Methods and compositions for selectively modulating gene expression in megakaryocytes and platelets

Abstract

Provided is a method for selectively modulating gene expression of one or more target genes in megakaryocytes, circulating platelets, and / or platelets produced by transfected megakaryocytes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a naked RNA oligonucleotide.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Nos. 63 / 391,002, filed July 21, 2022; 63 / 439,365, filed January 17, 2023; and 63 / 522,839, filed June 23, 2023, all of which are incorporated by reference herein in their entireties.

[0002] Reference to sequence listing The sequence listing submitted herewith as an xml file entitled "205961-7088WO1(00343)_Sequence Listing.xml", created on July 19, 2023, and having a size of 107.6 kilobytes, is hereby incorporated by reference in accordance with 37 CFR § 1.52(e)(5).

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under HL159006 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0004] background Megakaryocytes (MKs) are large nucleated cells present mainly in the bone marrow and spleen where they develop from precursor cells and eventually shed their cytoplasm in large numbers to generate platelets. Each mature megakaryocyte produces approximately 10 MKs per day in humans. 11At the rate at which new platelets are produced, the body produces and releases 1,000–2,000 platelets into the bloodstream. The primary function of platelets is to seal vascular wounds. However, overgrown platelet thrombi can occlude (block) blood vessels (thrombosis), which remains a leading cause of morbidity and mortality worldwide. Platelet thrombi form and stabilize under a variety of conditions, even in the absence of vascular wounds, such as atherothrombosis. This action is the underlying driver of adverse cardiac events, including ischemic stroke, myocardial infarction (heart attack), systemic arterial and venous thrombosis, and venous thromboembolism, e.g., pulmonary embolism and deep vein thrombosis. Therefore, a balance of platelet reactivity is essential to establish and maintain thrombus formation and prevent bleeding (hemostasis) without leading to thrombosis; platelet hypo- and hyper-reactivity underlie dysfunction of hemostasis or thrombosis. Platelet production from megakaryocytes (thrombopoiesis) also plays a direct and critical role in this balance: too many platelets (thrombocytosis) increases the risk of thrombosis, while too few platelets (thrombocytopenia) increases the risk of bleeding. Thrombosis is a major risk factor across many pathological conditions, including almost all types of cancer and many, if not most, inflammatory conditions, which together constitute a wide range of morbid and fatal conditions.

[0005] The current standard of care for thrombosis involves both anticoagulation and antiplatelet approaches. While the major antiplatelet drugs currently in widespread use have generally improved significantly in recent decades, they still pose a risk of clinical bleeding, which can be moderate to severe. Such severe bleeding includes intracranial hemorrhage. Furthermore, dysfunction of megakaryocyte development from stem and progenitor cells can lead not only to thrombocytopenia and downstream clinical symptoms, but also to myelodysplasia, which can progress to hematologic malignancies. Thus, there is a significant gap between the clinical control of bleeding and coagulation and the developmental control of blood cells, creating an urgent and long-term need for improved drugs and treatment methods. The present invention addresses this unmet need. Summary of the Invention

[0006] In one aspect, the present invention provides a method for selectively modulating gene expression of one or more target genes in cells of a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a naked RNA oligonucleotide, wherein the cells are at least one selected from the group consisting of megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide.

[0007] In another aspect, the present invention includes a method for selectively modulating gene expression of one or more target genes in a cell, ex vivo or in vitro, comprising transfecting the cell with a naked RNA oligonucleotide.

[0008] In yet another aspect, the present invention provides a method for selectively delivering a naked RNA oligonucleotide to a target tissue and / or target cell in a subject, the method comprising using platelets transfected with the naked RNA oligonucleotide as a vehicle for delivering the naked RNA oligonucleotide to the target tissue and / or target cell. In certain embodiments, the target cell is a tumor cell, a leukocyte, or an inflammatory cell. In certain embodiments, the target tissue comprises an endothelial cell.

[0009] In yet another aspect, the present invention provides a composition for selectively modulating gene expression of one or more target genes in cells of a subject, the cells being transfected with naked RNA oligonucleotides.

[0010] In yet another aspect, the present invention provides a composition for selectively modulating gene expression of one or more target genes in a cell, ex vivo or in vitro, comprising a naked RNA oligonucleotide in the cell.

[0011] In yet another aspect, the present invention provides a kit comprising a composition having a naked RNA oligonucleotide for selectively modulating the expression of at least one gene in a cell and instructions for its use.

[0012] In certain embodiments, the cells are at least one selected from the group consisting of megakaryocytes, platelets, and platelets generated from megakaryocytes transfected with naked RNA oligonucleotides.

[0013] In certain embodiments, the composition comprises a cell transfected with a naked RNA oligonucleotide, the cell being at least one selected from the group consisting of a megakaryocyte and a platelet.

[0014] In certain embodiments, the naked RNA oligonucleotide is selected from the group consisting of miRNA, siRNA, and any combination thereof.

[0015] In certain embodiments, the naked RNA oligonucleotide comprises a guide strand and a passenger strand.

[0016] In certain embodiments, the naked RNA oligonucleotide comprises a guide strand having a sequence selected from the guide strand sequences shown in Table 1.

[0017] In certain embodiments, the naked RNA oligonucleotide comprises a passenger strand having a sequence selected from the passenger strand sequences shown in Table 1.

[0018] In certain embodiments, the naked RNA oligonucleotide comprises at least one modification selected from the group consisting of locked nucleic acids (LNAs), 2'-fluorine bases, and 2'-O-methylated bases.

[0019] In certain embodiments, the naked RNA oligonucleotide is thermostable. In certain embodiments, the naked RNA oligonucleotide is non-immunogenic. In certain embodiments, the naked RNA oligonucleotide is resistant to cleavage by riboendonuclease (RNAase).

[0020] In certain embodiments, the composition is administered intravenously.

[0021] In certain embodiments, administration alters megakaryocyte development, platelet production, megakaryocyte function, and / or platelet function.

[0022] In certain embodiments, the subject is in need of: i. antiplatelet therapy, ii. anti-inflammatory therapy to treat thromboinflammation; iii. Treatment of thrombocytopenia, thrombocytosis, and other pathological conditions related to disrupted megakaryocyte development; iv. Platelet transfusion using platelets protected from platelet storage disorders; v. Treatment of thrombosis, vi. Treatment of acquired bleeding disorders, and / or vii. Treatment of inherited bleeding disorders.

[0023] In certain embodiments, the composition comprises saline.

[0024] In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human.

[0025] In certain embodiments, the cells are derived from at least one selected from the group consisting of a cell culture system, an concentrate, a cell suspension, a tissue homogenate, an organoid, a tissue, and an organ.

[0026] In certain aspects, the cell culture is a stem cell culture.

[0027] In certain embodiments, the concentrate is a platelet storage concentrate that includes autologous plasma.

[0028] In certain embodiments, transfection is performed without the use of synthetic carriers or adjuvants.

[0029] In certain embodiments, the naked RNA oligonucleotides are suspended in an aqueous sterile saline solution. [Brief explanation of the drawings]

[0030] The following detailed description of illustrative embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, non-limiting embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0031] [Figure 1A] Figures 1A-1C show that naked double-stranded miRNA mimics selectively transfect bone marrow megakaryocytes in vitro. (Figure 1A) Live, unfixed whole bone marrow cell suspensions captured on poly-L-lysine (PLL)-coated slides were imaged for AF488. Only megakaryocytes (MKs) exhibited green fluorescence, indicating MK-specific uptake. (Figure 1B) Fixed bone marrow cells were counterstained for the MK / platelet marker Cd41 and DAPI for nuclei. Only MKs, not other bone marrow cells, exhibited miLNA uptake. In this experiment, bone marrow cells were co-incubated with both miLNA-AF488 and an unmodified version of miR-223-3p conjugated to AlexaFluor 647. The bottom panel shows no fluorescence, indicating degradation of the unmodified miRNA mimic. (Fig. 1C) Similar cells as (Fig. 1B) counterstained for two MK / platelet markers, Cd42d and Cd41, showing uptake by MKs of different stages, including immature growth stages (small MKs). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A]Figures 2A–2F show that injected naked double-stranded miRNA mimics selectively transfect bone marrow megakaryocytes and platelets in vivo. (Figure 2A) Whole bone marrow cell suspensions were extracted 2 h after injection, captured on poly-L-lysine (PLL)-coated slides, and imaged live for AF488. Only megakaryocytes (MKs) exhibited green fluorescence, indicating MK-specific in vivo uptake. (Figure 2B) Fixed bone marrow cells were counterstained for MK / platelet markers Cd42d and Cd41 and DAPI for nuclei. At 18 and 72 h postinjection, only MKs and platelets, but not other bone marrow cells, exhibited miRNA uptake. (Figure 2C) The same cells as in (Figure 2B) were analyzed for miRNA uptake (by fluorescence) by flow cytometry, using Cd41 / Cd42d and size (forward scatter FSC) to identify MKs. In this experiment, spleens were also extracted, and splenocyte suspensions were analyzed at 72 hours. Combined, these findings demonstrated robust uptake and retention of miLNAs by MKs in bone marrow and splenic MKs. (Figure 2D) Peripheral blood cells from the same animals were fixed, captured on glass coverslips, and platelets were identified with Cd42d antibodies. The in vivo uptake efficiency of miLNAs by platelets approached 100%, similar to what was previously observed with washed in vitro platelets. Bar, 50 μm. (Figure 2E) High-magnification composite image of platelets derived from (Figure 2D). Bar, 10 μm. (Figure 2F) Blood cells and lung endothelium 2 hours after injection. Rapid miLNA uptake in platelets, but not in WBCs, RBCs, or endothelium (ECs). [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 3A]Figures 3A-3D show that injected naked RhoA siLNA selectively suppresses RhoA protein expression in bone marrow megakaryocytes and modulates megakaryocyte development and platelet production in vivo. (Figure 3A) RhoA siLNA selectively suppresses RhoA protein expression in bone marrow MKs after injection. RhoA levels were measured by flow cytometry in permeabilized whole bone marrow cell suspensions using RhoA antibodies in addition to Cd42d antibodies to label MKs and propidium iodide (PI) to label nuclear DNA. Left panel: RhoA expression levels in MKs as a function of ploidy class determined by PI staining. As expected, RhoA expression increased in larger, more mature MKs due to cytoplasmic expansion, but this increase was suppressed by RhoA siLNA. Right panel: Nucleated (PI+) non-MK bone marrow cells showed no change in RhoA protein levels. (Figure 3B) RhoA siLNA modulates RhoA-dependent MK function. RhoA knockout mice and mice treated with a RhoA inhibitor demonstrated that acute RhoA inhibition led to an increase in MK ploidy. RhoA siLNA caused an increase in MK ploidy compared with control siLNA, demonstrating the specific functional effect of RhoA inhibition on MK in vivo. (Figure 3C) RhoA siLNA increased peripheral blood platelet counts, corresponding to the increase in MK ploidy (Figure 3B). (Figure 3D) The lack of change in mean platelet volume by RhoA siLNA confirmed that increasing MK ploidy leads to a higher platelet count, but not to the generation of larger platelets. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4A]Figures 4A-4B show that infused naked P2y12 (P2ry12) siLNA reduces platelet ADP activation response and prevents injury-induced arterial thrombosis in WT mice, mimicking current first-line antiplatelet drugs that target P2Y12. (Figure 4A) Infusion of P2ry12 siLNA reprograms platelets with reduced ADP responsiveness. Platelet ADP responses were examined by FACS in whole blood cell suspensions 24 h after infusion of P2ry12 (open circles) or control scrambled (CTL, filled circles) siLNA for ADP-induced GpIIb / IIIa integrin activation using JonA antibody (upper panel) and alpha granule secretion using P-selectin antibody (lower panel). *p<0.05, n=3. (Figure 4B) At 96 hours (day 5), anesthetized mice were subjected to arterial injury ("injury") to the carotid artery by applying filter paper soaked in a solution of 7.5% FeCl3 for 90 seconds. Blood flow was monitored with a Doppler probe downstream of the injury site for up to 30 minutes after injury. As expected, in control mice, the artery became occluded and blood flow was blocked 5–12 minutes after injury. In contrast, arteries in mice treated with naked P2ry12 siLNA (which targets the P2y12 ADP receptor, the target of current first-line antiplatelet and antithrombotic drugs) remained patent, and no occlusion was observed over the experimental time frame. n = 3 for each. [Figure 4B] See legend to Figure 4A. [Figure 5A]Figures 5A–5D show that a single injection of naked miL-223-3p miRNA mimic transiently and potently increases platelet ADP reactivity and thrombotic responses. (Figure 5A) Injection of miL-223-3p led to a fourfold average increase in platelet ADP reactivity assessed 24 h postinjection, as measured by flow cytometry with the Jon / A antibody, which recognizes the activated form of integrin GpIIb / IIIa. However, reactivity was unaffected by control Cel-miL-67 or saline infusion. The increased reactivity was transient, indicating consumption of the targeting miLNA and subsequent reconstitution of the target protein. (Figure 5B) The entire population of circulating platelets showed a rightward shift in ADP reactivity. This result suggests a potent direct effect on circulating platelets, although an effect on MKs cannot be excluded. (Figure 5C) The effect of miL-223-3p infusion on injury-induced arterial thrombosis was examined by FeCl3 insult 24 hours after injection, as in Figure 4B. miL-223-3p infusion enhanced the thrombotic phenotype. Figure 5C shows the occlusion time in the carotid artery after injury and removal of the FeCl3 insult. n = 9 control, n = 6 miL-223-3p-treated mice. miL-223-3 infusion enhanced the thrombotic phenotype, demonstrating a direct and potent effect of naked miLNA on platelet physiological responses. (Figure 5D) Summary data from Figure 5C for the time to occlusion in the carotid artery. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A]Figures 6A-6B show that single or repeated addition of naked ADAM17 siRNA to platelets in storage concentrates prevents the loss of GP1bα. (Figure 6A) Single addition of naked ADAM17 siRNA directly to platelet storage concentrates consistently maintained global and surface expression of GP1bα in stored platelets for up to 48 hours. After 48 hours, GP1bα levels fell to low levels similar to those of control siRNA, indicating turnover of ADAM17 and GP1bα. (Figure 6B) Multiple additions of ADAM17 siRNA to stored platelet concentrates (by sedimenting, rinsing, and repeating the transfection of platelets, followed by reconstitution with stored plasma) given at 0 hours and again at 48 hours resulted in long-term maintenance of GP1bα in stored platelets. [Figure 6B] See legend to Figure 6A. [Figure 7A-1] Figures 7A-7B show that a single addition of naked ADAM17 siLNA to platelet storage concentrates prevents the loss of GP1bα function. (Figure 7A) Percent platelet agglutination (aggregation) over time after the addition of the indicated concentrations of ristocetin (Rs) is shown. A single addition of naked ADAM17 siLNA directly to platelet storage concentrates on day 0 maintained functional responsiveness of GP1bα over the 5-day experimental time frame. These data are the first to demonstrate this approach's improvement of cellular physiology in stored platelets. (Figure 7B) Expression of GP1bα in stored platelets. [Figure 7A-2] See legend to Figure 7A-1. [Figure 7B] See legend to Figure 7A-1. [Figure 8] Platelet-specific uptake of miLNA is demonstrated: platelets and platelet extracellular vesicles aggregate with leukocytes in peripheral blood, and uptake by leukocytes, for example, is not detected. [Figure 9] Normal CBC and megakaryocyte size (per ploidy class) with RhoA siLNA. [Figure 10]Normal CBC with miL-223-3p. [Figure 11] Normal CBC with P2ry12 siLNA. [Figure 12] 1 is a diagram outlining a general approach according to an embodiment of the present invention: intravenous injection modeling infusion of modified small RNAs to selectively transfect megakaryocytes and circulating blood platelets as shown herein. [Figure 13-1] The si / miLNA sequences designed and used for the experiments presented here are shown; some experiments used miR-223-3p mimics without fluorophore tags. Sequences are shown 5' to 3' for both the guide and passenger strands, which are hybridized prior to use to obtain double-stranded oligonucleotides with overhangs at each end. [Figure 13-2] See description of Figure 13-1. [Figure 14] Figures 14A-14C show miLNA uptake in MEG-01 cells only during megakaryocytic differentiation. MEG-01 pre-megakaryocytic cells were cultured + / - PMA for various days as indicated to induce megakaryocytic differentiation and then transfected with naked miL:AF488 in vitro for 1 hour. (Figure 14A) miLNA fluorescence and the percentage of miLNA-positive (miL+) cells are shown. miLNA uptake was only observed from day 4 of differentiation. (Figure 14B) The onset of CD41 expression (a megakaryocytic / platelet marker) coincides with the induction of miLNA uptake capacity on day 4. (Figure 14C) miLNA uptake evident in CD41+ cells. [Figure 15-1]Figure 15 shows the evaluation of the therapeutic window (TW) of P2ry12 siLNA and ticagrelor in mice. TW was assessed as the gap between antithrombotic and hemorrhagic effects across a 3-log dose range of either ticagrelor or P2ry12 siLNA after intravenous administration. Antithrombotic effects were also examined in mice treated with scrambled control siLNA at the highest dose tested for P2ry12 siLNA (500 μg / kg; n=5); results are shown by large black squares. Control siLNA had no protective effect against thrombosis, while P2ry12 siLNA had a dose-dependent protective effect against thrombosis. Thrombosis—squares and dashed line; inhibition of hemostasis—circles and dotted line. n=3-10. [Figure 15-2] See description of Figure 15-1. [Figure 16-1] FIG. 16 shows a representative example of an FeCl3 arterial injury assay for occlusive thrombosis. [Figure 16-2] See description of Figure 16-1. [Figure 16-3] See description of Figure 16-1. [Figure 17-1] Figure 17 shows representative Doppler blood flow tracings after carotid artery injury, 24 hours after IV administration of P2yl2 siLNA. The right panel shows the stability of occlusion as a function of P2yl2 inhibition as measured by FACS. The bottom left (continuation of Figure 17) shows the occlusion time as a function of siLNA IV dose. The experiment was stopped at 20 minutes (1200 seconds); any measurement at 1200 seconds indicates recovery of blood flow at the end of the experimental time frame. [Figure 17-2] See description of Figure 17-1. [Figure 17-3] See description of Figure 17-1. [Figure 17-4] See description of Figure 17-1. [Figure 17-5] See description of Figure 17-1. [Figure 18-1] FIG. 18 shows the diversity of reactivity among circulating platelet populations. [Figure 18-2] See description of Figure 18-1. [Figure 19]Figure 1 shows platelet hPAR4 suppression by F2RL3 siLNA in hPAR4 mice. 24 hours after a single injection of F2RL3 (hPAR4) siLNA into hPAR4 mice, platelet lysates were extracted and immunoblotted with PAR4 antibody. hPAR4 TG / mPar4 KO refers to mice transgenic for human PAR4 and lacking the mouse Par4 gene. [Figure 20] This shows that F2RL3 siLNA reprograms platelets with reduced responsiveness to PAR4 agonists. 0.5 mg / kg of F2RL3 or control siLNA was injected into hPAR4 mice, and platelet PAR4-activating peptide (PAR4-AP) responses were examined by flow cytometry 24 hours later in platelets from whole blood cell suspensions using Jon / A antibody (left), which indicates integrin activation, and P-selectin antibody (right), which indicates granule secretion. n=6. [Figure 21] Figure 1 shows that F2RL3 siLNA provides protection from thrombosis with minimal effect on bleeding. hPAR4 TG / mPar4 KO mice were injected with 0.5 mg / kg of F2RL3 or control siLNA and subjected to FeCl3 thrombosis (left) or tail-tip cut bleeding (right) at 24 hours. Bleeding is shown as the ratio of blood weight lost (g) to starting mouse body weight (g). n=3. [Figure 22A]Figures 22A-D illustrate in vivo siLNA-mediated silencing of Gsα (mRNA, Gnas) in mouse platelets at 24 hours, the hemostatic promoting effects of Gnas and Ptgir siLNAs, and rescue from acquired (ticagrelor-induced) hemorrhage by Gnas and Ptgir siLNAs. Figure 22A shows the suppression of Gsα by Gnas siLNA in mouse platelets 24 hours after tail vein injection of siLNA. n=3. Figure 22B shows the reduction in hemorrhage in mice treated with Gnas (Gsα) or Ptgir (IP, prostacyclin receptor) siLNA, similar to that measured in Figure 21. N is shown for each. Figure 22C shows accelerated hemostasis in mice with Gnas siLNA compared to controls after a 1.5 mm tail-tip amputation. The experiment was stopped at 20 minutes (1800 seconds). N = 6. Figure 22D shows rescue from acquired hemorrhage induced by 0.5 mg / kg ticagrelor in mice treated with Gnas and Ptgi combined siLNA 24 hours after siLNA injection. Ticagrelor or vehicle was given 5 minutes before the start of the bleeding assay. *, p < 0.04. n = 3-10. [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 22D] See legend to Figure 22A. [Figure 23] Dynamic Gsα expression over the platelet lifespan is shown. The ratio of Gsα expression in new (dotted line) and existing (solid line) platelets versus total platelets is shown ± standard error of the mean. Gsα was monitored by flow cytometry using a Gsα-specific antibody in fixed and permeabilized platelets extracted at the indicated times after pulse-labeling with Gp1bβ antibody. n=3. [Figure 24]Figures 24A-24B show the selective in vivo uptake of miLNA by (Figure 24A) platelets and (Figure 24B) bone marrow megakaryocytes. Cells were collected from uninjected WT and miR-223 KO mice (-) and from miR-223 KO mice (+) 18 hours after miL-223-3p injection, labeled with surface markers, and subjected to single-cell FACSort for each population as indicated. As a direct demonstration of cell-type-specific in vivo miLNA uptake in platelets / MKs, the presence of miR-223-3p was detected in each population by PCR from poly(dA)-tailed cDNA, as in previous studies. [Figure 25] We demonstrate that anti-miRs / antagomirs lack the unique property of platelet / megakaryocyte-specific internalization and utilization that miLNAs and siLNAs possess. We injected mice with eight-nucleotide antagomirs (anti-miRs) composed entirely of locked nucleic acids bearing a 3'-FAM fluorophore, and monitored FAM fluorescence in the indicated cell types by flow cytometry. [Figure 26] Figure 1 shows the targeting effect of siLNA administered subdermally using an osmotic pump in mice. Three-day release pumps containing 0.6 mg / kg P2ry12 (open red circles) or control (filled blue circles) siLNA were implanted subdermally in mice, and platelet ADP-induced integrin activation (left) and P-selectin exposure (right) were assessed daily by flow cytometry. Data are shown as the fold area under the curve for a range of ADP doses, ± standard error of the mean, n = 3, as in Figure 4. *, p < 0.03; **, p < 0.05. DETAILED DESCRIPTION OF THE INVENTION

[0032] Detailed Description of the Disclosure In the present invention, the overall approach is to introduce microRNA mimics (miRNA) and / or small interfering RNA (siRNA) into megakaryocytes and / or platelets to modulate the expression of their cognate target genes, which contribute to specific cellular, physiological, and pathophysiological functions as outlined above. mi / siRNA sequence-specifically suppresses the translation of its target mRNA, preventing the expression of its cognate protein. Therefore, mi / siRNA suppresses the protein expression of target genes.

[0033] definition As used herein, each of the following terms has the meaning associated with it in this section. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In general, the nomenclature used herein and laboratory methods in animal pharmacology, pharmacology, peptide chemistry, and organic chemistry are those well known and commonly used in the art. It should be understood that the order of steps or order for performing certain actions is not important, so long as the present teachings remain operable. Any use of section headings is intended to aid in the reading and comprehension of the document and should not be construed as limiting; the information associated with a section heading may be located within or outside of that particular section. All publications, patents, and patent documents mentioned in this document are incorporated by reference in their entirety, as if individually incorporated by reference.

[0034] In this application, when an element or component is said to be included in and / or selected from a recited list of elements or components, it is understood that the element or component may be any one of the recited elements or components, or may be selected from a group consisting of two or more of the recited elements or components.

[0035] In the methods described herein, acts may be performed in any order unless a temporal or operational order is expressly recited. Furthermore, specified acts may be performed simultaneously unless express claim language recites that they be performed separately. For example, a claimed act of performing X and a claimed act of performing Y may be performed simultaneously in a single operation, and the resulting process would be considered to fall within the literal scope of the claimed process.

[0036] In this document, the terms "a," "an," or "the" are used to include one or more than one, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. The phrases "at least one of A and B" or "at least one of A or B" have the same meaning as "A, B, or A and B."

[0037] As used herein, "about" when referring to a measurable value, e.g., an amount, a temporal duration, etc., is meant to encompass a variation of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, and in certain embodiments ±0.1% from the specified value, as such variations are appropriate for practicing the disclosed methods.

[0038] The terms "alteration" or "altering" or "modulating" refer to a change (increase or decrease) in the expression level or activity of a gene or polypeptide, as detected by standard methods known in the art, such as those described herein. In some embodiments, an alteration in expression levels includes a 10% change in expression levels, a 25% change, a 40% change, and a 50% or greater change in expression levels.

[0039] As used herein, the term "anti-miR" refers to a chemically modified single-stranded antisense oligonucleotide that inhibits the function of miRNA.The length of anti-miR ranges from seed targeting 8-mer oligonucleotides to anti-miRs that are completely complementary to mature miRNA.As a synthetic reverse complementary strand, it inhibits the activity of miRNA by competing with the target 3'UTR mRNA site for miRNA binding.

[0040] As used herein, the term "antagomir" refers to a 3' cholesterol-conjugated, 2'-O-methyl-modified antisense oligonucleotide that inhibits the function of a miRNA. Antagomir is perfectly complementary to the mature miRNA.

[0041] As used herein, the term cell refers to cells (such as, for example, megakaryocytes) and / or cell fragments (such as, for example, platelets).

[0042] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which the patient experiences such symptom, or both, is reduced.

[0043] The term "cleavage" refers to the destruction of covalent bonds, for example, in the backbone of a nucleic acid molecule.Cleavage can be caused by various methods, including but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds.Both single-strand and double-strand breaks can occur.Double-strand breaks can occur as a result of two different single-strand break events.RNA cleavage can generate either blunt ends or sticky ends.

[0044] In this disclosure, "comprises," "comprising," "containing," "having," and the like can have the meaning given them in U.S. patent law, and can mean "includes," "including," and the like, and "consisting essentially of" or "consisting essentially of" likewise have the meaning given them in U.S. patent law, and the terms are open-ended, permitting the presence of more than what is recited so long as the basic or novel characteristics of the recited items are not altered by the presence of more than what is recited, but excluding prior art aspects.

[0045] A "disease" is a state of an animal's health in which the animal is unable to maintain homeostasis and, if the disease does not go into remission, the animal's health continues to deteriorate.

[0046] An animal "disorder" is a state of health in which the animal is able to maintain homeostasis, but the animal's health status is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause further deterioration in the animal's health status.

[0047] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0048] As used herein, the term "modulating a gene" refers to modulating a gene and / or a gene product and / or any genetic material. In certain embodiments, "modulating a gene" refers to modulating, for example, mRNA, pre-mRNA, lncRNA, snRNA, snoRNA, tRNA, rRNA, YRNA, piRNA.

[0049] As used herein, "microRNA" or "miRNA" describes small, non-coding RNA molecules, generally about 15 to about 50 nucleotides in length, preferably 17 to 23 nucleotides, that can play a role in regulating gene expression, for example, through a process called RNA interference (RNAi). RNAi describes the phenomenon in which the presence of an RNA sequence that is complementary or antisense to the sequence of the target gene's messenger RNA (mRNA) results in the inhibition of target gene expression. miRNAs are processed from hairpin precursors of about 70 or more nucleotides (pre-miRNAs) derived from the primary transcript (pri-miRNA) through sequential cleavage by the RNAse III enzyme. miRBase is a comprehensive microRNA database located at www.mirbase.org, which is incorporated herein by reference in its entirety for all purposes.

[0050] As used herein, "homologous" refers to the identity of subunit sequences between two polymer molecules, for example, between two nucleic acid molecules, for example, two DNA molecules or two RNA molecules, or between two polypeptide molecules.If the subunit position in both molecules is occupied by the same monomer subunit; for example, if each position in two DNA molecules is occupied by adenine, they are homologous at that position.The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in two sequences (for example, 5 positions in a polymer with a length of 10 subunits) are homologous, the two sequences are 50% homologous; if 90% of the positions (for example, 9 out of 10) are matching or homologous, the two sequences are 90% homologous.

[0051] "Identity" as used herein refers to the identity of the subunit sequence between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules.If two amino acid sequences have the same residue at the same position; for example, if each position of two polypeptide molecules is occupied by arginine, they are identical at that position.The identity or degree to which two amino acid sequences have the same residue at the same position in alignment is often expressed as a percentage.The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions of the two sequences (for example, 5 positions in a polymer of 10 amino acids in length) are identical, the two sequences are 50% identical; if 90% of the positions (for example, 9 out of 10) are matching or identical, the two amino acid sequences are 90% identical.

[0052] As used herein, the term "locked nucleic acid (LNA)" refers to a modified RNA nucleotide in which the ribose moiety is modified with an additional bridge connecting the 2' oxygen to the 4' carbon.

[0053] As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the present invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells are modified through the introduction of nucleic acids.

[0054] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of treatment or compound, and / or compared to the level of the response in an otherwise identical, but untreated, subject. The term encompasses perturbing and / or affecting a native signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0055] As used herein, the term "newly generated platelets" refers to platelets generated by megakaryocytes between 0 and 48 hours following in vivo administration or ex vivo or in vitro transfection of siLNA or miLNA.

[0056] In the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0057] As used herein, the term "naked RNA oligonucleotide" refers to the RNA oligonucleotide that is not encapsulated in any kind of liposome or nanoparticle, and is not attached to any other molecule or adjuvant.Naked RNA oligonucleotides include, for example, microRNA mimics (miRNA) or small interfering RNA (siRNA).

[0058] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0059] The term "oligonucleotide" typically refers to a short polynucleotide, generally not exceeding about 60 nucleotides. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it will be understood to also include an RNA sequence in which "U" is replaced by "T" (i.e., A, U, G, C).

[0060] As used herein, "polynucleotide" includes cDNA, RNA, DNA / RNA hybrids, antisense RNA, siRNA, miRNA, snoRNA, tRNA, YRNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands. Polynucleotides can be chemically or biochemically modified to contain non-natural or derivatized, synthetic, or semi-synthetic nucleotide bases. Alterations of wild-type or synthetic genes, including, but not limited to, deletion, insertion, or substitution of one or more nucleotides, or fusion to other polynucleotide sequences, are also within the scope of the present invention. As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate administration of a compound to a patient. Multiple techniques for administering compounds exist in the art, including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical administration.

[0061] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0062] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, involved in carrying or transporting a compound useful within the present disclosure into or to a patient so that the compound can perform its intended function. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, e.g., the compound useful within the present disclosure, and not toxic to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, absorption delaying agents, and the like, that are compatible with the activity of the compound useful within the present disclosure and are physiologically acceptable to the patient. "Pharmaceutically acceptable carriers" can further include pharmaceutically acceptable salts of compounds useful within the present disclosure. Other additional ingredients that can be included in pharmaceutical compositions used in the practice of the present disclosure are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0063] As used herein, the phrase "pharmaceutically acceptable salts" refers to salts of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.

[0064] As used herein, a "pharmaceutically effective amount," "therapeutically effective amount," or "effective amount" of a compound is the amount of the compound that is sufficient to confer a beneficial effect on the subject to which the compound is administered.

[0065] As used herein, the term "prevent" or "prevention" means the absence of a disorder or disease occurring if none has occurred, or the absence of a further disorder or disease occurring if a disorder or disease has already occurred. The ability of something to prevent some or all of the symptoms associated with a disorder or disease is also considered.

[0066] " Small interfering RNA " or " siRNA " refers to double-stranded RNA. Optimally, siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a base overhang at its 3' end. These dsRNAs can be introduced into individual cells or whole animals; for example, they can be introduced systemically through the bloodstream. Such siRNAs are used to down-regulate mRNA levels or promoter activity.

[0067] The terms "siLNA" and "miLNA" refer to LNA-modified siRNA molecules and LNA-modified miRNA molecules, respectively.

[0068] As used herein, the terms "subject," "individual," and "patient" can be used interchangeably and can refer to a human or non-human mammal or bird. Non-human mammals include, for example, mammals such as livestock and pets, sheep, cows, pigs, dogs, cats, and mice. In certain embodiments, the subject is a human.

[0069] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Such a sequence is at least 60%, at least 80%, at least 85%, at least 90%, at least 95%, or even at least 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0070] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0071] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected with, transformed with, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0072] As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound useful within the present disclosure (alone or in combination with another pharmaceutical agent), to a patient, or to a tissue or cell line isolated from a patient with a disease or disorder and / or symptoms of a disease or disorder (e.g., for diagnostic or ex vivo applications), with the intent to cure, heal, alleviate, relieve, alter, cure, ameliorate, improve, or affect a disease or disorder and / or symptoms of a disease or disorder. Such treatments can be specifically tailored or modified based on knowledge obtained from the field of pharmacogenomics.

[0073] Ranges: Throughout this disclosure, various aspects of the present disclosure may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed not only individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, but also subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the breadth of the range.

[0074] explanation The objective of the present invention is to provide a platform for modulating megakaryocyte development, platelet production, and / or platelet reactivity in vivo, ex vivo, and in vitro to improve all of the clinical scenarios disclosed elsewhere herein, as well as to extend the lifespan and function of stored platelets along with reducing the inflammatory response in stored platelet units.

[0075] Without wishing to be limited by any theory, the cellular physiological basis of the present invention is the discovery that megakaryocytes and platelets have the unique ability to internalize naked, unencapsulated small RNAs due to the unique membrane structure of these cells. This feature provides the straightforward ability to transfect small regulatory RNAs into megakaryocytes and platelets in vivo, ex vivo, and in vitro with minimal off-target tissue transfection.

[0076] Importantly, although unassisted uptake of oligonucleotides (also known as "gymnosis") has been explored for therapeutic use, uptake efficiency and tissue targeting remain unresolved issues, which are resolved by the present invention for megakaryocytes and platelets.

[0077] composition In one aspect, the present invention provides a composition for selectively modulating gene expression in megakaryocytes (MK) and / or platelets and / or platelets produced by transfected megakaryocytes. The composition comprises a naked, e.g., unencapsulated, RNA oligonucleotide. In certain embodiments, the RNA oligonucleotide is suspended in an aqueous solution, but is not encapsulated in any kind of liposome or nanoparticle, or attached to any other molecule or adjuvant. In certain embodiments, the aqueous solution is, for example, sterile saline.

[0078] In certain embodiments, the RNA oligonucleotide is selected from the group consisting of double-stranded (ds) miRNA mimics, siRNAs, and any combination thereof, in which the guide strand comprises modified RNA bases corresponding to the nucleotide sequence of native miRNA. In certain embodiments, the RNA oligonucleotide can be easily and inexpensively synthesized.

[0079] In certain embodiments, RNA oligonucleotides are selectively transfected into megakaryocytes and platelets in vivo, ex vivo, or in vitro.In certain embodiments, in vivo transfection is used to modulate, for example, megakaryocyte development, function, and gene expression, platelet production, and platelet in vivo functions, such as hemorrhage, thrombosis, and inflammation control.In certain embodiments, ex vivo transfection is used to improve the functional lifespan of stored platelet concentrates and / or reduce inflammatory conditions.In certain embodiments, in vitro transfection is used for stem cell therapy approaches, such as modulating megakaryocytes derived from induced pluripotent stem cells.

[0080] In certain embodiments, RNA oligonucleotides are designed to avoid degradation by riboendonuclease (RNAse) in plasma and tissue.In certain embodiments, RNA oligonucleotides are non-immunogenic.In certain embodiments, RNA oligonucleotides are designed to avoid immune response or induce little or no immune response.

[0081] In certain other embodiments, the RNA oligonucleotides optionally include modified nucleotides, such as locked nucleic acids (LNAs) modified to include a methylene bridge between the 2' oxygen and the 4' carbon of the pentose ring, and 2'-OMe RNA bases that are methylated at the 2' oxygen and / or 2' fluorine (2'-F) bases.

[0082] In certain embodiments, when the 3'-terminal residue of the overhanging guide strand is an RNA nucleotide containing uracil (U), U is replaced with thymine (T) as LNA (T) (because uracil cannot be modified as LNA due to its structure).

[0083] LNA-based RNA oligonucleotides are known to confer strong resistance to RNA cleavage by RNAses, which are abundant in all tissues, including blood / plasma, bone marrow, spleen, and other hematopoietic niches, which are the target tissues of the RNA oligonucleotides of the present invention. Furthermore, the precise location of LNA and 2'-OMe RNA nucleotides within the double-stranded miRNA mimic, siRNA, or anti-miR confers several important properties, including: for the guide strand, a 3'-overhang consisting of two or three LNA bases to support the incorporation of the guide strand into the RNA-induced silencing complex (RISC) but avoid the loss of silencing activity; for the passenger strand, a truncated 5'-end plus a 5'-end LNA base to inactivate the strand, a moderate number of LNA bases (5-6) evenly distributed throughout the strand to confer RNase resistance, a 3'-end diuridine to create an overhang and further render the strand nonfunctional, and 2'-OMe modification of the 3'-end uridine and most or all adenosines to significantly reduce immunogenicity. The guide strand sequence of a miRNA mimic is determined by the native sequence of a given mature miRNA.

[0084] In certain embodiments, instead of 2'-OMe nucleotides, 2'-F nucleotides are also considered sufficient for those bases in the passenger strand of a synthetic double-stranded siLNA that would be substituted with 2'-OMe, providing a similar effect, i.e., protection from riboendonuclease and avoidance of inducing an immune response.

[0085] In certain embodiments, the siRNA is about 15 to about 30 nucleotides in length and is designed to target an mRNA of interest. In certain embodiments, the siRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 nucleotides in length.

[0086] In certain embodiments, the anti-miR comprises the reverse complement sequence of the 5' end of the target miRNA, which is typically, but not limited to, nucleotides 1 to 8. In certain embodiments, the anti-miR is composed entirely of LNA bases.

[0087] In certain embodiments, the RNA oligonucleotide comprises two strands: a guide strand and a passenger strand. In certain embodiments, the guide strand sequence and the passenger strand sequence are as listed in Table 1. In certain embodiments, the guide strand has a sequence substantially identical to the guide strand sequence shown in Table 1. In certain embodiments, the passenger strand has a sequence substantially identical to the passenger strand sequence shown in Table 1. In certain embodiments, the guide strand has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the guide strand sequence shown in Table 1. In certain embodiments, the passenger strand has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the passenger strand sequence shown in Table 1.

[0088] In certain embodiments, the naked RNA oligonucleotide comprises only unmodified nucleotides.

[0089] In certain embodiments, the RNA oligonucleotides are thermostable, ie, stable at freezing / deep-freezing temperatures, refrigeration temperatures, room temperature, and body temperature.

[0090] In certain embodiments, the composition is administered to a subject and enters the blood circulation. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human subject.

[0091] In another aspect, the present invention provides a composition for selectively modulating gene expression of one or more target genes in cells of a subject, the composition comprising cells transfected with naked RNA oligonucleotides, wherein the cells are at least one selected from the group consisting of megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with naked RNA oligonucleotides.

[0092] In yet another aspect, the present invention provides a composition for selectively modulating gene expression of one or more target genes in cells ex vivo or in vitro, the composition comprising cells transfected with naked RNA oligonucleotides, wherein the cells are at least one selected from the group consisting of megakaryocytes and platelets.

[0093] In certain embodiments, the naked RNA oligonucleotide is as described elsewhere herein.

[0094] method In another aspect, the present invention provides a method for selectively modulating gene expression of one or more target genes in cells of a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a naked RNA oligonucleotide. In certain embodiments, the cells are at least one selected from the group consisting of megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide. In certain embodiments, the composition comprising the naked RNA oligonucleotide is as described elsewhere herein.

[0095] Platelets do not contain a nucleus, but do have other forms of genetic material that regulate protein expression, and modulating gene expression involves interacting with downstream gene products, such as mRNA.

[0096] In certain embodiments, the term "modulating a gene" includes modulating a gene and / or a gene product and / or any genetic material. In certain embodiments, "modulating a gene" includes, but is not limited to, modulating mRNA, pre-mRNA, lncRNA, snRNA, snoRNA, tRNA, rRNA, YRNA, piRNA.

[0097] In certain embodiments, the composition is administered, for example, subcutaneously, intravenously, intramuscularly, or intraperitoneally. In certain embodiments, the composition is administered intravenously. In certain embodiments, the composition is administered topically.

[0098] In certain embodiments, administration results in alterations in, for example, megakaryocyte development, platelet production, megakaryocyte function, and / or platelet function.

[0099] In yet another aspect, the present invention provides methods for improved antiplatelet therapy over standard of care pharmacological agents for antithrombotic use with reduced risk of clinical bleeding. In certain embodiments, the methods comprise administering to a subject in need thereof a therapeutically effective amount of a composition described elsewhere herein.

[0100] In certain embodiments, the target of antiplatelet therapy includes, but is not limited to, P2Y12 (ADP receptor, gene P2RY12).This protein product is the target of current first-line antiplatelet agents, such as cangrelor, ticagrelor, and clopidogrel (Plavix), which are used worldwide as antithrombotic agents, but this leads to a variable risk of clinical bleeding, which can range from moderate to severe, such as life-threatening intracranial hemorrhage.MK / platelet-specific knockdown of P2Y12 by this platform can improve the usefulness of antithrombotic therapy, with reduced risk of bleeding compared to global pharmacological blockade, and affect many patients in many clinical scenarios of thrombosis risk reduction and management.

[0101] In certain embodiments, targets for antiplatelet therapy include, but are not limited to, PAR1 (thrombin receptor). This protein is the target of vorapaxar, which has been removed from most clinical use due to its bleeding-causing properties. MK / platelet-specific knockdown of PAR1 by this platform can provide an alternative to vorapaxar with improved antithrombotic efficacy, along with reduced bleeding risk, impacting many patients in many clinical scenarios for thrombosis risk reduction and management.

[0102] In yet another embodiment, the target of antiplatelet therapy includes, but is not limited to, FcγRIIa (gene, FCGR2A). This is an antibody receptor on platelets and other blood cells that mediates immunological thrombosis, e.g., thrombosis in response to increased immune activity. MK / platelet-specific knockdown of FcγRIIa by this platform can provide strong protection from immunological thrombosis across a variety of clinical settings of inflammation and for many patient cohorts, with improved specificity of action and therefore reduced side effects compared to current standard care.

[0103] In yet another embodiment, targets for antiplatelet therapy include, but are not limited to, platelet intracellular proteins involved in responsiveness. These include numerous putative targets, similar to those listed above, that can be modified by the present platform technology in a personalized medicine approach based on an individual patient's platelet expression profile. For example (one of many possibilities), a patient with overexpression of a cytoplasmic inhibitor of platelet receptor function, associated with reduced platelet responsiveness secondary to a bleeding diathesis, could be treated by targeting the overexpressed inhibitor protein.

[0104] In yet another aspect, the present invention provides a method for treating platelet-induced inflammation, thromboinflammation, comprising administering to a subject in need thereof a therapeutically effective amount of a composition described elsewhere herein.

[0105] In certain embodiments, targets for treating thromboinflammation include, but are not limited to, platelet-derived inflammatory cytokines and their upstream regulators. These include, but are not limited to, interleukins and their upstream inducers, such as NLRP3 and CCL lymphocyte-activating factor. Platelets contribute to inflammation by releasing inflammatory cytokines; for example, in sepsis, a major cause of mortality, they contribute to an exaggerated inflammatory response. However, in many scenarios, global blocking of inflammation impairs the patient's necessary immune response. MK / platelet-specific knockdown of inflammatory cytokines using this platform can improve anti-inflammatory efficacy while maintaining necessary immune function.

[0106] In other embodiments, targets for treating thrombo-inflammation include, but are not limited to, PAR4 (secondary thrombin receptor). PAR4 antagonists are also utilized as antiplatelet and anti-inflammatory drugs. PAR4 plays a major role in platelet production of inflammatory substances. However, in many scenarios, total blockage of PAR4 may be undesirable. MK / platelet-specific knockdown of PAR4 using this platform can improve anti-inflammatory efficacy while maintaining necessary functions.

[0107] In yet another aspect, targets for treating thromboinflammation include, but are not limited to, intracellular platelet proteins (there are multiple targets in this category) that mediate the production of platelet-derived microvesicles, an active process driven by the action of multiple proteins, which occurs, for example, as a result of platelet stimulation due to vascular injury or trauma, as a result of thrombolytic or thrombectomy treatment of ischemia, or in the setting of atherosclerosis and atherothrombosis. These microvesicles are both pro-inflammatory and pro-coagulant. This platform technology is believed to reduce platelet microvesicle production and / or reduce the expression of pro-inflammatory or pro-coagulant components, resulting in improved anti-inflammatory and anticoagulant outcomes compared to current standard of care.

[0108] In yet another aspect, the present invention provides treatments for acute bleeding and bleeding disorders, such as acquired and inherited bleeding disorders. These treatments can be considered antihemorrhagic or prohemostatic therapies. Bleeding disorders treated using the present invention include, but are not limited to, platelet reactivity disorders and coagulation disorders. Acquired bleeding disorders include, but are not limited to, those induced by antiplatelet or anticoagulant therapy. Targets for antihemorrhagic / prohemostatic therapy include, but are not limited to, endogenous negative regulatory proteins expressed in platelets, such as Gsα (gene name, GNAS), IP (prostacyclin receptor, gene name PTGIR), GSK3β, and other modulators of platelet reactivity.

[0109] In yet another aspect, the present invention provides treatment of thrombocytopenia, thrombocytosis, and other pathological conditions related to perturbed megakaryocyte development, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition described elsewhere herein.

[0110] These conditions are driven by altered gene expression in developing megakaryocytes, e.g., upregulation of transcription factors that suppress MK development in favor of the erythroid lineage, leading to thrombocytopenia, or conversely, factors that direct platelet production, leading to thrombocytopenia. Targeting these upregulated mediators of altered cell fate can be used to restore normal MK development and normal platelet production.

[0111] In yet another aspect, the present invention provides methods for improved treatment of platelet storage disorders, as outlined above. In certain embodiments, the methods comprise administering a therapeutically effective amount of a composition described elsewhere herein to a subject in need thereof. In certain embodiments, targets for treating platelet storage disorders include, but are not limited to, ADAM17 metalloproteinase, which is involved in cleavage of the platelet vWF receptor GP1bα. In certain embodiments, targets for treating platelet storage disorders include, but are not limited to, ADAM10 metalloproteinase, which is involved in cleavage of the platelet collagen receptor GPVI. In certain embodiments, targets for treating platelet storage disorders include, but are not limited to, miR-326 (using anti-miR-326) to derepress the anti-apoptotic master regulator BCLxL. In certain embodiments, targets for treating platelet storage disorders include, but are not limited to, neuraminidase I, which is involved in desialylation of platelet surface proteins, leading to accelerated clearance of stored platelets after transfusion. In certain embodiments, targets for treating platelet storage disorders include, but are not limited to, inflammatory cytokines, as described elsewhere herein, and proteins associated with microvesicles, as described elsewhere herein, each of which is released by platelets over time during storage, resulting in a pro-inflammatory state in the platelet storage concentrate. In yet other embodiments, other mediators of platelet death, dysfunction, and inflammatory states during storage are also targets of the platform technology.

[0112] In yet another aspect, the present invention provides improved treatments for thrombosis risk associated with hormone action, including gender differences in thrombosis risk, increased thrombosis risk with age and menopause, estrogen therapy, and hormone replacement therapy. In certain embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a composition described elsewhere herein. In certain embodiments, targets for treating thrombosis include, but are not limited to, estrogen receptor beta (ERβ, gene ESR2), which is the major estrogen receptor in MK / platelets that alters platelet reactivity and has established non-nuclear functions associated with thrombosis.

[0113] In yet another embodiment, targets for treating thrombosis include, but are not limited to, genes involved in hormone response, such as adrenoceptors, androgen receptors, and other hormone receptors expressed in platelets.In certain embodiments, targeting these genes reduces platelet reactivity without impairing the bodily function of hormones by specifically targeting MK / platelets.Together, this group of targets represents a major area for improvement beyond the current standard of care.

[0114] In yet another aspect, the present invention provides a method for selectively modulating the gene expression of one or more target genes in megakaryocytes or platelets from at least one selected from cell culture system, concentrate, cell suspension, tissue homogenate, organoid, tissue and organ in vitro or ex vivo, comprising transfecting megakaryocytes or platelets with naked RNA oligonucleotide.In certain embodiments, the target gene or target protein is at least one selected from those listed in Table 1.

[0115] In certain embodiments, platelets transfected with the naked RNA oligonucleotides of the present invention (in vivo or in vitro) can be used as delivery vehicles to deliver such naked RNA oligonucleotides to other target cells and / or tissues in vivo, including, for example, tumor cells, leukocytes, inflammatory cells, endothelial cells, etc. The delivery mechanism from transfected platelets to heterologous cells and tissues can include extracellular vesicles released from the transfected platelets, including microparticles (also known as microvesicles), exosomes, ectosomes, or apoptotic bodies, or other platelet release products containing the transfected naked RNA oligonucleotides. In the example of tumor cells, the leaky vasculature of solid tumor cells allows platelet microparticles carrying naked RNA oligonucleotides to reach the tumor. Therefore, the transfer of mi / siRNA from platelets may be more selective for tumor cells than for cells in normal tissues that are not easily accessible to platelet microparticles.

[0116] In certain embodiments, the present invention provides improved cell culture processing methods, e.g., for in vitro MKs, either directly isolated or derived from stem cell culture, for bone marrow reconstitution, and for industrial platelet production. In certain embodiments, the methods include transfecting MKs with at least one RNA oligonucleotide, as described elsewhere herein.

[0117] In certain embodiments, the cell culture is, for example, a stem cell culture.

[0118] In certain embodiments, the concentrate is a platelet storage concentrate, e.g., comprising autologous plasma. In certain embodiments, gene expression is modulated in platelets to extend the lifespan and function of stored platelets, as well as reduce inflammatory responses in stored platelet units.

[0119] In certain embodiments, transfection is achieved ex vivo, for example, by adding mi / siRNA / anti-miR to platelet storage units containing autologous plasma, or to any cell suspension containing megakaryocytes and platelets (including, but not limited to, blood, bone marrow cells, splenocytes, lung or other tissue homogenates, cell suspensions, organoids, tissues, or organs), or directly to in vitro cultured megakaryocytes or platelets. The mi / siRNA / anti-miR selectively transfects megakaryocytes and platelets and modulates their function as guided by changes in target gene expression.

[0120] In certain embodiments, transfection is performed without the use of synthetic carriers or adjuvants.

[0121] The compositions and methods described herein are an improvement over lentiviral or other approaches that have the risks associated with gene therapy approaches by using an active virus to increase platelet production.

[0122] In certain embodiments, the subject is a mammal.In certain embodiments, the subject is a human subject.In certain embodiments, the subject needs at least one selected from the group consisting of antiplatelet therapy, anti-inflammatory therapy for treating thromboinflammation, treatment of thrombocytopenia, thrombocytopenia and other pathological conditions related to disordered megakaryocyte development, platelet transfusion with platelets that are protected from platelet storage disorders, treatment of thrombosis, treatment of acquired bleeding disorders, and treatment of inherited bleeding disorders.

[0123] kit In yet another aspect, the present invention provides a kit comprising a composition comprising a naked RNA oligonucleotide for selectively modulating the expression of at least one gene in megakaryocytes and / or platelets and instructional materials for its use. In certain embodiments, the composition is used to modulate gene expression in vivo and / or ex vivo and / or in vitro.

[0124] In yet another aspect, the present invention provides a kit comprising a composition comprising cells transfected with a naked RNA oligonucleotide for selectively modulating the expression of at least one gene in megakaryocytes and / or platelets, and an instructional material for its use. In certain embodiments, the composition is used to modulate gene expression in vivo and / or ex vivo and / or in vitro. In certain embodiments, the composition is as described elsewhere herein. In certain embodiments, the naked RNA oligonucleotide is as described elsewhere herein.

[0125] Pharmaceutical Compositions The composition or pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The dosage and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0126] The pharmaceutical compositions of the present invention can be administered in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, emulsions, etc. The pharmaceutical compositions of the present invention can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by nasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes. In some embodiments, the compositions can be applied to the nose, pharynx, or bronchi, for example, by inhalation.

[0127] The dosage of the above treatments administered to a patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment, and can be determined based on physical and physiological factors such as body weight, severity of the condition, previous or concurrent therapeutic interventions, and the route of administration. Scaling of dosages for human administration can be performed according to art-recognized practices. Doses of miR mimics, for example, will generally be in the range of 1 to about 100 mg for an adult patient, usually administered monthly for a period of 1 to 12 months. A preferred monthly dose is 1 to 10 mg per month, although larger doses exceeding 10 mg per month can be used in some cases.

[0128] Dosages for humans can be initially determined by extrapolating the amount of compound used in mice, as those skilled in the art will recognize that modifying dosages for humans compared to animal models is routine. In certain embodiments, it is expected that dosages can include effective amounts between about 1 microgram / kg / body weight, 5 micrograms / kg / body weight, 10 micrograms / kg / body weight, 50 micrograms / kg / body weight, 100 micrograms / kg / body weight, 200 micrograms / kg / body weight, 350 micrograms / kg / body weight, 500 micrograms / kg / body weight, 1 milligram / kg / body weight, 5 milligrams / kg / body weight, 10 milligrams / kg / body weight, 50 milligrams / kg / body weight, 100 milligrams / kg / body weight, 200 milligrams / kg / body weight, 350 milligrams / kg / body weight, or 500 milligrams / kg / body weight to 1000 mg / kg / body weight or more per administration, and any range derivable therein. In other embodiments, an effective amount can be about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 mg / Kg of body weight. In other embodiments, it is contemplated that an effective amount may range from about 1 microgram of compound to about 100 mg of compound. In other embodiments, an effective amount may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg per single dose. In other embodiments, an effective amount includes less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 mg per day. In an exemplary embodiment, an effective amount includes less than about 50 mg per day.Of course, the single dose or daily dose can be adjusted upward or downward, as is routine in such treatment protocols, depending on the results of initial clinical trials and the needs of the particular subject. Those skilled in the art will recognize conditions and circumstances that warrant dosage modifications.

[0129] The precise determination of what constitutes an effective amount will depend on factors unique to each subject, including the subject's size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by one of ordinary skill in the art from this disclosure and knowledge in the art.

[0130] Optionally, the methods of the present invention provide for administering the compositions of the present invention to an appropriate animal model to identify the dosage of the composition, the concentration of components therein, and the timing of administering the composition that induces tissue repair, reduces cell death, or induces another desired biological response. Such determinations are routine and can be ascertained without undue experimentation.

[0131] Biologically active agents can be conveniently provided to a subject as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which may be buffered to a selected pH. The cells and agents of the present invention can be provided as a liquid or viscous formulation. For some applications, a liquid formulation is desirable because it is convenient for administration, particularly by injection. When prolonged contact with tissue is desired, a viscous composition may be preferred. Such compositions are formulated within an appropriate viscosity range. Liquid or viscous compositions can contain a carrier, which can be a solvent or dispersion medium, such as water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0132] Sterile injectable solutions are prepared by suspending talampanel and / or perampanel in the required amount of an appropriate solvent with various amounts of other ingredients, as desired. Such compositions may be mixed with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, dextrose, or the like. The compositions may also be lyophilized. Depending on the desired route of administration and preparation, the compositions may contain auxiliary substances, such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, coloring agents, and the like. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th edition, 1985, incorporated herein by reference, can be consulted to prepare suitable preparations without undue experimentation.

[0133] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of injectable pharmaceutical forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the present invention, it is considered that any vehicle, diluent, or additive used must be compatible with the agents present in cells or their conditioned medium.

[0134] The composition can be isotonic, i.e., the composition can have the same osmotic pressure as blood and tear fluid.The desired isotonicity of the composition of the present invention can be achieved by using sodium chloride or other pharmaceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes.For the buffer containing sodium ions, sodium chloride is particularly preferred.

[0135] If desired, the viscosity of the composition can be maintained at a selected level by using a pharmaceutically acceptable thickener, such as methylcellulose.Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc.The selection of suitable carriers and other additives will depend on the exact route of administration and the specific dosage form, for example, the nature of the liquid dosage form (for example, whether the composition is formulated into a solution, suspension, gel, or other liquid form, for example, sustained release form or liquid-filled form).Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert.

[0136] It is understood that the methods and compositions contemplated as useful in this invention are not limited to the particular formulations described in the examples. The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, expansion and culture methods, and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0137] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of those in the art. Such techniques are fully explained in the literature, e.g., "Molecular Cloning: A Laboratory Manual," second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and therefore may be considered in making and practicing the invention. Techniques that are particularly useful for particular embodiments are discussed in the following sections.

[0138] Wherever values and ranges are provided herein, it should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, all values and ranges encompassed within these values and ranges are meant to be encompassed within the scope of the invention. Moreover, all values falling within these ranges and upper or lower limits of value ranges are also contemplated by this application. The description of a range should be considered to specifically disclose not only individual numbers within that range, and, where appropriate, partial integers of numerical values within the range, but also all possible subranges. For example, the description of a range such as 1 to 6 should be considered to specifically disclose not only individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6, but also subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This applies regardless of the breadth of the range.

[0139] The following examples further illustrate aspects of the present invention, but are in no way a limitation of the teachings or disclosure of the present invention set forth herein. [Example]

[0140] Experimental Example The present invention will be further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless so specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0141] Without further description, it is believed that one of ordinary skill in the art can, using the foregoing description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0142] material and method Design and synthesis of double-stranded RNA and preparation of "naked" dsRNA Approximately 19-29 nucleotide (nt) guide and passenger strand RNAs for double-stranded siRNA duplexes were designed from the mature (spliced) complete target mRNA sequence using siDesign (sidirect2.rnai.jp / ) according to the parameters outlined in Amarzguioui, M et al., Biochem Biophys Res Commun 316, 1050-1058, (2004). PMID: 15044091; Reynolds, A. et al., Nature biotechnology 22, 326-330, (2004). PMID: 14758366; Ui-Tei, K. et al., Nucleic Acids Res 32, 936-948, (2004). PMID: 14769950; PMCID: PMC373388, using a minimum score of 14. Therapy 23, 73-82, (2016). Using the scoring method of PMID: 26987292, the best candidates were selected from the highest-scoring candidates. Candidate sequences were designed by software to incorporate a 2-nt overhang at each 3' end of the duplex. The double-stranded miRNA mimic was designed as the full-length sequence of the mature miRNA as the guide strand, and the passenger strand was the exact reverse complement sequence truncated by the first two or three 5' nt to generate a 3' overhang in the guide strand, and diuridine (UU) was added to the 3' end to create a UU overhang in the duplex. Dithymidine (TT) can also be substituted for UU. The sequences were then modified according to the following rules: 1) the 2-nt 3'-overhang sequence in the passenger strand for siRNA was replaced with diuridine (UU) (for miRNA mimics, UU was added to the passenger strand sequence as described above); 2) the 2- or 3-nt 3'-overhang of the guide strand was converted to the corresponding locked nucleic acid (LNA). (Note that the 3'-terminal nt in the guide strand must be converted to the corresponding LNA, while the others do not need to be converted, but are preferably converted.)Furthermore, U residues generally cannot be LNA, so whenever a U appears in an overhang, it is considered not to be LNA. Furthermore, the nt added to the 3' end could never be U, since the design rules for sequence selection force either G or C at the terminal position; 3) the first 5'-terminal nt in the passenger strand was converted to the corresponding LNA; 4) adenosines (As) in the passenger strand were converted to the corresponding 2'-oxymethyl (2'-OMe)An nts, with a minimum of five 2'-OMeAn nts, distributed as evenly as possible throughout the passenger strand sequence. When A nts were limited, U nts were converted to 2'-OMe U nts to obtain a total of at least five 2'-OMe A or U nts; 5) the second U in the diuridine (UU) overhang at the 3' end of the passenger strand was also converted to 2'-OMe U; 6) in addition to the 5'-terminal LNA nt in the passenger strand (which could not be U), five C, G, or A nts in the passenger strand were converted to the corresponding LNA nts, distributed as evenly as possible throughout the passenger strand sequence. siRNAs and miRNA mimics with the above modifications were synthesized, annealed as duplexes, purified, and validated by standard oligonucleotide synthesis. Purified RNA duplexes were suspended in a buffer consisting of sterile deoxygenated / deionized water, aliquoted, and stored at -20°C or -80°C for up to one year. Freeze / thaw cycles are not recommended, but are unlikely to substantially reduce efficiency if kept to a minimum. For multiple treatments as needed for a particular indication, the RNA duplexes can be combined by mixing the suspensions.

[0143] In vivo transfection Naked dsRNA duplex in sterile liquid suspension is administered into the blood circulation of the subject at an empirically determined concentration.Administration can be via any route that reaches peripheral blood circulation, including but not limited to intravenous or intraarterial, via needle, catheter, or any other direct administration route to blood vasculature, or via indirect route, such as intramuscular, subdermal, direct or transdermal application absorbed into patch, intraocular, intranasal, or intraperitoneal.Oral administration can also be feasible.Dosage and frequency of administration depend on target gene and specific indication.Typically, naked dsRNA is given at approximately 0.2mg / kg per day, but this can vary.The overall duration of administration also varies depending on indication.

[0144] Ex vivo and in vitro transfection Naked dsRNA duplexes in sterile liquid suspension were added directly to stored platelet concentrates or in vitro cultured megakaryocytes or platelets at empirically determined concentrations for each desired target mRNA(s). Concentrations typically range from approximately 100 to 1000 nM. Transfection typically begins at or near the time of collection of platelet concentrates from the donor or at or near the time of megakaryocyte or platelet culture, but may be initiated at any time based on the indication and can be repeated as frequently as once per day throughout the duration of storage, prior to transfusion or cell culture, as needed for the indication.

[0145] Table 1 shows target genes and RNA oligonucleotide sequences for targeting these genes. TIFF2025525584000001.tif152161TIFF2025525584000002.tif227161TIFF20255255840 00003.tif223161TIFF2025525584000004.tif223161TIFF2025525584000005.tif205161 Sequence Guide: Bold italics = locked nucleic acid (LNA) modifications Bold = 2'-OMe nucleotides or 2'-F modifications All sequences are 5'→3'

[0146] Evaluation of the therapeutic window of P2ry12 siLNA and ticagrelor in mice The therapeutic window (TW) was assessed in age-matched wild-type C57Bl6 / J mice as the gap between antithrombotic and hemorrhagic effects across a 3-log dose range of either ticagrelor or P2ry12 siLNA (gene name, P2ry12; protein name, P2y12) after intravenous administration. A scrambled siLNA lacking a predicted human mRNA target was used as a control. Thrombosis was assessed using FeCl3 intimal injury to the carotid artery and Doppler monitoring of blood flow downstream from the injury site, and hemorrhage was assessed by tail clip amputation, blood collection, and weight measurement to measure blood loss. Tests were performed in anesthetized mice 5 minutes after ticagrelor administration and 24 hours after siLNA administration.

[0147] thrombosis: The left carotid artery was exposed, cleaned of excess tissue, and placed on a Doppler probe connected to a blood flow monitor. Injury was induced by lifting the artery (temporarily occluding blood flow) and placing a 2 x 2 mm square piece of filter paper soaked in 4 μL of 7.5% FeCl3 on the exposed carotid artery for 90 seconds. Blood flow was monitored for 20 minutes after removal of the filter paper. In a typical experimental situation, without antithrombotic treatment, an occlusive clot forms (thrombosis) within approximately 9–11 minutes after vascular injury, causing downstream blood flow blockage. In some cases, treatments that only moderately reduce clot growth may be observed as incomplete restoration of blood flow through the narrowed vessel. Thrombosis is represented by a square as the percentage of blood flow blockage at the 20-minute endpoint compared to the initial flow velocity, calculated from the average flow velocity over the initial and final 100 seconds.

[0148] bleeding:A new razor blade was used to amputate a 1 mm diameter tip of the mouse tail, measured with a caliper, and the tail was immersed in warm saline for 20 minutes. The increase in weight of the saline tube after the assay gave the weight of blood loss during the assay time frame. Inhibition of hemostasis, indicated by a circle, was calculated as weight of blood loss / initial mouse body weight (wt, g / g) and expressed as the observed fold change relative to the mean weight of blood loss / mouse body weight of the vehicle control (n=18).

[0149] The therapeutic window (TW) was calculated as the ratio of the dose resulting in a 3-fold increase in blood loss compared to control (inhibition of hemostasis, blue dotted line) to the dose resulting in 50% blood flow recovery (thrombosis, red dashed line). Results are shown ± standard error of the mean. n = 3–8, respectively. ns, not significantly different from control.

[0150] Example 1: Naked double-stranded miRNA mimics selectively transfect bone marrow megakaryocytes in vitro Freshly extracted mouse bone marrow cell suspensions from femurs were incubated, without washing (e.g., with existing plasma and RNases, modeling in vivo conditions), with the indicated concentrations of AF488 (green)-conjugated miL-223-3p ds mimics for 1 hour, and then imaged live or fixed and processed using poly- L Live, unfixed whole bone marrow cell suspensions captured on polylysine (PLL)-coated slides were imaged for AF488 (Figure 1A). Only megakaryocytes (MKs) exhibited green fluorescence, indicating MK-specific uptake. Fixed bone marrow cells were counterstained for the MK / platelet marker Cd41 and DAPI for nuclei (Figure 1B). Only MKs, not other bone marrow cells, exhibited miLNA uptake. In this experiment, bone marrow cells were co-incubated with both miLNA-AF488 and the unmodified version of miR-223-3p conjugated to AlexaFluor 647. The bottom panel shows no fluorescence, indicating degradation of the unmodified miRNA mimic. Similar cells (Figure 1C) were counterstained for two MK / platelet markers, Cd42d and Cd41, indicating uptake by MKs at different stages of development, including immature developmental stages (small MKs).

[0151] Example 2: Injected naked double-stranded miRNA mimics selectively transfect bone marrow megakaryocytes and platelets in vivo As in (Figure 1A), miL-223-3p conjugated to AlexaFluor488 was injected into the tail vein of WT mice. Bone marrow cell suspensions were extracted and processed as in (Figures 1A-1C). Whole bone marrow cell suspensions were extracted 2 h postinjection, captured on poly-L-lysine (PLL)-coated slides, and imaged live for AF488 (Figure 2A). Only megakaryocytes (MKs) exhibited green fluorescence, indicating MK-specific in vivo uptake. Fixed bone marrow cells were counterstained for MK / platelet markers Cd42d and Cd41 and DAPI for nuclei (Figure 2B). At 18 and 72 h postinjection, only MKs and platelets, but not other bone marrow cells, exhibited miLNA uptake. Similar cells as in (Figure 2B) were analyzed for miL uptake (by fluorescence) by flow cytometry, using Cd41 / Cd42d and size (forward scatter FSC) to identify MKs (Figure 2C). In this experiment, spleens were also extracted, and splenocyte suspensions were analyzed at 72 hours. Combined, these results demonstrated robust uptake and retention of miLNAs by MKs in bone marrow and splenic MKs. Peripheral blood cells from the same animals were fixed, captured on glass coverslips, and platelets were identified with the Cd42d antibody. The in vivo uptake efficiency of miLNAs by platelets approached 100%, similar to what was previously observed with washed in vitro platelets (Figure 2D).

[0152] Example 3: Injected naked RhoA siLNA selectively suppresses RhoA protein expression in bone marrow megakaryocytes and modulates megakaryocyte development and platelet production in vivo These experiments demonstrate the effect of injected naked siLNAs on target gene expression and cellular function in megakaryocytes (MKs). RhoA or Cel-miL-67 siLNAs were injected into WT mice as previously detailed. Peripheral blood samples were collected daily to assess blood cell counts, and bone marrow was extracted 96 hours after the start of the experiment. Cel-miL-67 was used as a negative control. RhoA siLNAs selectively suppress RhoA protein expression in bone marrow MKs after injection (Figure 3A). RhoA levels were measured by flow cytometry in permeabilized whole bone marrow cell suspensions using a RhoA antibody in addition to a Cd42d antibody to label MKs and propidium iodide (PI) to label nuclear DNA. (Left panel) RhoA expression levels in MKs as a function of ploidy class, as determined by PI staining. As expected, RhoA expression increased in larger, more mature MKs due to cytoplasmic expansion, but this increase was suppressed by RhoA siLNAs. (Right panel) Nucleated (PI+) non-MK bone marrow cells showed no change in RhoA protein levels. RhoA siLNA modulates RhoA-dependent MK function (Figure 3B). RhoA knockout mice and mice treated with a RhoA inhibitor demonstrated that acute RhoA inhibition led to an increase in MK ploidy. RhoA siLNA caused an increase in MK ploidy compared with control siLNA, demonstrating the specific functional effect of RhoA inhibition on MK in vivo. Figure 3C shows an increase in peripheral blood platelet counts by RhoA siLNA, which corresponds to the increase in MK ploidy (Figure 3B). The lack of change in mean platelet volume by RhoA siLNA confirmed that increasing MK ploidy leads to a higher platelet count but not to the generation of larger platelets (Figure 3D).

[0153] Example 4: Injected naked P2y12 (P2ry12) siLNA reduces platelet ADP reactivity and prevents injury-induced arterial thrombosis in WT mice, mimicking current first-line antiplatelet drugs that target P2Y12 (e.g., Plavix, Cangrelor, Ticagrelor). Figures 4A-4B show that infused naked P2y12 (P2ry12) siLNA reduces platelet ADP activation response and prevents injury-induced arterial thrombosis in WT mice, mimicking current first-line antiplatelet drugs that target P2Y12. (Figure 4A) Infusion of P2ry12 siLNA reprograms platelets with reduced ADP responsiveness. Platelet ADP responses were examined by flow cytometry (FACS) in whole blood cell suspensions 24 h after infusion of P2ry12 or control scrambled (CTL) siLNA for ADP-induced GpIIb / IIIa integrin activation using JonA antibody (upper panel) and alpha granule secretion using P-selectin antibody (lower panel). *p<0.05, n=3. (Figure 4B) WT mice were infused daily for 4 days via tail vein injection with either saline (control) or 0.2 mg / kg of naked P2ry12 siLNA. At 96 hours (day 5), anesthetized mice were subjected to arterial injury ("injury") to the carotid artery by applying filter paper soaked in 4 μL of 7.5% FeCl3 solution for 30 seconds. The paper was then removed and blood flow was restored. Blood flow was monitored with a Doppler probe downstream of the injury site for up to 30 minutes after injury.

[0154] As expected, in control mice, arteries became occluded and blood flow was blocked 5–12 minutes after injury, as shown in Figure 4B. In contrast, arteries in mice treated with naked P2ry12 siLNA (which targets the P2y12 ADP receptor, the target of current first-line antiplatelet and antithrombotic drugs) remained patent, and no occlusion was observed over the experimental time frame (n=3 each).

[0155] Example 5: A single injection of naked miL-223-3p miRNA mimic transiently and potently increases platelet ADP responsiveness These experiments demonstrate the effects of infused naked miLNAs on circulating platelets. miR-223-3p (miL-223-3p) was used because this miRNA was found to target key negative regulators of the platelet response to ADP agonists; importantly, these targets must be rapidly turned over and constitutively translated in platelets. We hypothesized that overexpression of miR-223-3p with miL-223-3p would lead to direct platelet effects independent of the MK effect by suppressing its target negative regulator proteins in platelets.

[0156] As shown in Figure 5A, injection of miL-223-3p led to a fourfold average increase in platelet ADP reactivity assessed 24 hours postinjection, as measured by flow cytometry with the Jon / A antibody, which recognizes the activated form of integrin GpIIb / IIIa; however, reactivity was unaffected by control Cel-miL-67 or saline infusion. The increased reactivity was transient, indicating consumption of the targeting miLNA and subsequent reconstitution of the target protein. As shown in Figure 5B, the entire population of circulating platelets exhibited a rightward shift in ADP reactivity. This result suggests a strong direct effect on circulating platelets, although an effect on MKs cannot be excluded.

[0157] Example 6: Single or repeated addition of naked ADAM17 siRNA to platelets in storage concentrates prevents loss of GP1bα For initial studies, ADAM17 or control (Cel-miR-67) siRNA (unmodified; the Cel-miR-67 sequence is identical to the Cel-miR-67 sequence used elsewhere) was obtained from Dharmacon and added to freshly isolated, washed human platelets for 1 hour (as previously reported). Platelet-rich plasma was then reconstituted with stored autologous plasma to generate platelet-rich plasma, which was then maintained at room temperature with gentle agitation for up to 96 hours, reflecting typical storage conditions for platelet concentrates for transfusion recipients. The ADAM17 metalloproteinase is known to cause progressive proteolytic cleavage of the GP1bα vWF receptor on platelets, leading to reduced hemostatic function of stored platelets, a key component of impaired platelet storage. We hypothesized that ADAM17 siRNA would prevent this degradation. Note: Platelet counts remained comparable over the experimental time frame (results not shown). Figure 6A shows that a single addition of naked ADAM17 siRNA directly to platelet storage concentrates consistently maintained global and surface expression of GP1bα in stored platelets for up to 48 hours. After 48 hours, GP1bα levels fell to low levels similar to those of control siRNA, indicating turnover of ADAM17 and GP1bα. Figure 6B shows that multiple additions of ADAM17 siRNA to stored platelet concentrates (by sedimenting, rinsing, and repeating the transfection of platelets, followed by reconstitution with stored plasma), given at 0 hours and again at 48 hours, resulted in long-term maintenance of GP1bα in stored platelets. Based on these data, we designed and produced an ADAM17 siLNA (Figure 13).

[0158] Example 7: A single addition of naked ADAM17 siLNA to platelet storage concentrates prevents loss of GP1bα function ADAM17 or control (Cel-miL-67) siLNA (modified as shown in Figure 13) was added directly to freshly isolated human platelet-rich plasma, which was then kept at room temperature with gentle agitation for up to 96 hours, reflecting typical storage conditions for platelet concentrates for transfusion recipients.

[0159] At the indicated times, a fraction of the platelet-rich plasma was transferred to an aggregometer at 37°C, and 0.5, 1.0, or 1.5 mg / mL of the GP1bα cofactor ristocetin (Rs) was added to crosslink plasma vWF, thereby initiating GP1b-dependent platelet aggregation, which was monitored over time by light transmission. Another fraction was separated for Western blot to assess the expression level of GP1bα in platelets.

[0160] Figure 7A shows percent platelet aggregation (clumping) over time after the addition of the indicated concentrations of ristocetin (Rs). A single addition of naked ADAM17 siLNA directly to the platelet storage concentrate on day 0 maintained functional GP1bα reactivity over the 5-day experimental time frame. These data provide the first demonstration of this approach for improving cellular physiology in stored platelets. Figure 7B, Expression of GP1bα in stored platelets. These results support the use of naked siLNA to modulate platelet storage disorders. It is predicted that precisely timed addition of key targeting siLNAs, timed for maximal hemostatic efficacy in transfusion recipients, will result in platelets with a reactivity profile similar to freshly isolated platelets.

[0161] Example 8: Therapeutic window (TW) of P2ry12 siLNA and ticagrelor in mice In the first series of experiments, we tested siLNAs against mouse P2y12 (mRNA, P2ry12; human P2Y12), the primary ADP receptor on platelets (also expressed on several other cells). In response to other stimuli, partially stimulated platelets secrete ADP, which then stimulates a secondary response via P2Y12, leading to so-called "secondary" feedback stimulation through autocrine and paracrine signaling. The ADP-driven P2Y12 secondary activation response is a critical turning point for excessive clot growth leading to thrombosis. Current antiplatelet SOC non-aspirin monotherapy is primarily centered on P2Y12 inhibitors (clopidogrel, prasugrel, cangrelor, and ticagrelor). Dual antiplatelet therapy typically includes a P2Y12 inhibitor and aspirin. While beneficial and generally safe, aspirin has not resolved clinical thrombosis and is currently being used less frequently, especially in elderly patients, where adverse effects are often present. The unmet need addressed herein is the well-documented and unresolved induction of clinical bleeding in patients receiving P2Y12 mono- or dual therapy.

[0162] The data in Figure 15 represent a test of the therapeutic window for P2y12 siLNA compared to the P2Y12 antagonist ticagrelor in a mouse model, i.e., a safe dose range that provides antithrombotic effects (therapeutic benefit) but does not cause excessive bleeding. Like all siLNAs, P2y12 siLNA is designed to function as an siRNA once internalized in platelets and / or megakaryocytes. The function of siRNA is to specifically bind to target mRNA (in this case, P2y12) and prevent translation of the cognate protein (P2y12) via the cell's native RNA-induced silencing mechanism.

[0163] As can be seen in Figure 15, the TW for ticagrelor was determined to be 6.8, while the TW for P2ry12 siLNA was determined to be >100. Therefore, the TW ratio of P2ry12 siLNA / ticagrelor is >14-fold. The TW observed for ticagrelor in mice closely matches the TW previously established in rats using a similar approach and reflects clinical findings in humans. The actual upper limit of the TW for P2ry12 siLNA remains unclear, as no increase in bleeding was observed at the highest dose tested. Furthermore, lower doses of 1-10 μg / kg may also provide therapeutic benefit (inhibition of thrombosis). As a control, thrombosis was evaluated in mice injected with scrambled siLNA at the highest dose tested to date (0.5 mg / kg). Unlike P2ry12 siLNA, the scrambled control siLNA had no protective effect against thrombosis, as occlusion was observed in all mice receiving scrambled siLNA, as well as in untreated mice subjected to thrombotic insult.

[0164] Example 9: P2ry12 siLNA destabilizes thrombi and prevents thrombosis in a manner dependent on the surface density of P2y12 Twenty-four hours after a single IV injection of a series of P2y12 or scrambled siLNAs, we examined the inhibition of thrombosis as a function of P2y12 surface expression, assessed by flow cytometry using a fluorophore-conjugated P2y12 antibody. Similar to Figure 17, a threshold level was observed for siLNA-mediated P2y12 inhibition to destabilize thrombi and result in an antithrombotic effect. Approximately 20% P2y12 inhibition was sufficient to destabilize thrombi and restore blood flow, while approximately 35-40% inhibition appeared sufficient to prevent the formation of any blockages despite vascular injury.

[0165] Example 10: P2ry12 siLNA selectively normalizes high ADP responsiveness and P2y12 levels in newly generated platelets Platelet reactivity was monitored as a function of in vivo platelet lifespan. As shown in Figure 18, newly generated platelets are hyperresponsive to ADP (the physiological ligand for the P2y12 receptor) and, to a lesser extent, thromboxane (another "secondary" agonist, blocked by aspirin) compared to the average reactivity of the entire population, whereas responsiveness to thrombin and convulxin (the main "primary" activating agonists) is similar throughout lifespan. The surface density of P2y12 is also highest on new platelets compared to the entire population (Figure 18, right), and this high level of P2y12 was found to decline over time, indicating synthesis of P2y12 primarily in megakaryocytes and / or newly generated platelets. P2y12 siLNA normalized this increase in the high-expressing subpopulation but did not affect existing P2y12 levels, as expected for siRNA-mediated gene silencing. Thus, P2ry12 siLNAs reduce thrombosis by disproportionately suppressing elevated P2y12 levels in young platelets and normalizing ADP responsiveness, whereas older platelets with reduced P2y12 synthesis are refractory to siLNA-mediated silencing because pre-existing P2y12 protein is not targeted, thereby maintaining hemostasis.

[0166] Using pulse-chase labeling with a single injection of Gp1bβ-X488 antibody, we monitored (Figure 18, left) reactivity and (Figure 18, right) P2y12 surface expression in ex vivo mouse platelets throughout their lifespan. Gp1bβ antibodies conjugated to fluorophores were injected into the bloodstream for flow cytometric tracking, where they specifically label platelets but not other cells. However, these antibodies are inactive and do not activate or destroy platelets. In vivo antibody labeling saturates within 1 hour. Therefore, when tracked by flow cytometry from blood samples, Gp1bβ+ platelets (fluorophore-positive, shown by the solid line) represent the entire platelet population at the time of pulse labeling. When platelets are extracted at subsequent time points, in this case at 24-hour intervals after pulse labeling, Gp1bβ- platelets (fluorophore-negative, shown by the dotted line) represent newly generated platelets that were not subjected to pulse labeling at the "0" time point. Over time, as previously present platelets are gradually cleared (murine platelets have a lifespan of approximately 5 days), these Gp1bβ-platelets comprise an increasingly larger percentage of the total platelet pool, as evidenced by the convergence of the red and blue lines at later time points.

[0167] In Figure 18, results are shown as two independent measures of platelet activation: relative integrin activation (top row) and α-granule secretion (bottom row), or P2y12 levels in new (Gp1bβ-, dotted line) and existing (Gp1bβ+, solid line) platelets compared to the total population. Top right, control; bottom right, 24 hours after injection of P2y12 siLNA. All ± standard error of the mean. n = 3–6.

[0168] Example 11: PAR4 as a second (new) siLNA target in platelets PAR4 is another platelet GPCR important for platelet activation. It is one of two major thrombin receptors on human platelets, and thrombin is the primary agonist for so-called "primary" platelet activation, which initiates clot formation. Mouse platelets have only Par4 as their sole thrombin receptor. PAR4 small molecule antagonists have been developed as putative antithrombotic drugs. It has been investigated whether knockdown of PAR4 could be beneficial as an alternative antithrombotic target to P2Y12, both as a single target and potentially in combination with P2Y12 antagonists or P2Y12 siLNAs. Unlike P2Y12 / P2Y12, mouse and human Par4 / PAR4 genes are not sufficiently similar, so clinically relevant functional studies are best focused on the human gene, if possible. Therefore, we developed humanized PAR4 mice, which carry a human PAR4 transgene, including all surrounding regulatory sequences, deleted for mouse PAR4, and backcrossed them for several generations onto a WT C57Bl / 6J background. Platelet PAR4 has been previously studied, demonstrating that the human PAR4 transgene, expressed in mouse platelets at levels similar to those in human platelets, supports PAR4 function in these mice within human platelet physiology. These mice, hereafter referred to as "hPAR4" mice, therefore provide a unique resource for testing the inhibition of human PAR4 using a designed and engineered human-targeting siLNA (gene and siLNA name, F2RL3). IV injection of F2RL3 siLNA into these mice resulted in substantial inhibition of human PAR4 in their platelets within 24 hours.

[0169] Silencing platelet hPAR4 modestly reduced platelet responsiveness to a receptor-specific peptide agonist known as PAR4-activating peptide or PAR4-AP, which stimulates neither other PARs nor receptors.

[0170] Because PAR4 is the only reactive thrombin receptor on these platelets, it was predicted that moderate inhibition of PAR4 would be sufficient to achieve an antithrombotic effect. Similar to Figure 20, IV injection of naked F2RL3 siLNA at the same dose that resulted in a moderate reduction in platelet reactivity to PAR4 agonist stimulation was sufficient to prevent thrombosis in the FeCl3 carotid artery injury model at 24 h. However, despite the antithrombotic effect of F2RL3 siLNA, achieved at 500 μg / kg, the highest dose used to test P2y12 siLNA to date, no increase in bleeding was observed in these mice compared with control hPAR4 or WT mice. The full therapeutic window of F2RL3 siLNA remains to be determined. These PAR4 data indicate that multiple siLNAs targeting distinct genes (mRNAs) are functional, as indicated by reduced target protein expression and the expected functional effects.

[0171] Example 12: siLNA targeting of non-receptor cytosolic proteins and antihemorrhagic / prohemostatic effects Gsα is a cytosolic small G protein involved in platelet signaling. Although P2y12 and PAR4 are distinct genes, their protein products are both members of the G protein-coupled receptor family. These data for Gsα in Figure 22 demonstrate that siLNA silences the expression of various types of protein targets. Notably, Gsα protein also exhibits different expression kinetics in circulating platelets compared with P2y12. Gsα appears to be most expressed in older platelets. Thus, siLNA normalizes target protein expression across the entire platelet population in a target-specific manner. Furthermore, the data in Figure 22 demonstrate the antihemorrhagic / prohemostatic in vivo effects of siLNA-mediated inhibition of endogenous negative regulatory proteins in platelets, such as Gsα (siLNA, Gnas) and IP (siLNA, Ptgir). The data in Figure 22 further demonstrate the rescue of acquired bleeding diathesis by Gnas / Ptgir combination siLNA in mice, which were briefly treated with a medium dose of ticagrelor, which caused increased bleeding, and the increased bleeding was rescued by these siLNAs.

[0172] Example 13: Platelet- and megakaryocyte-specific uptake of miLNA / siLNA demonstrated by PCR : As an orthogonal approach to examine the megakaryocyte / platelet-selective in vivo uptake of naked mi / siLNA, we used miR-223 knockout (KO) mice to monitor the internalization of ectopically injected naked miL-223-3p by PCR in isolated cell populations subjected to FACSort analysis using specific markers. miR-223-3p was detected in all examined BM and blood cells of WT mice but, as expected, was absent in cells from miR-223 KO mice. Upon injection of miL-223-3p into miR-223 KO mice, miR-223-3p was observed to be present in platelets and bone marrow megakaryocytes, but the injected miLNA was not detected in either blood or bone marrow white blood cells / leukocytes (WBCs, which include CD45+ WBC progenitors). All of these populations were single-cell sorted based on surface markers (and size for megakaryocytes), eliminating heterotypic cell aggregates. These results demonstrate that only megakaryocytes and platelets, but not other cells in hematopoietic tissues or blood, possess the unique ability to internalize infused naked double-stranded miLNAs.

[0173] Example 14: Broad Blood Cell Targeting with Anti-miRs In contrast to the platelet / megakaryocyte-specific in vivo targeting by double-stranded miLNAs and siLNAs, an eight-nucleotide single-stranded anti-miR (anti-miR-223-3p), composed of locked nucleic acids (LNAs) modeling the McKenzie / Tsygankov anti-miR-148a and bearing a 3'-FAM fluorophore (anti-miR), was found to be internalized by platelets in vivo within 1 h of intravenous administration in WT mice, as well as by red blood cells (RBCs) and white blood cells / white blood cells (WBCs), as shown in Figure 25. Notably, RBCs have substantially larger volumes than platelets, and WBCs are much larger than RBCs; these larger volumes compared to platelets explain the observed apparently lower relative mean fluorescence intensity (per cell). Thus, all blood cells exhibit a similar ability to internalize anti-miRs modeled on anti-miR-148a. Therefore, anti-miRs / antagomirs do not have the unique property of platelet / megakaryocyte-specific internalization and utilization that miLNAs and siLNAs have.

[0174] Example 15: Long-term modulation of platelet reactivity by siLNA using subdermal osmotic pumps As shown in Figure 26, modulation of platelet reactivity can be achieved by subdermal administration, providing an alternative route to parenteral administration. Platelet ADP responses were suppressed in mice with subdermal osmotic pumps constantly releasing P2ry12 siLNA, demonstrating subdermal administration of siLNA to deliver the siLNA payload to the bloodstream and platelets, and for modulation of platelet function.

[0175] List of aspects: The following exemplary aspects are provided, the numbering of which should not be construed as designating levels. Aspect 1 provides the following: 1. A method for selectively modulating gene expression of one or more target genes in a cell of a subject, comprising: administering to said subject a therapeutically effective amount of a composition comprising a naked RNA oligonucleotide. Including, The cells are at least one selected from the group consisting of megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide. method. Aspect 2 provides the following: The method of embodiment 1, wherein said naked RNA oligonucleotide comprises a guide strand having a sequence selected from the guide strand sequences listed in Table 1 and a passenger strand having a sequence selected from the passenger strand sequences listed in Table 1. Aspect 3 provides the following: The method of embodiment 1 or 2, wherein the naked RNA oligonucleotide is selected from the group consisting of miRNA, siRNA, and any combination thereof. Aspect 4 provides the following: The method of any one of embodiments 1 to 3, wherein the naked RNA oligonucleotide does not comprise any modified nucleotides / bases. Aspect 5 provides the following: The method of any one of embodiments 1 to 4, wherein the transfection is performed without the use of synthetic carriers or adjuvants. Aspect 6 provides the following: The method of embodiments 1 to 5, wherein said naked RNA oligonucleotide optionally comprises at least one modification selected from the group consisting of locked nucleic acids (LNAs), 2'-fluorine bases, and 2'-O-methylated bases. Aspect 7 provides the following: The method of any one of embodiments 1 to 6, wherein the naked RNA oligonucleotide is thermostable. Aspect 8 provides the following: The method of any one of embodiments 1 to 7, wherein the naked RNA oligonucleotide is non-immunogenic. Aspect 9 provides the following: The method of any one of embodiments 1 to 8, wherein the naked RNA oligonucleotide is resistant to cleavage by a riboendonuclease (RNAase). Aspect 10 provides the following: The method of any one of embodiments 1 to 9, wherein the composition is administered intravenously. Aspect 11 provides the following: The method of any one of embodiments 1 to 10, wherein said administering alters megakaryocyte development, platelet production, megakaryocyte function, and / or platelet function. Aspect 12 provides the following: The subject is: i. antiplatelet therapy, ii. anti-inflammatory therapy to treat thromboinflammation; iii. Treatment of thrombocytopenia, thrombocytosis, and other pathological conditions related to disrupted megakaryocyte development; iv. Platelet transfusions, where the subject requires protection from a platelet storage disorder or where the subject requires a transfusion due to a platelet disorder. v. Treatment of thrombosis, vi. Treatment of acquired bleeding disorders, and vii. Treatment of Inherited Bleeding Disorders The method of any one of embodiments 1 to 11, further comprising at least one selected from the group consisting of: Aspect 13 provides the following: The method of any one of embodiments 1 to 12, wherein the composition comprises saline. Aspect 14 provides the following: The method of any one of embodiments 1 to 13, wherein the subject is a mammal. Aspect 15 provides the following: The method of any one of aspects 1 to 14, wherein the mammal is a human. Aspect 16 provides the following: 1. A method for selectively modulating gene expression of one or more target genes in a cell, ex vivo or in vitro, comprising: transfecting said cells with naked RNA oligonucleotides. Including, The cells are at least one selected from megakaryocytes and platelets. method. Aspect 17 provides the following: 17. The method of embodiment 16, wherein said naked RNA oligonucleotide comprises a guide strand having a sequence selected from the guide strand sequences listed in Table 1 and a passenger strand having a sequence selected from the passenger strand sequences listed in Table 1. Aspect 18 provides the following: The method of embodiments 16 to 17, wherein the cells are derived from at least one selected from the group consisting of a cell culture system, a concentrate, a cell suspension, a tissue homogenate, an organoid, a tissue, and an organ. Aspect 19 provides the following: The method of any one of aspects 16 to 18, wherein the cell culture is a stem cell culture. Aspect 20 provides the following: 20. The method of any one of embodiments 16 to 19, wherein the concentrate is a platelet storage concentrate comprising autologous plasma. Aspect 21 provides the following: The method of any one of embodiments 16 to 20, wherein the transfecting step comprises transfecting without the use of a synthetic carrier or adjuvant. Aspect 22 provides the following: 22. The method of any one of embodiments 16 to 21, wherein the naked RNA oligonucleotide is thermostable. Aspect 23 provides the following: 23. The method of any one of embodiments 16 to 22, wherein the naked RNA oligonucleotide is non-immunogenic. Aspect 24 provides the following: The method of any one of embodiments 16 to 23, wherein the naked RNA oligonucleotide is resistant to cleavage by a riboendonuclease (RNAase). Embodiment 25 provides the following: 1. A method for delivering naked RNA oligonucleotides to target tissues and / or cells in a subject, comprising: using platelets transfected with said naked RNA oligonucleotide as a vehicle for delivering said naked RNA oligonucleotide to said target tissue and / or said target cell. A method comprising: Embodiment 26 provides the following: 26. The method of embodiment 25, wherein said naked RNA oligonucleotide comprises a guide strand having a sequence selected from the guide strand sequences listed in Table 1 and a passenger strand having a sequence selected from the passenger strand sequences listed in Table 1. Aspect 27 provides the following: The method of any one of embodiments 25 to 26, wherein the target cells are selected from tumor cells, leukocytes, or inflammatory cells. Embodiment 28 provides the following: 28. The method of any one of embodiments 25 to 27, wherein the target tissue comprises endothelial cells. Aspect 29 provides the following: 1. A composition for selectively modulating gene expression of one or more target genes in a cell of a subject, comprising: Cells transfected with naked RNA oligonucleotides Including, The cells are at least one selected from the group consisting of megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide. composition. Aspect 30 provides the following: 30. The composition of embodiment 29, wherein said naked RNA oligonucleotide is suspended in aqueous sterile saline. Aspect 31 provides the following: 1. A composition for selectively modulating gene expression of one or more target genes in a cell, ex vivo or in vitro, comprising: Cells transfected with naked RNA oligonucleotides Including, The cells are at least one selected from the group consisting of megakaryocytes and platelets. composition. Aspect 32 provides the following: The composition of embodiment 31, wherein said cells are derived from a cell culture, a concentrate, a cell suspension, a tissue homogenate, an organoid, a tissue, and an organ. Aspect 33 provides the following: 1. A kit comprising a composition having a naked RNA oligonucleotide for selectively modulating the expression of at least one gene in a cell and instructions for its use, The cells are at least one selected from the group consisting of megakaryocytes, platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide. kit.

[0176] Other Aspects The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0177] The disclosures of any and all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety.

[0178] While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. A method for selectively modulating the gene expression of one or more target genes in target cells, A step of administering a therapeutically effective amount of a composition containing naked RNA oligonucleotides to the subject. Includes, The cell is at least one selected from the group consisting of megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide. method.

2. The method according to claim 1, wherein the naked RNA oligonucleotide comprises a guide strand having a sequence selected from the guide strand sequences listed in Table 1 and a passenger strand having a sequence selected from the passenger strand sequences listed in Table 1.

3. The method according to claim 1, wherein the naked RNA oligonucleotide is selected from the group consisting of miRNA, siRNA, and any combination thereof.

4. The method according to claim 1, wherein the naked RNA oligonucleotide contains no modified nucleotides / bases.

5. The method according to claim 1, wherein the naked RNA oligonucleotide optionally comprises at least one modification selected from the group consisting of locked nucleic acid (LNA), 2'-fluorinated bases, and 2'-O-methylated bases.

6. The method according to claim 1, wherein the composition is administered intravenously.

7. The method according to claim 1, wherein the administration step alters megakaryocyte development, platelet production, megakaryocyte function, and / or platelet function.

8. The aforementioned subjects are as follows: i. Antiplatelet therapy, ii. Anti-inflammatory therapy to treat thrombotic inflammation, iii. Treatment of other pathological symptoms relating to thrombocytopenia, thrombocytosis, and confused megakaryocyte development. iv. Platelet transfusions in patients who require protection from platelet preservation disorders or who require transfusion due to the aforementioned platelet disorders. v. Treatment of thrombosis, vi. Treatment of acquired hemorrhagic disorders, and vii. Management of hereditary bleeding disorders The method according to claim 1, which requires at least one selected from the group consisting of the following.

9. A method for selectively modulating the gene expression of one or more target genes in cells, either ex vivo or in vitro, The process of transfecting the cells with naked RNA oligonucleotides. Includes, The cell is at least one selected from megakaryocytes and platelets. method.

10. The method according to claim 9, wherein the naked RNA oligonucleotide comprises a guide strand having a sequence selected from the guide strand sequences listed in Table 1 and a passenger strand having a sequence selected from the passenger strand sequences listed in Table 1.

11. The method according to claim 9, wherein the cells are derived from at least one selected from the group consisting of cell culture systems, concentrates, cell suspensions, tissue homogenates, organoids, tissues, and organs.

12. The method according to claim 11, wherein the cells are derived from stem cell culture.

13. The method according to claim 11, wherein the cells are derived from a platelet-preserving concentrate containing autologous plasma.

14. The method according to claim 9, wherein the transfecting step includes transfecting without using a synthetic carrier or adjuvant.

15. The method according to claim 9, wherein the naked RNA oligonucleotide is resistant to cleavage by riboendonucleases (RNAases).

16. A method for delivering naked RNA oligonucleotides to target tissues and / or cells, The process of using platelets transfected with the naked RNA oligonucleotide as a vehicle for delivering the naked RNA oligonucleotide to the target tissue and / or target cells. Methods that include...

17. The method according to claim 16, wherein the naked RNA oligonucleotide comprises a guide strand having a sequence selected from the guide strand sequences listed in Table 1 and a passenger strand having a sequence selected from the passenger strand sequences listed in Table 1.

18. The method according to claim 16, wherein the target cells are selected from tumor cells, leukocytes, or inflammatory cells.

19. The method according to claim 16, wherein the target tissue includes endothelial cells.

20. A composition for selectively modulating the gene expression of one or more target genes in target cells, Cells transfected with naked RNA oligonucleotides Includes, The cell is at least one selected from the group consisting of megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide. composition.

21. The composition according to claim 20, wherein the naked RNA oligonucleotide is suspended in aqueous sterile saline solution.

22. A composition for selectively modulating the gene expression of one or more target genes in cells, ex vivo or in vitro, Cells transfected with naked RNA oligonucleotides Includes, The cell is at least one selected from the group consisting of megakaryocytes and platelets. composition.

23. The composition according to claim 22, wherein the cells are derived from a selection of the group consisting of cell culture systems, concentrates, cell suspensions, tissue homogenates, organoids, tissues, and organs.

24. A kit comprising a composition having a naked RNA oligonucleotide for selectively modulating the expression of at least one gene in a cell, and explanatory materials for its use, The cell is at least one selected from the group consisting of megakaryocytes, platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide. kit.

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