mg / dose, about 2.5 mg / dose, or about 3 mg / dose. The compound is administered in an amount of about 3.5 mg / dose, about 4 mg / dose, about 4.5 mg / dose, about 5 mg / dose, about 5.5 mg / dose, about 6 mg / dose, about 6.5 mg / dose, about 7 mg / dose, about 7.5 mg / dose, about 8 mg / dose, about 8.5 mg / dose, about 9 mg / dose, about 9.5 mg / dose, about 10 mg / dose, about 11 mg / dose, about 12 mg / dose, about 13 mg / dose, about 14 mg / dose, about 15 mg / dose, about 16 mg / dose, about 17 mg / dose, about 18 mg / dose, about 19 mg / dose, or about 20 mg / dose.

[0231] In some embodiments, the present disclosure provides a method for preventing, inhibiting, suppressing, or delaying the onset of symptoms and / or sequelae of a neuromuscular or neurodegenerative disease or condition, such as multiple sclerosis, in a subject in need thereof, comprising administering an aptamer (e.g., ApTOLL) of the present disclosure to the subject. In some embodiments, the aptamer (e.g., ApTOLL) of the present disclosure is administered to the subject about 24 hours after the onset of symptoms of the neuromuscular or neurodegenerative disease or condition. In some embodiments, a single dose of the aptamer (e.g., ApTOLL) of the present disclosure is administered to the subject. In some embodiments, multiple doses (e.g., 2, 3, 4, or 5 doses) of the aptamer (e.g., ApTOLL) of the present disclosure are administered to the subject.

[0232] In some embodiments, administering an aptamer of the present disclosure (e.g., ApTOLL) to a subject having a neuromuscular or neurodegenerative disease or condition results in (i) improved clinical score, (ii) reduced weight loss (weight regain), (iii) remyelination, (iv) reduced axonal damage, (v) reduced inflammation, (vi) reduced demyelination, (vii) increased myelin area, (viii) increased neurofilaments, or (ix) any combination. Thus, in some embodiments, the present disclosure provides a method of (i) improving clinical score, (ii) reducing weight loss (weight regain), (iii) remyelination, (iv) reduced axonal damage, (v) reduced inflammation, (vi) reduced demyelination, (vii) increased myelin area, (viii) increased neurofilaments, or (ix) any combination in a subject having a neuromuscular or neurodegenerative disease or condition, the method comprising administering an aptamer of the present disclosure (e.g., ApTOLL) to the subject. In some embodiments, remyelination can be determined by measuring the levels of biomarkers such as PDGFRα, CC1, Oligo2, or a combination thereof.

[0233] In some embodiments, an aptamer of the disclosure (e.g., ApTOLL) is administered to a human subject having a neuromuscular or neurodegenerative disease or condition at a dose of about 0.007 mg / kg to about 0.20 mg / kg.Thus, in some embodiments, an aptamer of the disclosure (e.g., ApTOLL) is administered to a human subject having a neuromuscular or neurodegenerative disease or condition at a dose of about 0.007 mg / kg per dose, about 0.008 mg / kg per dose, about 0.009 mg / kg per dose, about 0.010 mg / kg per dose, about 0.011 mg / kg per dose, about 0.012 mg / kg per dose, about 0.013 mg / kg per dose, about 0.014 mg / kg per dose, about 0.015 mg / kg per dose, about 0.016 mg / kg per dose, about 0.017 mg / kg per dose, about 0.018 mg / kg per dose, about 0.019 mg / kg per dose, about 0.020 mg / kg per dose, about 0.021 mg / kg per dose, about 0.022 mg / kg per dose, about 0.023 mg / kg per dose, about 0.024 mg / kg per dose, about 0.025 mg / kg per dose, about 0.026 mg / kg per dose, about 0.027 mg / kg per dose, about 0.028 mg / kg per dose, about 0.029 mg / kg per dose, about 0.030 mg / kg per dose, about 0.031 mg / kg per dose, about 0.032 mg / kg per dose, about 0.033 mg / kg per dose, about 0.034 mg / kg per dose, about 0.035 mg / kg per dose, about 0.036 mg / kg per dose, about 0.037 mg / kg per dose, about 0.038 mg / kg per dose, about 0.039 mg / kg per dose, about 0.040 mg / kg per dose, about 0.041 mg / kg per dose, about 0.042 mg 0.017mg / kg, about 0.018mg / kg per dose, about 0.019mg / kg per dose, about 0.020mg / kg per dose, about 0.021mg / kg per dose, about 0.022mg / kg per dose, about 0.023mg / kg per dose, about 0.024mg / kg per dose, about 0.025mg / kg per dose, about 0.030mg / kg per dose, about 0.035mg / kg per dose, about 0.040mg / kg per dose, about 0.045mg / kg per dose, about 0.050mg / kg per dose, about 0.055mg / kg, about 0.060mg / kg per dose, about 0.065mg / kg per dose, about 0.070mg / kg per dose, about 0.075mg / kg per dose, about 0.080mg / kg per dose, about 0.085mg / kg per dose, about 0.090mg / kg per dose, about 0.095mg / kg per dose, about 0.100mg / kg per dose, about 0.105mg / kg per dose, about 0.110mg / kg per dose, about 0.115mg / kg per dose, about 0.120mg / kg per dose, about 0.125mg / kg per dose, about 0.130mg / kg per dose, about 0.135 ... The medicament is administered at a dosage of about 0.130 mg / kg per dose, about 0.135 mg / kg per dose, about 0.140 mg / kg per dose, about 0.145 mg / kg per dose, about 0.150 mg / kg per dose, about 0.155 mg / kg per dose, about 0.160 mg / kg per dose, about 0.165 mg / kg per dose, about 0.170 mg / kg per dose, about 0.175 mg / kg per dose, about 0.180 mg / kg per dose, about 0.185 mg / kg per dose, about 0.190 mg / kg per dose, or about 0.2 mg / kg per dose.

[0234] According to the above disclosure, considering a dose range of about 0.007 mg / kg to about 0.20 mg / kg, and considering a standard body weight of about 70 kg for a human subject with a neuromuscular or neurodegenerative disease or condition, a standard single dose amount is about 0.5 mg / dose to about 10 mg / dose. Thus, in some embodiments, the aptamer (e.g., ApTOLL) of the present disclosure is administered to a human subject with a neuromuscular or neurodegenerative disease or condition at doses of about 0.5 mg / dose, about 0.6 mg / dose, about 0.7 mg / dose, about 0.8 mg / dose, about 0.9 mg / dose, about 1 mg / dose, about 1.1 mg / dose, about 1.2 mg / dose, about 1.3 mg / dose, about 1.4 mg / dose, about 1.5 mg / dose, about 1.6 mg / dose, about 1.7 mg / dose, about 1.8 mg / dose, about 1.9 mg / dose, about 2 mg / dose, about 2.5 mg / dose. g / dose, about 3 mg / dose, about 3.5 mg / dose, about 4 mg / dose, about 4.5 mg / dose, about 5 mg / dose, about 5.5 mg / dose, about 6 mg / dose, about 6.5 mg / dose, about 7 mg / dose, about 7.5 mg / dose, about 8 mg / dose, about 8.5 mg / dose, about 9 mg / dose, about 9.5 mg / dose, about 10 mg / dose, about 11 mg / dose, about 12 mg / dose, about 13 mg / dose, about 15 mg / dose, about 16 mg / dose, about 17 mg / dose, about 18 mg / dose, about 19 mg / dose, about 20 mg / dose.

[0235] Also provided is a method for preventing, inhibiting, suppressing, or delaying the onset of symptoms and / or sequelae of pathologies involving primary and secondary demyelination, such as stroke or craniocerebral trauma (traumatic brain injury), in a subject, comprising administering to the subject an aptamer of the present disclosure (e.g., ApTOLL).

[0236] In some embodiments, an aptamer of the disclosure (eg, ApTOLL) is administered to a human subject who has suffered a stroke or traumatic brain injury at a dose of about 0.007 mg / kg to about 0.20 mg / kg.Thus, in some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) is administered to a human subject suffering from stroke or traumatic brain injury at a dose of about 0.007 mg / kg per dose, about 0.008 mg / kg per dose, about 0.009 mg / kg per dose, about 0.010 mg / kg per dose, about 0.011 mg / kg per dose, about 0.012 mg / kg per dose, about 0.013 mg / kg per dose, about 0.014 mg / kg per dose, about 0.015 mg / kg per dose, about 0.016 mg / kg per dose, about 0.017 mg / kg per dose, about 0.018 mg / kg per dose, about 0.019 mg / kg per dose, about 0.020 mg / kg per dose, about 0.021 mg / kg per dose, about 0.022 mg / kg per dose, about 0.023 mg / kg per dose, about 0.024 mg / kg per dose, about 0.025 mg / kg per dose, about 0.026 mg / kg per dose, about 0.027 mg / kg per dose, about 0.028 mg / kg per dose, about 0.029 mg / kg per dose, about 0.030 mg / kg per dose, about 0.031 mg / kg per dose, about 0.032 mg / kg per dose, about 0.033 mg / kg per dose, about 0.034 mg / kg per dose, about 0.035 mg / kg per dose, about 0.036 mg / kg per dose, about 0.037 mg / kg per dose, about 0.038 mg / kg per dose, about 0.039 mg / kg per dose, about 0.040 mg / kg per dose, about 0.041 mg / kg per dose, about 0.042 mg / kg per dose 7mg / kg, about 0.018mg / kg per dose, about 0.019mg / kg per dose, about 0.020mg / kg per dose, about 0.021mg / kg per dose, about 0.022mg / kg per dose, about 0.023mg / kg per dose, about 0.024mg / kg per dose, about 0.025mg / kg per dose, about 0.030mg / kg per dose, about 0.035mg / kg per dose, about 0.040mg / kg per dose, about 0.045mg / kg per dose, about 0.050mg / kg per dose, about 0.055mg / kg per dose , about 0.060mg / kg per dose, about 0.065mg / kg per dose, about 0.070mg / kg per dose, about 0.075mg / kg per dose, about 0.080mg / kg per dose, about 0.085mg / kg per dose, about 0.090mg / kg per dose, about 0.095mg / kg per dose, about 0.100mg / kg per dose, about 0.105mg / kg per dose, about 0.110mg / kg per dose, about 0.115mg / kg per dose, about 0.120mg / kg per dose, about 0.125mg / kg per dose about 0.130 mg / kg, about 0.135 mg / kg per dose, about 0.140 mg / kg per dose, about 0.145 mg / kg per dose, about 0.150 mg / kg per dose, about 0.155 mg / kg per dose, about 0.160 mg / kg per dose, about 0.165 mg / kg per dose, about 0.170 mg / kg per dose, about 0.175 mg / kg per dose, about 0.180 mg / kg per dose, about 0.185 mg / kg per dose, about 0.190 mg / kg per dose, or about 0.2 mg / kg per dose.

[0237] According to the above disclosure, considering a dose range of about 0.007 mg / kg to about 0.20 mg / kg, and considering the standard body weight of a human subject suffering from stroke or traumatic brain injury of about 70 kg, the amount of a standard single dose is about 0.5 mg / dose to about 10 mg / dose. Thus, in some embodiments, the aptamer (e.g., ApTOLL) of the present disclosure is administered to a human subject suffering from stroke or traumatic brain injury at doses of about 0.5 mg / dose, about 0.6 mg / dose, about 0.7 mg / dose, about 0.8 mg / dose, about 0.9 mg / dose, about 1 mg / dose, about 1.1 mg / dose, about 1.2 mg / dose, about 1.3 mg / dose, about 1.4 mg / dose, about 1.5 mg / dose, about 1.6 mg / dose, about 1.7 mg / dose, about 1.8 mg / dose, about 1.9 mg / dose, about 2 mg / dose, about 2.5 mg / dose, about 3 mg / dose. g / dose, about 3.5 mg / dose, about 4 mg / dose, about 4.5 mg / dose, about 5 mg / dose, about 5.5 mg / dose, about 6 mg / dose, about 6.5 mg / dose, about 7 mg / dose, about 7.5 mg / dose, about 8 mg / dose, about 8.5 mg / dose, about 9 mg / dose, about 9.5 mg / dose, about 10 mg / dose, about 11 mg / dose, about 12 mg / dose, about 13 mg / dose, about 14 mg / dose, about 15 mg / dose, about 16 mg / dose, about 17 mg / dose, about 18 mg / dose, about 19 mg / dose, about 20 mg / dose.

[0238] III. TLR-4 specific aptamers The aptamers used in the methods of the present disclosure specifically bind to at least one epitope located on the extracellular domain of TLR-4 and have the ability to inhibit TLR-4. Specific examples of aptamers of the present disclosure are provided in Table 1. In some embodiments, the aptamers of the present disclosure are variants and / or derivatives of the aptamers disclosed in Table 1. [Table 1]

[0239] The aptamers in Table 1 have lengths of 45 to 78 nucleotides. The A content ranges from about 17% to about 27%. The T content ranges from about 17% to about 28%. The G content ranges from about 21% to about 33%. The C content ranges from about 20% to about 34%.

[0240] In some embodiments, the aptamers of the present disclosure are chemically modified aptamers as disclosed below. In some embodiments, the aptamers of the present disclosure are DNA and / or RNA aptamers (e.g., ssDNA aptamers) that can specifically bind to and inhibit TLR-4 with at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the ability of the aptamers disclosed in Table 1 to specifically bind to and inhibit TLR-4.

[0241] In some embodiments, an aptamer of the disclosure comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more nucleotides 5' of a sequence disclosed in Table 1, and the aptamer is capable of specifically binding to and inhibiting TLR-4.

[0242] In some embodiments, an aptamer of the disclosure comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more nucleotides 3' of a sequence disclosed in Table 1, and the aptamer is capable of specifically binding to and inhibiting TLR-4.

[0243] In some embodiments, an aptamer of the present disclosure comprises a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to a sequence disclosed in Table 1, and the aptamer is capable of specifically binding to and inhibiting TLR-4.

[0244] In some embodiments, the aptamer of the present disclosure consists of a nucleic acid sequence (e.g., ssDNA) of about 30 to about 200 nucleotides, about 35 to about 150 nucleotides, about 40 to about 100 nucleotides, about 45 to about 80 nucleotides, about 40 to about 50 nucleotides, about 35 to about 55 nucleotides, 30 to about 60 nucleotides, about 35 to about 65 nucleotides, about 40 to about 70 nucleotides, about 75 to about 85 nucleotides, about 70 to about 90 nucleotides, about 65 to about 95 nucleotides, about 60 to about 100 nucleotides, about 55 to about 95 nucleotides, about 50 to about 90 nucleotides, about 45 to about 85 nucleotides, about 50 to about 80 nucleotides, about 55 to about 75 nucleotides, or about 60 to about 75 nucleotides.

[0245] In some embodiments, the aptamer of the present disclosure can be covalently or non-covalently attached to at least one biologically active molecule. In some embodiments, the biologically active molecule can specifically bind to TLR-4. In some embodiments, the biologically active molecule includes, for example, an antibody or its antigen-binding fragment, a small molecule, a peptide, an aptamer, a lipid, a lipopolysaccharide, a polysaccharide, an enzyme, or a nucleic acid. In some embodiments, the biologically active molecule includes an anti-inflammatory agent.

[0246] In some embodiments, the biologically active molecule is a TLR-4 antagonist selected from the group consisting of naloxone, (+)-naloxone, naltrexone, (+)-naltrexone, lipopolysaccharide (LPS), ibudilast, propentofylline, amitriptyline, ketotifen, cyclobenzaprine, mianserin, imipramine, lipid A analogues (e.g., eritoran or E5531), pinocembrin, palmitoylethanolamide, tapentadol, polypropyletherimine dendrimer glucosamine (DG), aminoalkyl glucosaminide 4-phosphate (e.g., CRX-526), ​​IAXO-102, Rs-LPS, TLR-IN-C34, TAK-242, E5564, or any combination thereof.

[0247] In some embodiments, the biologically active molecule comprises an antiplatelet agent, such as aspirin or clopidogrel. In some embodiments, the biologically active molecule comprises an anticoagulant, such as heparin, acenocoumarol, warfarin, dabigatran, or rivaroxaban. In some embodiments, the biologically active molecule comprises an antioxidant, such as edaravone. In some embodiments, the biologically active molecule is tissue plasminogen activator.

[0248] In some embodiments, the biologically active molecule is a beta-blocker, such as metoprolol or cabedilol, an ACE inhibitor, a statin, or an aldosterone antagonist, such as spironolactone or eplerenone.

[0249] In some embodiments, biologically active molecules include nucleic acids (e.g., antisense RNA, antisense DNA, and small interfering RNA) capable of silencing the expression of genes involved in pathologies characterized by increased expression of TLR-4 and / or increased activation of TLR-4, including, but not limited to, NFKB1, RIPK3, IFNB1, LY96 (MD-2), IRF3, TLR3, TIRAP (MaI), TICAM1 (TRIF), RIPK1, TRAF6, CD14, TRAM, IKBKG (IKK-γ), IFNA1, and TLR4 genes. The term "antisense RNA" in the context of this disclosure refers to a single-stranded RNA whose nucleotide sequence is complementary to a target messenger RNA, thereby interfering with or silencing the expression of the respective gene. The term "antisense DNA" in the context of this disclosure refers to a single-stranded DNA whose nucleotide sequence is complementary to a target messenger RNA, thereby interfering with or silencing the expression of the respective gene. The term "small interfering RNA" or "siRNA" in the context of the present disclosure refers to a double-stranded RNA having a length of 20 to 25 nucleotides that is highly specific for the nucleotide sequence of its target messenger RNA, thereby interfering with the expression of the respective gene.

[0250] In some embodiments, the aptamers of the present disclosure (e.g., ApTOLL) are resistant to degradation by λ-exonuclease, e.g., after incubation with the nuclease for at least about 5 minutes, at least about 15 minutes, at least about 30 minutes, at least about 1 hour, at least 2 hours, or at least about 4 hours.

[0251] In some embodiments, an aptamer of the present disclosure (e.g., ApTOLL) inhibits or reduces LPS (lipopolysaccharide)-mediated TLR-4 activation, as measured, for example, using HEK-blue-hTLR-4 cells expressing hTLR-4 and the TLR-4 coactivator proteins MD2 and CD14, using methods known in the art. In some embodiments, such reduction in activation is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%, compared to the effect observed under control conditions, e.g., without administration of an aptamer of the present disclosure (e.g., ApTOLL).

[0252] In some embodiments, the aptamers of the present disclosure (e.g., ApTOLL) have a binding affinity for human TLR-4 of 30-60 nM, as measured using methods known in the art and cynomolgus monkey and human monocytes. In some embodiments, the aptamers of the present disclosure have a binding affinity for human TLR-4 of at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, or at least about 70 nM.

[0253] In some embodiments, the aptamers of the present disclosure (e.g., ApTOLL) inhibit TLR-4 activation induced by damage-associated molecular patterns (DAMPs), as measured, for example, using methods known in the art, using HEK-blue-hTLR-4 cells expressing hTLR-4 and the TLR-4 coactivator proteins MD2 and CD14. DAMPs (damage-associated molecular patterns) are tissue molecules, such as heat shock proteins, nucleic acids, fibronectin, or hyaluronic acid, that are released into the brain parenchyma under conditions of injury. Thus, in some embodiments, the aptamers of the present disclosure can inhibit TLR-4 activation by an endogenous TLR-4 agonist (e.g., a DAMP) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% compared to control conditions (e.g., without administration of the aptamer of the present disclosure).

[0254] In some embodiments, the aptamers of the present disclosure induce a reduction in downstream TLR-4 cellular effectors, such as NOx levels, in mouse peritoneal macrophages stimulated with LPS, as measured, for example, using methods known in the art. In some embodiments, administration of an aptamer of the present disclosure induces a reduction in NOx levels of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, or at least about 75% compared to control conditions (e.g., without administration of an aptamer of the present disclosure).

[0255] In some embodiments, the aptamers of the disclosure have no detectable agonist effect on TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9 human Toll receptors and no antagonist effect on TLR2 and TLR5.

[0256] In some embodiments, the TLR-4 receptor is internalized after binding to an aptamer of the present disclosure, e.g., as measured in human macrophages using methods known in the art. In some embodiments, the TLR-4 receptor comprising a binding aptamer of the present disclosure is internalized into the cytoplasm approximately 20 minutes after the aptamer binds to TLR-4.

[0257] In some embodiments, the new TLR-4 receptor capable of binding to the aptamers of the present disclosure is detected on the cell surface following TLR-4 internalization after binding of the aptamers of the present disclosure to TLR-4 (i.e., internalized TLR-4 is recycled to the plasma membrane), e.g., as measured in human macrophages using methods known in the art.

[0258] In some embodiments, the new TLR-4 receptor capable of binding to the aptamers of the present disclosure is detected on the cell surface approximately 5 hours after TLR-4 internalization following binding of the aptamers of the present disclosure to TLR-4.

[0259] In some embodiments, administration of aptamers of the present disclosure to iPSC-derived cortical glutamatergic (80%) and GABAergic (20%) neurons results in no detectable toxicity to the neurons.

[0260] In some embodiments, administration of an aptamer of the present disclosure to a subject in need thereof results in a reduction of inflammatory cytokines, hi some embodiments, the inflammatory cytokines are selected from the group consisting of interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin-12p70 (IL-12p70), and any combination thereof.

[0261] In one embodiment, administration of an aptamer of the present disclosure may result in a reduction in interferon-γ (IFN-γ) levels of at least about 5%, at least about 10%, at least 15%, at least about 20%, or at least about 25% compared to a control condition (e.g., without administration of an aptamer of the present disclosure).

[0262] In one embodiment, administration of an aptamer of the present disclosure results in a reduction in interleukin-12p70 (IL-12p70) levels of at least about 5%, at least about 10%, at least 15%, at least about 20%, at least about 25%, at least about 30%, or at least about 35% compared to a control condition (e.g., without administration of an aptamer of the present disclosure).

[0263] In one embodiment, administration of an aptamer of the present disclosure may result in a reduction in tumor necrosis factor alpha (TNF-α) levels of at least about 5%, at least about 10%, or at least about 15% compared to a control condition (e.g., without administration of an aptamer of the present disclosure).

[0264] In one embodiment, administration of an aptamer of the present disclosure may result in a reduction in interleukin-6 (IL-6) levels of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least 45%, or at least about 50% compared to a control condition (e.g., without administration of an aptamer of the present disclosure).

[0265] In some embodiments, the aptamer of the present disclosure can be transported across the blood-brain barrier (BBB).In some embodiments, the aptamer of the present disclosure can be transported across the BBB after the BBB is damaged, for example, by hemorrhagic or ischemic events.Therefore, in some embodiments, the aptamer of the present disclosure cannot pass through the BBB in healthy subjects.

[0266] In one specific embodiment, the aptamer of the present disclosure is ApTOLL. As used herein, the term "ApTOLL" refers to a nucleic acid (single-stranded DNA, ssDNA) aptamer that specifically binds to TLR-4 and comprises the sequence of SEQ ID NO: 1. In a specific embodiment, the term ApTOLL refers to a structured nucleic acid aptamer of SEQ ID NO: 1. As used herein, the term "structured nucleic acid aptamer" or "structured aptamer" refers to a nucleic acid aptamer that has been linearized by exposure to denaturing conditions (e.g., at a high temperature such as 95°C, for example, for 10 minutes) and then refolded at a low temperature (e.g., by immersion in ice, for example, for 10 minutes), thereby acquiring a tertiary structure that allows interaction between the structured aptamer, e.g., ApTOLL, and its target, e.g., an epitope on the extracellular domain of TLR-4. See Figure 1.

[0267] The chemical formula of ApTOLL is C 575 H 723 N 223 O 351 P 58 The molecular weight of ApTOLL is 18,170.80 Da. The molecular sequence of ApTOLL has been confirmed by controlled enzymatic digestion followed by MS-MS (mass spectrometry) sequencing. The correct structure has been verified by confirming the predicted biological activity in an in vitro assay. To adopt the biologically active conformation, the aptamer must be dissolved in PBS-1 mM MgCl2. After dissolution, the aptamer must be heated to 95°C for approximately 10 minutes and then quickly chilled on ice for approximately 10 minutes. This buffer and conditions support the aptamer structure and its biological activity.

[0268] The dosage form of investigational medicinal product (IMP) ApTOLL corresponds to a powder for infusion concentrate, consisting of a lyophilized powder that is reconstituted with water for injection and further diluted with saline for intravenous administration.

[0269] ApTOLL has demonstrated specific binding to human TLR-4 and TLR4 antagonism. ApTOLL has demonstrated long-term protective effects against brain injury induced by, for example, middle cerebral artery occlusion (MCAO). Furthermore, its efficacy in models of cerebral ischemia-reperfusion supports the use of this aptamer in patients undergoing arterial recanalization induced by pharmacological and / or mechanical intervention.

[0270] Preclinical pharmacokinetic studies have demonstrated the C value of ApTOLL in rats. max We demonstrated that the dose-dependent kinetics of ApTOLL appeared to be characterized by dose-independent (linear) kinetics over the dose range of 0.45 to 2 mg / kg, and that the extent of systemic exposure in female rats to ApTOLL appeared to be characterized by nonlinear (dose-dependent) kinetics over the dose range of 0.45 to 2 mg / kg. Increasing the dose of ApTOLL beyond 0.45 mg / kg may result in lower systemic exposure than predicted by a linear relationship, consistent with the potential for increased plasma clearance of ApTOLL at higher dose levels. Nonclinical pharmacodynamic, safety pharmacology, pharmacokinetics, and toxicology studies were conducted to characterize ApTOLL in three species: mice (C57Bl6, ICR), rats (Wistar and Sprague Dawley (SD)), and nonhuman primates (NHPs, cynomolgus monkeys). These species were chosen due to the human homology of the receptor and TLR4 pharmacology.

[0271] Pharmacodynamic characterization performed in vitro and in vivo also shows that the binding of ApTOLL to TLR-4 from humans and non-human primates (NHPs) has a Ka of approximately 30–60 nM, and there is no binding of ApTOLL to other TLRs.

[0272] Pharmacodynamic characterization of the disclosed aptamers, for example, in vivo aptamers, indicates that a reduction in infarct volume of up to 65.5% can be observed after administration of the aptamer to subjects suffering from acute ischemic stroke. A therapeutic window of up to 12 hours has been observed. Administration of multiple doses of the disclosed aptamers, for example, ApTOLL, generally provides better protection than administration of a single dose. Administration of the disclosed aptamers, for example, ApTOLL, to subjects suffering from acute ischemic stroke results in improved neurological outcomes in both the short and long term. Experimental observations have confirmed that administration of the disclosed aptamers, for example, ApTOLL, to subjects in need thereof results in blocking the inflammatory cascade. Furthermore, administration of the disclosed aptamers, for example, ApTOLL, did not exhibit any drug-drug interactions with intravenous rt-PA.

[0273] Biodistribution studies have shown that ApTOLL is primarily present in the kidney, spleen, and liver 1 hour after intravenous injection in both naive and ischemic subjects. 24 hours after injection, ApTOLL levels are barely detectable. Under physiological conditions, ApTOLL cannot cross the BBB in healthy subjects. However, ApTOLL can cross the BBB in individuals who have experienced an ischemic event. When administered after an ischemic event, ApTOLL is primarily present in the ipsilateral hemisphere of the subject's brain (i.e., the hemisphere affected by the ischemic event).

[0274] The metabolism and distribution of ApTOLL have been determined both in vitro and in vivo. ApTOLL is degraded by exonucleases in plasma within minutes of administration. No drug interactions or inhibition of transporters or cytochromes were detected. In vivo controlled pharmacokinetic studies performed in SD rats demonstrated that T max was achieved within 1 minute after administration, and C max showed linear kinetics over the dose range of 0.45 mg / kg to 2 mg / kg, whereas exposure (AUCt) showed nonlinear kinetics over the same dose range.

[0275] In some specific embodiments, ApTOLL is presented as a vial of 7 mg lyophilized powder that is reconstituted with 3 mL of water to produce ApTOLL concentrate, which is further diluted with 100 mL of 0.9% sodium chloride solution. The resulting solution can be administered intravenously, for example, via an infusion pump. In some embodiments, ApTOLL administration is performed as a single dose. In other embodiments, multiple doses are administered. In some embodiments, ApTOLL infusion has a duration of approximately 30 minutes.

[0276] In some embodiments, when ApTOLL infusion is administered as part of a thrombectomy procedure, ApTOLL infusion is administered immediately after intravenous thrombolysis, including rt-PA (recombinant tissue plasminogen activator, alteplase) administration, if appropriate, and prior to thrombectomy.

[0277] IV. Chemically Modified Aptamers The aptamers of the present disclosure (e.g., ApTOLL) can be chemically modified to make them highly stable or further truncated to eliminate oligonucleotide sequences that are not critical for target interaction or the correct three-dimensional aptamer structure. The aptamers of the present disclosure can be in the form of unmodified single-stranded DNA (ssDNA) aptamers for the treatment of, for example, acute ischemic stroke and other diseases and conditions disclosed herein due to their rapid pharmacokinetics and low toxicity profile. However, to extend the therapeutic and / or protective effects of the aptamers of the present disclosure, the aptamers can be modified, for example, to increase their resistance to degradation by nucleases and / or their half-life in circulation.

[0278] In some embodiments, the aptamer of the present disclosure (e.g., ApTOLL) comprises at least one chemically modified nucleoside and / or nucleotide. When the aptamer of the present disclosure is chemically modified, the aptamer can be referred to as a "modified aptamer."

[0279] "Nucleoside" refers to a compound comprising a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase").

[0280] "Nucleotide" refers to a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized by any useful method, e.g., chemically, enzymatically, or recombinantly, and can include one or more modified or non-natural nucleosides.

[0281] The aptamers of the present disclosure can comprise one or more regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester bonds, in which case the aptamer would comprise a region of nucleotides.

[0282] The modified aptamers disclosed herein can include a variety of distinct modifications. In some embodiments, the modified aptamers include one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified aptamers can exhibit one or more desirable properties, such as improved thermal or chemical stability, reduced immunogenicity, reduced degradation, increased binding to the TLR-4 target epitope, or reduced non-specific binding to other regions of TLR-4 or other molecules, such as other Toll-like receptors, compared to the corresponding unmodified aptamers.

[0283] In some embodiments, polynucleotides (e.g., aptamers such as ApTOLL) of the present disclosure are chemically modified. As used herein with respect to polynucleotides, the term "chemically modified" or, where appropriate, "chemically modified" refers to a modification of one or more of adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides in their position, pattern, percentage, or population, including, but not limited to, their nucleobase, sugar, backbone, or any combination thereof.

[0284] In some embodiments, polynucleotides (e.g., aptamers such as ApTOLL) of the disclosure can have a uniform chemical modification of all or any of the same nucleoside types, or a population of modifications resulting from a downward titration of the same starting modifications of all or any of the same nucleoside types, or a measured percentage of chemical modifications of either all but random incorporation of the same nucleoside types. In another embodiment, polynucleotides (e.g., aptamers such as ApTOLL) of the disclosure can have two, three, or four uniform chemical modifications of the same nucleoside type throughout the polynucleotide (e.g., all adenosines and / or all cytidines, etc., are modified in the same way).

[0285] Modified nucleotide base pairing includes not only standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides containing non-standard or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between non-standard and standard bases, or between two complementary non-standard base structures. One example of such non-standard base pairing is base pairing between the modified nucleobase inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker can be incorporated into the polynucleotides of the present disclosure (e.g., aptamers such as ApTOLL).

[0286] In some embodiments, the nucleobases, sugars, backbone linkages, or any combination thereof in a polynucleotide (e.g., an aptamer such as ApTOLL) of the disclosure are at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% modified.

[0287] 1. Base modification In certain embodiments, the chemical modification is in a nucleobase of a polynucleotide (e.g., an aptamer such as ApTOLL) of the present disclosure. In some embodiments, at least one chemically modified nucleoside is a modified uridine (e.g., pseudouridine (ψ), 2-thiouridine (s2U), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), or 5-methoxy-uridine (mo5U)), a modified cytosine (e.g., 5-methyl-cytosine (m5C)), a modified adenosine (e.g., 1-methyl-adenosine (m1A), N6-methyl-adenosine (m6A), or 2-methyl-adenine (m2A)), a modified guanosine (e.g., 7-methyl-guanosine (m7G) or 1-methyl-guanosine (m1G)), or a combination thereof.

[0288] In some embodiments, the polynucleotides of the present disclosure (e.g., aptamers such as ApTOLL) are uniformly modified for specific modifications (e.g., completely modified, modified throughout the entire sequence).For example, polynucleotides can be uniformly modified with the same type of base modification, such as 5-methylcytidine (m5C), which means that all cytosine residues in the polynucleotide sequence are replaced with 5-methylcytidine (m5C).Similarly, polynucleotides can be uniformly modified for any type of nucleoside residue present in the sequence, for example, by replacing them with any of the above-mentioned modified nucleosides.

[0289] In some embodiments, a polynucleotide of the disclosure (e.g., an aptamer such as ApTOLL) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 11 and / or more than 80 modified nucleobase combinations. In some embodiments, at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% of nucleobases of a certain type in a polynucleotide of the disclosure (e.g., an aptamer such as ApTOLL) are modified nucleobases.

[0290] 2. Skeletal modification In some embodiments, the polynucleotides (e.g., aptamers such as ApTOLL) of the present disclosure comprise any useful modification to the internucleoside linkage. Such linkages, including backbone modifications useful in the compositions of the present disclosure, include, but are not limited to, 3'-alkylene phosphonates, 3'-amino phosphoramidates, alkene-containing backbones, aminoalkyl phosphoramidates, aminoalkyl phosphotriesters, boranophosphates, -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, -CH2-NH-CH2-, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene(methylimino), methyleneformacetyl, and Thioformacetyl backbone, methyleneimino and methylenehydrazino backbone, morpholino linkage, -N(CH3)-CH2-CH2-, oligonucleosides with heteroatom internucleoside linkages, phosphinates, phosphoramidates, phosphorothioates, phosphorothioate internucleoside linkages, phosphorothioates, phosphotriesters, PNAs, siloxane backbone, sulfamate backbone, sulfide sulfoxide and sulfone backbone, sulfonate and sulfonamide backbone, thionoalkylphosphonates, thionoalkylphosphotriesters, and thionophosphoramidates. [ka]

[0291] In some embodiments, the presence of the backbone linkages disclosed above increases the stability (e.g., thermal stability) and / or resistance to degradation (e.g., enzymatic degradation) of the polynucleotides of the present disclosure (e.g., aptamers such as ApTOLL).

[0292] In some embodiments, stability and / or resistance to degradation (e.g., degradation by nucleases) is increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in modified polynucleotides of the disclosure compared to the corresponding polynucleotide without the modification (reference or control aptamer).

[0293] In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the backbone linkages in a polynucleotide of the disclosure (e.g., an aptamer such as ApTOLL) are modified (e.g., all of them are phosphorothioate).

[0294] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 1, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or more than 80 backbone linkages are modified (e.g., phosphorothioate).

[0295] In some embodiments, the backbone comprises linkages selected from the group consisting of phosphodiester linkages, phosphotriester linkages, methylphosphonate linkages, phosphoramidate linkages, phosphorothioate linkages, and combinations thereof.

[0296] 3. Sugar modification Modified nucleosides and nucleotides that can be incorporated into polynucleotides (e.g., aptamers such as ApTOLL) of the present disclosure can be modified on the sugar of the nucleic acid. Thus, in some embodiments, an aptamer (e.g., ApTOLL) of the present disclosure includes at least one nucleoside analog (e.g., a nucleoside having a sugar modification).

[0297] In some embodiments, sugar modifications increase the affinity of binding of a polynucleotide of the present disclosure (e.g., an aptamer such as ApTOLL) to its target epitope. Incorporation of affinity-enhancing nucleotide analogs, such as LNA or 2'-substituted sugars, into a polynucleotide of the present disclosure (e.g., an aptamer such as ApTOLL) can reduce the length of a polynucleotide of the present disclosure (e.g., an aptamer such as ApTOLL) and can reduce the upper size limit of a polynucleotide of the present disclosure (e.g., an aptamer such as ApTOLL) before nonspecific or aberrant binding occurs.

[0298] In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the nucleotides in a polynucleotide of the disclosure (e.g., an aptamer such as ApTOLL) comprise a sugar modification (e.g., LNA).

[0299] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 1, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or more than 80 nucleotide units are sugar-modified (e.g., LNA).

[0300] Generally, RNA contains the sugar group ribose, which is a five-membered ring containing oxygen. Exemplary modified nucleotides include, but are not limited to, substitution of oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexene); ring contraction of ribose (e.g., to form a four-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a six- or seven-membered ring with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also has a phosphoramidate backbone); polycyclic forms (e.g., tricyclo, and glycol These include "unlocked" forms such as nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs in which the ribose is replaced by a glycol unit attached to a phosphodiester bond), threose nucleic acids (TNAs in which the ribose is replaced by α-L-threofuranosyl-(3'→2')), and peptide nucleic acids (PNAs in which a 2-amino-ethyl-glycine bond replaces the ribose and phosphodiester backbone). The sugar group may also contain one or more carbons with a stereochemical configuration opposite that of the corresponding carbon in ribose. Thus, polynucleotide molecules may include, for example, nucleotides containing arabinose as the sugar.

[0301] The 2' hydroxyl group (OH) of ribose can be modified or replaced with several different substituents. Exemplary substitutions at the 2' position include H, halo, optionally substituted C 1~6 Alkyl; optionally substituted C 1~6 Alkoxy; optionally substituted C 6~10 Aryloxy; optionally substituted C 3~8 Cycloalkyl; optionally substituted C 3~8 Cycloalkoxy; optionally substituted C 6~10 Aryloxy; optionally substituted C 6~10 Aryl-C 1~6 Alkoxy, optionally substituted C 1~12(heterocyclyl)oxy; sugars (e.g., ribose, pentose, or any described herein); polyethylene glycol (PEG), -O(CH2CH2O) n CHCHOR (wherein R is H or optionally substituted alkyl and n is an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20)); 1~6 Alkylene or C 1~6 These include, but are not limited to, "locked" nucleic acids (LNAs) that are connected to the 4'-carbon of the same ribose sugar by a heteroalkylene bridge (exemplary bridges include methylene, propylene, ether, amino bridge, aminoalkyl, aminoalkoxy, amino, and amino acid).

[0302] In some embodiments, the nucleoside analogs present in the polynucleotides of the present disclosure (e.g., aptamers such as ApTOLL) comprise, for example, 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-O-alkyl-SNA, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabinonucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (intercalating nucleic acid) units, 2'MOE units, or any combination thereof. In some embodiments, the LNA is, for example, oxy-LNA (such as β-D-oxy-LNA or α-L-oxy-LNA), amino-LNA (β-D-amino-LNA or α-L-amino-LNA), thio-LNA (such as β-D-thio-LNA or α-L-thio-LNA), ENA (such as β-D-ENA or α-L-ENA), or any combination.

[0303] In some embodiments, nucleoside analogs present in polynucleotides of the disclosure (e.g., aptamers such as ApTOLL) comprise locked nucleic acids (LNA); 2'-O-alkyl-RNA; 2'-amino-DNA; 2'-fluoro-DNA; arabinonucleic acid (ANA); 2'-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), constrained ethyl nucleosides (cEt), 2'-O-methyl nucleic acid (2'-OMe), 2'-O-methoxyethyl nucleic acid (2'-MOE), or any combination thereof.

[0304] In some embodiments, polynucleotides (e.g., aptamers such as ApTOLL) of the present disclosure can contain both modified RNA nucleotide analogs (e.g., LNA) and DNA units. See, e.g., U.S. Patent Nos. 8,404,649, 8,580,756, 8,163,708, and 9,034,837 (all of which are incorporated by reference in their entirety).

[0305] V. Methods of Manufacturing and Formulation The present disclosure also provides methods for producing the aptamers of the present disclosure (e.g., ApTOLL). Generally, the aptamers of the present disclosure can be obtained using the methods disclosed in U.S. Patent No. 10,196,642 and synthesized using the methods described therein or methods generally known in the art.

[0306] The production of the aptamers of the present disclosure (e.g., ApTOLL) can be carried out according to conventional methods in the art. Non-limiting examples of techniques for producing aptamers include enzymatic techniques such as transcription, recombinant expression systems, and standard solid-phase (or liquid-phase) chemical synthesis, all of which are commercially available. Where appropriate, for example, when the aptamers of the present disclosure include nucleic acid variants such as those described above, nucleotide analogs such as analogs with chemically modified bases or sugars, backbone modifications, etc., the aptamers of the present disclosure can be produced by chemical synthesis. Alternatively, recombinant expression may be a preferred technique for producing the aptamers of the present disclosure, for example, when the aptamer has a length of 200 nucleotides or more. Aptamers produced by any of the aforementioned techniques can optionally be purified by methods well known in the art.

[0307] As used herein, the term "synthesize" refers to assembling an aptamer using polynucleotide synthesis methods known in the art. The term "synthesize" also encompasses the assembly of a conjugate or complex comprising an aptamer of the present disclosure (e.g., ApTOLL) and at least one biologically active molecule (e.g., a small molecule drug covalently or non-covalently attached to the aptamer). For example, a peptide or small molecule component can be prepared recombinantly, chemically, or enzymatically, and then conjugated to an aptamer (e.g., ApTOLL) in one or more synthesis steps (e.g., conjugation of a linker to the aptamer of the present disclosure, followed by conjugation of a small molecule to the linker). In some embodiments, each of the components of a conjugate or complex comprising at least one aptamer (e.g., ApTOLL) of the present disclosure can be prepared using methods known in the art, such as recombinant protein production, solid-phase peptide or nucleic acid synthesis, chemical synthesis, enzymatic synthesis, or any combination thereof, and the resulting components can be conjugated using chemical and / or enzymatic methods known in the art.

[0308] Aptamers of the present disclosure (e.g., ApTOLL) can be purified, for example, by filtration, to remove contaminants. In some embodiments, manufacturing of aptamers of the present disclosure (e.g., ApTOLL) involves lyophilization or any other form of dry storage suitable for reconstitution. In some embodiments, preparation of the aptamer in a dry form occurs after combining the aptamer (e.g., ApTOLL) with a biologically active molecule (e.g., a small molecule drug), i.e., both therapeutic agents can be co-lyophilized.

[0309] In some embodiments, a method for preparing a composition comprising an aptamer (e.g., ApTOLL) of the present disclosure together with a biologically active molecule (e.g., a small molecule drug) comprises mixing the aptamer with the biologically active molecule (e.g., a small molecule drug) in solution. In some embodiments, after combining the aptamer (e.g., ApTOLL) and the biologically active molecule (e.g., a small molecule drug) in solution, the resulting solution is lyophilized or dried. In some embodiments, the combination of the aptamer (e.g., ApTOLL) with the biologically active molecule (e.g., a small molecule drug) is performed in a dry form.

[0310] In some embodiments, aptamers of the present disclosure (eg, ApTOLL) can be purified, for example, to remove contaminants and / or to generate a homogenous population of aptamers.

[0311] The present disclosure also provides a formulation comprising an aptamer of the present disclosure, e.g., ApTOLL. The aptamer of the present disclosure can be formulated according to the method shown schematically in Figure 20. The aptamer API (Active Pharmaceutical Ingredient) is combined with a solution containing pre-filtered excipients. After the structuring step, the solution containing the aptamer (e.g., ApTOLL) and excipients is subjected to two filtration steps, transferred to a vial, and lyophilized. The structuring step is a key step in the preparation of an aptamer (such as ApTOLL). The structuring process involves dissolving the aptamer in a suitable solvent. In some embodiments, the solvent contains a divalent ion. In some embodiments, the divalent ion is Mg 2+ In some embodiments, the solvent is phosphate buffered saline (PBS) containing MgCl. In some embodiments, the solvent is PBS containing 1 mM MgCl. After the aptamer (e.g., ApTOLL) is dissolved, it is heated to a denaturing temperature (e.g., 95°C) for a short period of time (e.g., approximately 10 minutes), followed by rapid cooling (e.g., by transferring to ice, e.g., for approximately 10 minutes). In some embodiments, the aptamer (e.g., ApTOLL) does not function in the absence of the heating and cooling steps.

[0312] After synthesis, the aptamers of the present disclosure (e.g., ApTOLL) are linear. Raising the temperature completely linearizes the aptamer, but subsequent cooling allows the aptamer to properly fold and yield a functional aptamer. In some embodiments, the aptamers of the present disclosure (e.g., ApTOLL) are prepared by heating and cooling the aptamer after the heating and cooling steps, which removes divalent ions, e.g., Mg 2+ In certain embodiments of the present disclosure, the aptamers of the present disclosure (e.g., ApTOLL) do not function unless performed in the presence of Mg 2+ It has no therapeutic function unless it is dissolved in a buffer containing (e.g., 1 mM MgCl2), heated to 95°C for 10 minutes, and then cooled to 0°C in ice for 10 minutes.

[0313] The manufacturing process for aptamers of the present disclosure (e.g., ApTOLL) involves two lyophilization steps. In the first step, a structured aptamer (e.g., an aptamer of the present disclosure in PBS) is lyophilized. The lyophilized aptamer (e.g., ApTOLL) is redissolved in a buffer, e.g., PBS, and refrozen. The second lyophilization increases the stability of the aptamer of the present disclosure (e.g., ApTOLL) relative to the same aptamer undergoing a single lyophilization step.

[0314] In some embodiments, the aptamers of the present disclosure (e.g., ApTOLL) are formulated in doses containing 7 mg of aptamer, e.g., structured and lyophilized aptamer. In other embodiments, the aptamers of the present disclosure are formulated in doses containing at least about 1 mg, at least about 2 mg, at least about 3 mg, at least about 4 mg, at least about 5 mg, at least about 6 mg, at least about 7 mg, at least about 8 mg, at least about 9 mg, or at least about 10 mg of the aptamers of the present disclosure (e.g., ApTOLL).

[0315] In some embodiments, the aptamers of the present disclosure can be formulated into nanoparticles, such as polymeric nanoparticles, lipid nanoparticles (e.g., liposomes or micelles), or metal nanoparticles, comprising an aptamer of the present disclosure covalently or non-covalently attached to the nanoparticle (e.g., encapsulated in the nanoparticle). See, e.g., U.S. Patent No. 10,196,642, which is incorporated herein by reference in its entirety.

[0316] As described above, the aptamers of the present disclosure can be covalently or non-covalently attached to biologically active molecules and / or nanoparticles (e.g., formed nanoparticles or components of nanoparticles). Covalent attachment between the aptamers of the present disclosure (e.g., ApTOLL) and biologically active molecules and / or nanoparticles can be carried out by conjugation techniques well known to those skilled in the art. The result is a covalent bond between the aptamers of the present disclosure and the biologically active molecules and / or nanoparticles or components thereof. Conjugation can involve binding of the primary amine at the 3' or 5' end of the aptamer of the present disclosure to a functional group during chemical synthesis of the aptamer.

[0317] Conjugation can also be carried out by conventional cross-linking reaction, which has the advantage that the chemical reactivity of primary alkylamine labels is much higher than that of the arylamines of nucleotides themselves.Conjugation methods are well known in the art and are based on the use of cross-linking reagents.The cross-linking reagents comprise at least two reactive groups that target groups such as primary amines, sulfhydryls, aldehydes, carboxyls, hydroxyls, azides, etc. in the biologically active molecules and / or nanoparticles that are conjugated to the aptamers of the present disclosure.

[0318] Crosslinkers vary in their chemical specificity, spacer arm length, spacer arm composition, cleavable spacer arm, and structure. For example, conjugation of biologically active molecules and / or nanoparticles or their components to the aptamers of the present disclosure can be carried out directly or via a linking moiety through one or more non-functional groups in the aptamer and / or functional groups (such as amine, carboxyl, phenyl, thiol, or hydroxyl groups). More selective binding can be achieved by using heterobifunctional linkers. Conventional linkers such as diisocyanates, diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides, maleimide-hydroxysuccinimide esters, glutaraldehyde, or hydrazides such as hydrazine and 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH) can be used.

[0319] In some embodiments, conjugation can occur subsequent to production of the aptamers of the present disclosure by recombinant or enzymatic methods.

[0320] In some embodiments, the aptamer (e.g., ApTOLL) of the present disclosure is formulated in a vial, with each dose vial containing about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10 mg of the aptamer (e.g., ApTOLL) of the present disclosure per vial. In one particular embodiment, each dose vial contains 7 mg of the aptamer (e.g., ApTOLL) of the present disclosure per vial. In some embodiments, the contents of the vial are lyophilized aptamer (e.g., ApTOLL) of the present disclosure.

[0321] VI. Pharmaceutical Compositions The present disclosure also provides pharmaceutical compositions comprising one or more aptamers (e.g., ApTOLL) of the present disclosure suitable for administration to a subject according to the methods disclosed herein (e.g., methods for treating any of the diseases or conditions disclosed herein, e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia, or ischemic stroke).

[0322] Pharmaceutical compositions generally comprise one or more aptamers of the present disclosure (e.g., ApTOLL) with a desired purity, and a pharmaceutically acceptable excipient or carrier in a form suitable for administration to a subject. Pharmaceutically acceptable excipients or carriers are determined in part by the specific composition being administered and the specific method used to administer the composition. Thus, there are a wide variety of suitable formulations of pharmaceutical compositions comprising one or more aptamers of the present disclosure (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th ed. (1990)). Pharmaceutical compositions are generally formulated aseptically and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0323] In some embodiments, a pharmaceutical composition comprises one or more aptamers (e.g., ApTOLL) of the present disclosure. In certain embodiments, an aptamer (e.g., ApTOLL) of the present disclosure is co-administered with one or more additional therapeutic agents in a pharmaceutically acceptable carrier and / or a surgical procedure (e.g., thrombectomy in the case of myocardial infarction). In some embodiments, a pharmaceutical composition comprising an aptamer (e.g., ApTOLL) of the present disclosure is administered prior to administration of the additional therapeutic agent and / or a surgical procedure (e.g., thrombectomy in the case of myocardial infarction).

[0324] In other embodiments, a pharmaceutical composition comprising an aptamer of the present disclosure (e.g., ApTOLL) is administered after administration of an additional therapeutic agent and / or a surgical procedure (e.g., thrombectomy in the case of myocardial infarction). In further embodiments, a pharmaceutical composition comprising an aptamer of the present disclosure (e.g., ApTOLL) is administered simultaneously with an additional therapeutic agent and / or a surgical procedure (e.g., thrombectomy in the case of myocardial infarction).

[0325] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0326] The example of carrier or diluent includes but is not limited to water, physiological saline, Ringer's solution, dextrose solution and 5% human serum albumin.The use of such media and compounds for pharmaceutically active substances is well known in the art.Except when any conventional media or compounds are incompatible with the aptamer of the present disclosure, they are contemplated to be used in composition.

[0327] Supplementary therapeutic agents suitable for treating or preventing (e.g., suppressing, inhibiting, or delaying) any of the diseases or conditions disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke), or for improving homeostasis in a subject suffering from, having suffered from, or at risk for any of the diseases or conditions disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke) can also be incorporated into the compositions of the present disclosure.

[0328] Typically, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Aptamers of the present disclosure (e.g., ApTOLL) can be administered, for example, parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intraperitoneally, intranasally, or intramuscularly, or as an inhalant.

[0329] In certain embodiments, pharmaceutical compositions comprising the aptamers of the present disclosure (e.g., ApTOLL) are administered intravenously or intraarterially, for example, by injection. The aptamers described herein (e.g., ApTOLL) can optionally be administered in combination with other therapeutic agents that are at least partially effective in treating any of the diseases or conditions disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia, or ischemic stroke) targeted by the aptamers described herein (e.g., ApTOLL).

[0330] The solution or suspension may contain the following components: a sterile diluent such as water, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial compound such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating compound such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and a compound for adjusting isotonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The preparation can be placed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0331] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders. For intravenous or intraarterial administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The composition is generally sterile and fluid to the extent that easy injectability exists. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal compounds, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. If necessary, isotonic compounds, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, can be added to the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, for example, aluminum monostearate and gelatin.

[0332] The pharmaceutical compositions of the present disclosure can be sterilized by conventional, well-known sterilization techniques. Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.

[0333] Sterile injectable solutions can be prepared by incorporating the aptamer of the present disclosure (e.g., ApTOLL) in an effective amount with one or a combination of the ingredients listed herein, as needed, in a suitable solvent. Generally, dispersions are prepared by incorporating the aptamer of the present disclosure (e.g., ApTOLL) into a sterile vehicle containing a basic dispersion medium and any other desired ingredients. In the case of sterile powders for preparing sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, which obtain a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution. The aptamers described herein (e.g., ApTOLL) can be administered in the form of depot injection or implant preparations, which can be formulated in a way that allows for sustained or pulsatile release of the aptamer of the present disclosure.

[0334] Systemic administration of compositions comprising the aptamer (e.g., ApTOLL) described herein can also be via transmucosal means. For transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, surfactants, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved, for example, by using nasal sprays.

[0335] In certain embodiments, a pharmaceutical composition comprising an aptamer of the present disclosure (e.g., ApTOLL) is administered intravenously or intraarterially to a subject who will benefit from the pharmaceutical composition. In certain other embodiments, the composition is administered to the lymphatic system, for example, by intralymphatic injection, intranodal injection (see, e.g., Senti et al., PNAS 105(46):17908(2008)), intramuscular injection, intraperitoneal injection, or subcutaneous administration.

[0336] In certain embodiments, the pharmaceutical composition comprising the aptamer of the present disclosure (for example, ApTOLL) is administered as a liquid suspension.In certain embodiments, the pharmaceutical composition is administered as a formulation that can form a depot after administration.In certain preferred embodiments, the depot slowly releases the aptamer into circulation or remains in depot form.

[0337] Typically, a pharmaceutically acceptable composition is highly purified to be free of contaminants, biocompatible, non-toxic, and suitable for administration to a subject. When water is a component of the carrier, the water is highly purified and treated to be free of contaminants, such as endotoxins.

[0338] Pharmaceutically acceptable carriers can be, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and / or mineral oil. Pharmaceutical compositions can further include lubricants, wetting agents, sweeteners, flavorings, emulsifiers, suspending agents, and / or preservatives.

[0339] The pharmaceutical compositions described herein comprise an aptamer (e.g., ApTOLL) described herein and optionally a pharmaceutically active agent or therapeutic agent. The therapeutic agent can be, for example, a biological agent (e.g., a peptide or nucleic acid), a small molecule agent, or a combination thereof.

[0340] Dosage forms comprising the aptamers (e.g., ApTOLL) or pharmaceutical compositions described herein are provided for use in accordance with the methods disclosed herein. In some embodiments, the dosage forms are formulated as liquid suspensions for intravenous or intraarterial injection.

[0341] The aptamers (e.g., ApTOLL) of the present disclosure or pharmaceutical compositions comprising the aptamers of the present disclosure can be used in conjunction with other therapies, such as drugs and / or surgery. Specifically, the aptamers (e.g., ApTOLL) or pharmaceutical compositions of the present disclosure can be used together with pharmaceuticals commonly used to treat any of the diseases or conditions disclosed herein (e.g., myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia, or ischemic stroke), or in combination with pharmacological and / or surgical procedures known in the art used to treat such diseases or conditions (e.g., thrombectomy in the case of myocardial infarction).

[0342] VII. Kit The present disclosure also provides kits or articles of manufacture comprising an aptamer of the present disclosure (e.g., an isolated aptamer of the present disclosure or an aptamer of the present disclosure conjugated or complexed to a biologically active molecule, e.g., ApTOLL) and optionally instructions for use according to the methods of the present disclosure.

[0343] In some embodiments, the kit or article of manufacture comprises a pharmaceutical composition of the present disclosure comprising at least one aptamer of the present disclosure (e.g., ApTOLL) in one or more containers, and optionally instructions for use according to the methods of the present disclosure.

[0344] In some embodiments, the kit or article of manufacture includes an aptamer of the present disclosure (e.g., ApTOLL), or a pharmaceutical composition of the present disclosure, and a pamphlet. In some embodiments, the kit or article of manufacture includes an aptamer of the present disclosure (e.g., ApTOLL), or a pharmaceutical composition of the present disclosure, and instructions for use. Those skilled in the art will readily recognize that an aptamer (e.g., ApTOLL) or a pharmaceutical composition of the present disclosure, or a combination thereof, can be easily incorporated into one of the established kit formats well known in the art.

[0345] In some embodiments, the kit or article of manufacture comprises a dried form of an aptamer of the present disclosure (e.g., ApTOLL) in a container (e.g., a glass vial), and optionally a vial with a solvent suitable for hydrating the aptamer, and optionally instructions for using the product reconstituted according to the methods disclosed herein. In some embodiments, the kit or article of manufacture further comprises at least one additional container (e.g., a glass vial) containing a biologically active molecule (e.g., a second TLR-4 antagonist).

[0346] Those skilled in the art will readily recognize that the aptamers of the present disclosure (e.g., ApTOLL), pharmaceutical compositions comprising the aptamers of the present disclosure (e.g., ApTOLL), or combinations thereof, can be readily incorporated into one of the established kit formats well known in the art.

[0347] In some embodiments, the kit includes reagents for conjugating a biologically active molecule to an aptamer of the present disclosure (e.g., ApTOLL), instructions for performing the conjugation, and instructions for using the conjugate according to the methods of the present disclosure.

[0348] In some embodiments, the kit includes a biologically active molecule and an aptamer of the present disclosure (e.g., ApTOLL), instructions for combining them to form a complex, and instructions for using the complex obtained according to the method of the present disclosure.

[0349] In some embodiments, the kit or article of manufacture comprises an aptamer of the present disclosure (e.g., ApTOLL) in solution and instructions for use according to the methods of the present disclosure. In some embodiments, the kit or article of manufacture comprises an aptamer of the present disclosure (e.g., ApTOLL) in dry form and instructions for use (e.g., instructions for reconstitution and administration according to the methods disclosed herein).

[0350] VIII. Embodiments E1. A method of treating a TLR-4 mediated disease or condition in a subject in need thereof, comprising administering to the subject at least one dose of a nucleic acid aptamer 40 to 80 nucleobases in length, wherein the aptamer binds to an epitope on the extracellular domain of TLR-4, and wherein binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation.

[0351] E2. The method of embodiment E1, further comprising administering an additional therapy or combination thereof.

[0352] E3. The method of embodiment E2, wherein the additional therapy is a second TLR-4 antagonist.

[0353] E4. The method of embodiment E3, wherein the additional treatment is surgical intervention.

[0354] E5. The method of embodiment E2, wherein the additional treatment comprises administration of an anti-inflammatory agent, a nucleic acid, a peptide, or a combination thereof.

[0355] E6. The method of embodiment E5, wherein the peptide comprises an antibody or an antigen-binding fragment thereof.

[0356] E7. The method of embodiment E5, wherein the nucleic acid comprises an antisense oligonucleotide, an antimir, an siRNA, or an shRNA.

[0357] E8. The method of embodiment E1, wherein the nucleic acid aptamer comprises a sequence at least 70% identical to SEQ ID NO: 1, 2, 3, or 4, or a combination thereof.

[0358] E9. The method of embodiment E1, wherein the nucleic acid aptamer further comprises a biologically active molecule covalently or non-covalently attached to the aptamer.

[0359] E10. The method of embodiment E1, wherein the nucleic acid aptamer cross-competes with or binds to the same TLR-4 epitope as the nucleic acid aptamer of SEQ ID NO: 1, 2, 3, or 4.

[0360] The method of embodiment E1, wherein the nucleic acid aptamer cross-competes with or binds to an epitope that overlaps with the TLR-4 epitope recognized by the nucleic acid aptamer of SEQ ID NO: 1, 2, 3, or 4.

[0361] E12. The method of embodiment E1, wherein the nucleic acid aptamer is administered in a dose regimen comprising multiple doses.

[0362] E13. The method of embodiment E12, wherein multiple doses are administered simultaneously, sequentially, or a combination thereof.

[0363] E14. The method of embodiment E12, wherein the multiple doses comprise 2, 3, 4, or 5 doses.

[0364] E15. The method of embodiment E1, wherein each dose comprises 0.007-0.45 mg / kg of nucleic acid aptamer.

[0365] E16. The method of embodiment E1, wherein the nucleic acid aptamer is administered intravenously, intraarterially, or intraperitoneally.

[0366] E17. The method of embodiment E1, wherein the TLR-4-mediated disease or condition is an ischemic disease or condition.

[0367] E18. The method of embodiment E17, wherein the ischemic condition is myocardial infarction or ischemic stroke.

[0368] E19. The method of embodiment E1, wherein the TLR-4-mediated disease or condition is a hemorrhagic condition.

[0369] E20. The method of embodiment E19, wherein the bleeding condition is hemorrhagic stroke or hemorrhagic transformation.

[0370] E21. The method of embodiment E1, wherein the TLR-4-mediated disease or condition is a neuromuscular disease or condition.

[0371] E22. The method of embodiment E21, wherein the neuromuscular disease or condition is a neurodegenerative disease or condition.

[0372] E23. The method of embodiment E22, wherein the neurodegenerative disease or condition is multiple sclerosis.

[0373] E24. A method of ameliorating or ameliorating at least some symptoms or sequelae of acute myocardial infarction in a subject in need thereof, comprising administering to the subject an aptamer during, before, or immediately after acute myocardial infarction, (a) the aptamer has a length of 40 to 100 nucleotides and is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4; (i) the aptamer specifically binds to an epitope on the extracellular domain of TLR-4; (ii) binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation, or (b) the aptamer is a functionally equivalent variant of the aptamer of (a) having at least 85% sequence identity to SEQ ID NO: 1, 2, 3, or 4, wherein the functionally equivalent variant is derived from SEQ ID NO: 1, 2, 3, or 4 and specifically binds to TLR-4; and maintaining the ability to reduce and / or inhibit its activation.

[0374] E25. The method of embodiment 24, wherein administration of the aptamer causes a reduction in infarct area.

[0375] E26. The method of embodiment E25, wherein administration of the aptamer causes a reduction in infarct size of at least 25% compared to control conditions.

[0376] E27. The method of embodiment E24, wherein administration of the aptamer causes a reduction in fibrosis and / or necrosis caused by acute myocardial infarction.

[0377] E28. Administration of aptamers (i) improved cardiac function; (ii) reduced extracellular matrix degradation; (iii) improvement of cardiac remodeling; (iv) preservation of ventricular anatomy; (v) reduction in infarct progression; or (vi) The method of embodiment E24, resulting in any combination thereof.

[0378] E29. A method of ameliorating or ameliorating at least some symptoms or sequelae of a neuromuscular or neurodegenerative disease or condition, comprising administering to a subject an aptamer during, before, or after the onset of the neuromuscular or neurodegenerative disease or condition, (a) the aptamer has a length of 40 to 100 nucleotides and is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4; (i) the aptamer specifically binds to an epitope on the extracellular domain of TLR-4; (ii) binding of the aptamer to the epitope reduces and / or inhibits TLR-4 activation, or (b) the aptamer is a functionally equivalent variant of the aptamer of (a) having at least 85% sequence identity to SEQ ID NO: 1, 2, 3, or 4, wherein the functionally equivalent variant is derived from SEQ ID NO: 1, 2, 3, or 4 and specifically binds to TLR-4; and maintaining the ability to reduce and / or inhibit its activation.

[0379] E30. Administration of aptamers (i) reduced demyelination, (ii) reduction of axonal damage, or (iii) The method of embodiment E29, causing their combination.

[0380] E31. The method of embodiment E30, wherein administration of the aptamer causes an inhibition of demyelination by at least 20-80% compared to a control condition (e.g., administration of a placebo).

[0381] E32. The method of embodiment E30, wherein administration of the aptamer causes a reduction in axonal damage of at least 10-30% compared to a control condition (e.g., administration of a placebo).

[0382] E33. The method of embodiment E29, wherein the neuromuscular or neurodegenerative disease or condition is selected from the group consisting of myocardial infarction, hemorrhagic stroke, hemorrhagic transformation, multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, Alzheimer's disease, vascular dementia disease, or ischemic stroke.

[0383] E34. The method of embodiment E24 or E29, wherein the aptamer is ApTOLL.

[0384] E35. The method of embodiment E24 or E29, wherein the aptamer is administered in a dose range of between about 0.5 mg / dose and about 14 mg / dose.

[0385] E36. The method of embodiment E24 or E29, wherein the aptamer is administered in a dose range of between about 0.007 mg / kg per dose and about 0.2 mg / kg per dose.

[0386] E37. The method of embodiment E24 or E29, wherein the aptamer is formulated in PBS (sodium chloride, potassium chloride, disodium hydrogen phosphate dihydrate, and potassium dihydrogen phosphate) pH 7.4, containing magnesium chloride hexahydrate, and optionally containing A-trehalose dihydrate.

[0387] E38. The method of embodiment E24 or E29, wherein the aptamer is administered intravenously by infusion.

[0388] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of the art and are fully explained in the literature. For example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press), Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY), DNGlover ed., (1985) DNA Cloning, Volumes I and II, Gait, ed. (1984) Oligonucleotide. Synthesis, Mullis et al. U.S. Patent No. 4,683,195, Hames and Higgins, eds. (1984) Nucleic Acid Hybridization, Hames and Higgins, eds. (1984) Transcription And Translation, Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986), Perbal (1984) A Practical Guide To Molecular Cloning;the treatise,Methods In Enzymology(Academic Press,Inc.,NY), Miller and Calos eds.(1987)Gene Transfer Vectors For Mammalian Cells,(Cold Spring Harbor Laboratory),Wu et al.,eds.,Methods In Enzymology,Vols.154 and 155, Mayer and Walker,eds.(1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London), Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986), Crooke, Antisense drug Technology: Principles, Strategies and See Applications, 2nd Ed. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.). .

[0389] The contents of all cited references (including literature references, patents, patent applications, and websites) that may be cited throughout this application, as well as the references cited therein, are expressly incorporated by reference in their entirety for all purposes.

[0390] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0391] Methods for selecting, characterizing, and optimizing the aptamers of the present disclosure are disclosed in detail in US Pat. No. 10,196,642, which is incorporated herein by reference in its entirety.

[0392] Example 1. In vitro primary pharmacodynamics Antagonism of aptamers against hTLR-4 activation hTLR-4 activation assays were performed in HEK-blue-hTLR4 cells. Because ultra-pure LPS (0.1 ng / ml) was used, important aspects related to the mechanism of kidney stone formation can also be derived from such studies.

[0393] TLR-4 agonists to activate cells and the natural LPS antagonist (LPS-RS, 200 ng / ml) were used as a positive control for antagonist activity against hTLR-4. hTLR-4 activation was quantified by measuring secreted embryonic alkaline phosphatase (SEAP) 24 h after addition of the ligand to the incubation medium. Poly-AG nucleotide (38x) (38x(AG)) was used as a control ssDNA (scrambled). 38x(AG) is an oligonucleotide ssDNA, with a fixed sequence, 38 times that of AG. Because it was designed in the laboratory, it is a control aptamer with no 3D structure or very limited, unstable structural diversity. It does not specifically recognize any targets; in fact, when interacting with proteins, it is only recognized by weak coupling. The results showed that both ApTLR#1R and ApTLR#4F partially inhibited hTLR-4 activation induced by LPS (Figure 2). The concentration-response curves showed that the maximum antagonistic activity was obtained at 20 nM concentrations (ApTLR#1R) and 200 nM (ApTLR#4F), indicating a 30% reduction in hTLR-4 activation mediated by LPS. When the concentration was increased due to receptor saturation, no further effect was observed. No agonist activity of the aptamers was observed during the assay.

[0394] Lead optimization of aptamers with hTLR-4 antagonistic activity The sequences and induced secondary structures of the aptamers ApTLR#1R and ApTLR#4F were modified by deleting regions at both ends of each molecule that do not contribute to the secondary structure and are not expected to affect specific binding properties, in order to improve the bioavailability and biodistribution of the molecules. The resulting truncated forms of the aptamers were designated ApTLR#1RT and ApTLR#4FT (Figure 3).

[0395] To test whether ApTLR#1RT and ApTLR#4FT maintain the same affinity for hTLR-4 displayed by the parent molecules, flow cytometry assays were performed using ApTLR#1RT and ApTLR#4FT (20 nM) conjugated with Alexa Fluor 488 and incubated with 293-hTLR4A cells, using HEK-293 cells without TLR-4 expression as a control.

[0396] Both aptamers bound to 293-hTLR4 cells (Figure 4, Panel A, right panel) but not to HEK-293 cells (Figure 4, Panel A, left panel), showing higher binding affinity to ApTLR#4FT (Figure 4, Panel A, blue line) than to ApTLR#1RT (Figure 4, Panel A, red line). When cells were previously activated with LPS, the increase in FL-1 signal was slightly higher in 293-hTLR4A cells (average increase of 9.9) than in HEK-293 cells (average increase of 9.04) (Figure 4, Panel B, left vs. right panel).

[0397] Quantification of the antagonistic activity of the truncated aptamers by the SEAP assay showed that both aptamers maintained the properties of the parent molecule at 20 nM concentrations (Figure 5, Panel A). Furthermore, in the case of ApTLR#4FT, this inhibitory activity was demonstrated after 96 hours of administration (Figure 5, Panel B).

[0398] Antagonism of hTLR-4 activation by DAMPs The antagonistic profiles of ApTLR#1R, ApTLR#4F, and their corresponding truncated forms were also tested against endogenous TLR-4 ligands to mimic the molecular environment of TLR-4 activation similar to that of ischemic brain tissue. Endogenous TLR-4 agonists, also known as DAMPs (damage-associated molecular patterns), are tissue molecules such as heat shock proteins, nucleic acids, fibronectin, and hyaluronic acid that are released into the brain parenchyma under conditions of injury. To simulate TLR-4 activation by DAMPs, HEK-blue-hTLR4 cells (which express SEAP in response to TLR-4 activation) were incubated with HEK-293 cell lysates containing cell-derived DAMPs. Previous experiments determined that a 1:1 dilution of cell lysate was equivalent to 0.2 ng of LPS for TLR-4 activation. Cell lysate dilutions were added to the incubation medium in the presence or absence of several concentrations of aptamer. All four aptamers partially abolished hTLR-4 activation induced by DAMPs at all concentrations tested (Figure 6). AGA (38xAG) was used as a control ssDNA (scrambled).

[0399] Therefore, starting from two candidate aptamers (ApTLR#1R and ApTLR#4F) with confirmed antagonistic activity against TLR-4, optimized truncated forms were generated for additional in vitro and pharmacological testing. The development of aptamers for the treatment of ischemic stroke is focused on ApTLR#4F and ApTLR#4FT for further characterization. To identify the best candidate aptamer, a series of studies aimed at characterizing the pharmacodynamic, pharmacokinetic, and toxicological properties of both aptamers was initiated. Because they exhibited similar pharmacokinetic and toxicological profiles, pharmacodynamic criteria were used to select the lead molecule. In this regard, ApTLR#4FT demonstrated a better dose-response curve in the mouse pMCAO model and superior efficacy in the rat tMCAO model, covering a wider range of ischemic models in vivo. Furthermore, the smaller size of ApTLR#4FT indicates better distribution of the molecule within body compartments, an interesting feature in indications such as ischemic stroke, where one of the potential target organs is the brain. It is well known that under ischemic conditions, the blood-brain barrier is more permissive than under normal conditions; however, a smaller molecular size can further improve brain distribution after intravenous or intra-arterial administration. Together, these evidences indicate ApTLR#4FT as a candidate aptamer with a better pharmacological profile for stroke indications. ApTLR#4FT (designated ApTOLL) was selected for further development toward its clinical positioning.

[0400] Pharmacodynamic effects of ApTOLL on biologically relevant inflammatory endpoints The antagonistic effect of ApTOLL was further confirmed in mouse peritoneal macrophages stimulated with LPS (500 ng / ml). ApTOLL (20 nM and 200 nM) was added to the incubation medium 1 hour after LPS stimulation, and after 24 hours, NOx concentrations were measured by the Griess reaction (Figure 7, Panel A) as an endpoint parameter of the enzymatic activity of inducible nitric oxide synthase, one of the major target proteins expressed in response to TLR-4 activation. The aptamer induced a decrease in NOx levels in the incubation medium (Figure 7, Panel B).

[0401] In vitro binding characterization To characterize the affinity of the TLR-4 receptor for ApTOLL, affinity studies were performed on monocytes. For this purpose, cells were obtained from cynomolgus monkey and human blood samples and cultured in RPMI 1640 medium supplemented with 2% FBS (2–4 million cells / ml). ApTOLL-488 (0–100 nM) and LPS (50 nM) were added to the medium, and cells were analyzed by flow cytometry. A total of 10,000 viable cells were counted. Propidium iodide staining was performed to exclude nonviable populations. The results sho...

Claims

1. 1. An aptamer for use in ameliorating or ameliorating at least some symptoms or sequelae of acute myocardial infarction, comprising: (a) the aptamer has a length of 40-100 nucleotides and is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4; (i) the aptamer specifically binds to an epitope on the extracellular domain of TLR-4; (ii) binding of said aptamer to said epitope reduces and / or inhibits TLR-4 activation; or (b) the aptamer is a functionally equivalent variant of the aptamer of (a) having at least 85% sequence identity to SEQ ID NO: 1, 2, 3, or 4, wherein the functionally equivalent variant is derived from SEQ ID NO: 1, 2, 3, or 4 and retains the ability to specifically bind to and reduce and / or inhibit activation of TLR-4; The aptamer, wherein the aptamer is administered during, before, or immediately after the acute myocardial infarction.

2. The aptamer of claim 1 , wherein said administration of said aptamer causes a reduction in infarct area.

3. The aptamer of claim 2, wherein said administration of said aptamer causes a reduction in infarct area of ​​at least 25% compared to control conditions.

4. The aptamer of claim 1 , wherein the administration of the aptamer causes a reduction in fibrosis and / or necrosis caused by the acute myocardial infarction.

5. said administering said aptamer (i) improving cardiac function; (ii) reducing extracellular matrix degradation; (iii) amelioration of cardiac remodeling; (iv) preservation of ventricular anatomy; (v) reducing the progression of the infarction; or (vi) The aptamer of claim 1, which provides any combination thereof.

6. 1. An aptamer for use in ameliorating or ameliorating at least some symptoms or sequelae of a neuromuscular or neurodegenerative disease or condition, comprising: (a) the aptamer has a length of 40-100 nucleotides and is selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4; (i) the aptamer specifically binds to an epitope on the extracellular domain of TLR-4; (ii) binding of said aptamer to said epitope reduces and / or inhibits TLR-4 activation; or (b) the aptamer is a functionally equivalent variant of the aptamer of (a) having at least 85% sequence identity to SEQ ID NO: 1, 2, 3, or 4, wherein the functionally equivalent variant is derived from SEQ ID NO: 1, 2, 3, or 4 and retains the ability to specifically bind to and reduce and / or inhibit activation of TLR-4; The aptamer, wherein the aptamer is administered during, before, or after the onset of the neuromuscular or neurodegenerative disease or condition.

7. administering the aptamer (i) reduction of demyelination; (ii) reducing axonal damage, or (iii) An aptamer according to claim 6 that causes their combination.

8. The aptamer of claim 7, wherein said administration of said aptamer causes an inhibition of demyelination by at least 20-80% compared to a control condition (e.g., administration of a placebo).

9. The aptamer of claim 7, wherein the administration of the aptamer causes a reduction in (i.e., protection against) axonal damage by at least 10-30% compared to a control condition (e.g., administration of a placebo).

10. The aptamer of claim 1 or 6, wherein the aptamer is ApTOLL.

11. The aptamer of claim 1 or 6, wherein the aptamer is administered in a dose range of about 0.5 mg / dose to about 10 mg / dose.

12. 10. The aptamer of claim 1 or 6, wherein the aptamer is administered in a dose range of about 0.007 mg / kg per dose to about 0.14 mg / kg per dose.

13. 10. The aptamer of claim 1 or 6, wherein the aptamer is formulated in PBS (sodium chloride, potassium chloride, disodium hydrogen phosphate dihydrate, and potassium dihydrogen phosphate) pH 7.4, containing magnesium chloride hexahydrate and optionally containing A-trehalose dihydrate.

14. The aptamer of claim 1 or 6, wherein the aptamer is administered intravenously by infusion.

15. 7. The aptamer of claim 6, wherein the neuromuscular or neurodegenerative disease or condition is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, Alzheimer's disease, and vascular dementia.

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