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

Abstract

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

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 391,002, filed on July 21, 2022; U.S. Provisional Application No. 63 / 439,365, filed on January 17, 2023; and U.S. Provisional Application No. 63 / 522,839, filed on June 23, 2023, under 35 U.S.C. § 119(e). All of these applications are hereby incorporated by reference in their entirety.

[0003] Sequence Listing Reference

[0004] The Sequence Listing, submitted herewith on July 19, 2023, and having a size of 107.6 kilobytes, is an xml file named "205961 - 7088WO1(00343)_Sequence Listing.xml", which is incorporated by reference herein in accordance with 37 C.F.R. § 1.52(e)(5).

[0005] Statement Regarding Federally Sponsored Research or Development

[0006] This invention was made with government support under Grant No. HL159006 awarded by the National Institutes of Health. The government has certain rights in the invention. Background of the Invention

[0007] Megakaryocytes (MKs) are large, nucleated cells that are mainly present in the bone marrow and spleen. They develop from progenitor cells and ultimately shed their large amount of cytoplasm to generate platelets. Each mature megakaryocyte produces 1000 - 2000 platelets and releases them into the blood, with a daily production rate in humans of approximately 10 11A new platelet. The main function of platelets is to seal blood vessel wounds. However, the growth of platelet plugs may become too large and occlude (block) blood vessels (thrombosis), which remains the leading cause of morbidity and mortality worldwide. In the absence of blood vessel trauma, platelet plugs can also form and stabilize under various conditions, such as atherosclerotic thrombosis. This effect is a fundamental driving force for adverse cardiac events, including ischemic stroke, myocardial infarction (heart attack), systemic arterial and venous thrombosis, and venous thromboembolism (including pulmonary embolism and deep vein thrombosis). Therefore, the balance of platelet reactivity is crucial for establishing and maintaining embolus formation and preventing bleeding (hemostasis) without causing thrombosis, and too low and too high platelet reactivity are the roots of hemostasis or thrombosis dysfunction. Megakaryocytes also directly and importantly participate in this balance in the production of platelets (thrombopoiesis): 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 for many disease states, including almost all types of cancer and many (if not most) inflammatory states, and these disease states together constitute a large number of morbid and fatal conditions.

[0008] Current standards of care for thrombosis include both anticoagulant methods and antiplatelet methods. The main antiplatelet drugs currently in widespread use, although generally much improved in recent decades, still carry a risk of clinically significant bleeding, including intracranial bleeding. In addition, dysfunction in the development of stem cells and progenitor cells into megakaryocytes can not only lead to thrombocytosis or thrombocytopenia and downstream clinical manifestations, but also to myelodysplasia, which may progress to hematological malignancies. Therefore, there is a significant gap in the clinical control of bleeding and coagulation and in the control of blood cell development, which poses an urgent and long-term need for improved drugs and treatment methods. The present invention addresses this unmet need. Summary of the Invention

[0009] In one aspect, the present invention provides a method for selectively regulating the 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 megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide.

[0010] In another aspect, the present invention includes a method for selectively regulating the gene expression of one or more target genes in cells in vitro or ex vivo, the method comprising transfecting the cells with a naked RNA oligonucleotide.

[0011] On the other hand, the present invention provides a method for delivering a naked RNA oligonucleotide to a target tissue and / or target cell of a subject, wherein the method comprises delivering the naked RNA oligonucleotide to the target tissue and / or target cell using platelets transfected with the naked RNA oligonucleotide as a vehicle. In certain embodiments, the target cell is a tumor cell, a leukocyte, or an inflammatory cell. In certain embodiments, the target tissue comprises endothelial cells.

[0012] On the other hand, the present invention provides a composition for selectively regulating the gene expression of one or more target genes in cells of a subject, wherein the composition is a cell transfected with a naked RNA oligonucleotide.

[0013] On the other hand, the present invention provides a composition for selectively regulating the gene expression of one or more target genes in a cell ex vivo or in vitro, wherein the composition comprises a naked RNA oligonucleotide within the cell.

[0014] On the other hand, the present invention provides a kit comprising a composition having a naked RNA oligonucleotide for selectively regulating the expression of at least one gene in a cell and instructional materials for its use.

[0015] In certain embodiments, 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.

[0016] In certain embodiments, the composition comprises a cell transfected with a naked RNA oligonucleotide, wherein the cell is at least one selected from the group consisting of megakaryocytes and platelets.

[0017] In certain embodiments, the naked RNA oligonucleotide is selected from miRNA, siRNA, and any combination thereof.

[0018] In certain embodiments, the naked RNA oligonucleotide comprises a guide strand and a passenger strand.

[0019] In certain embodiments, the naked RNA oligonucleotide comprises a guide strand having a sequence selected from the guide strand sequences shown in Table 1.

[0020] In certain embodiments, the naked RNA oligonucleotide comprises a passenger strand having a sequence selected from the passenger strand sequences shown in Table 1.

[0021] In certain embodiments, the naked RNA oligonucleotide comprises at least one modification selected from locked nucleic acid (LNA), 2'-fluorinated base, and 2'-O-methylated base.

[0022] In certain embodiments, the naked RNA oligonucleotides shown are thermostable. In certain embodiments, the naked RNA oligonucleotides shown are non-immunogenic. In certain embodiments, the naked RNA oligonucleotides are resistant to endoribonuclease (RNase) cleavage.

[0023] In certain embodiments, the composition is administered intravenously.

[0024] In certain embodiments, the administration alters megakaryocyte development, platelet production, megakaryocyte function, and / or platelet function.

[0025] In certain embodiments, the subject requires:

[0026] i. Antiplatelet therapy,

[0027] ii. Anti-inflammatory therapy for treating thromboinflammation,

[0028] iii. Treatment of thrombocytopenia, thrombocytosis, and other pathological manifestations of disrupted megakaryocyte development,

[0029] iv. Platelet transfusion with platelets protected from platelet storage lesion,

[0030] v. Treatment of thrombosis,

[0031] vi. Treatment of acquired bleeding disorders, and / or

[0032] vii. Treatment of inherited bleeding disorders.

[0033] In certain embodiments, the composition comprises a saline solution.

[0034] In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human.

[0035] In certain embodiments, the cells are derived from at least one selected from cell culture systems, concentrates, cell suspensions, tissue homogenates, organoids, tissues, and organs.

[0036] In certain embodiments, the cell culture is a stem cell culture.

[0037] In certain embodiments, the concentrate is a platelet storage concentrate containing autologous plasma.

[0038] In certain embodiments, the transfection is performed without using a synthetic carrier or adjuvant.

[0039] In certain embodiments, the naked RNA oligonucleotides are suspended in a sterile saline solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following detailed description of example embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, non-limiting embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact arrangements and instrumentalities shown in the embodiments of the drawings.

[0041] Figures 1A - 1C Showing selective transfection of bone marrow megakaryocytes by naked double-stranded miRNA mimics in vitro. ( Figure 1A ) Real-time imaging of AF488 was performed on whole unfixed bone marrow cell suspensions captured on poly-L-lysine (PLL)-coated slides. Only megakaryocytes (MKs) showed green fluorescence, indicating MK-specific uptake. ( Figure 1B ) Fixed bone marrow cells were counterstained to examine the MK / platelet markers Cd41, and the nuclear dye was DAPI. Only MKs showed miR-LNA uptake, while other bone marrow cells did not. In this experiment, bone marrow cells were co-incubated with both miR-LNA-AF488 and the unmodified form of miR-223-3p conjugated to Alexa Fluor 647. No fluorescence was shown in the figures below, indicating degradation of the unmodified miRNA mimics. ( Figure 1C ) The cells in ( Figure 1B ) were counterstained to examine two MK / platelet markers, Cd42d and Cd41, showing uptake by MKs at different stages including the immature growth stage (small MKs).

[0042] Figures 2A - 2F Showing selective transfection of bone marrow megakaryocytes and platelets by infused naked double-stranded miRNA mimics in vivo. ( Figure 2A ) Whole bone marrow cell suspensions extracted 2 hours after infusion were captured on poly-L-lysine (PLL)-coated slides and imaged in real-time for AF488. Only megakaryocytes (MKs) showed green fluorescence, indicating MK-specific in vivo uptake. ( Figure 2B ) Fixed bone marrow cells were counterstained to examine the MK / platelet markers Cd42d and Cd41, and the nuclear dye was DAPI. At 18 hours and 72 hours after infusion, only MKs and platelets showed miR-LNA uptake, while other bone marrow cells did not. ( Figure 2C ) Analysis by flow cytometry ( Figure 2B) miL uptake (by fluorescence) of cells in , and MK was identified using Cd41 / Cd42d and size (forward scatter FSC). In this experiment, the spleen was also harvested, and the splenocyte suspension was analyzed at 72 h, jointly showing robust MK uptake and miLNA retention in both bone marrow and splenic MK. Figure 2D ) Peripheral blood cells from the same animals as above were fixed and captured on coverslips, and platelets were identified with Cd42d antibody. The in vivo uptake efficiency of platelet miLNA was close to 100%, as previously observed for washed in vitro platelets. Scale bar, 50 μm. Figure 2E ) From Figure 2D ) Composite high magnification image of platelets from . Scale bar, 10 μm. Figure 2F ) Blood cells and lung endothelium at 2 h post-infusion. There was rapid miLNA uptake in platelets, but not in WBC, RBC, or endothelium (EC).

[0043] Figures 3A - 3D Infused naked RhoAsiLNA was shown to selectively repress RhoA protein expression in bone marrow megakaryocytes and regulate megakaryocyte development and platelet production in vivo. Figure 3A ) RhoA siLNA selectively repressed RhoA protein expression in bone marrow MK after infusion. Using RhoA antibody, combined with Cd42d antibody to label MK, and propidium iodide (PI) to label nuclear DNA, RhoA levels were measured by flow cytometry in permeabilized whole bone marrow cell suspensions. Left panel, RhoA expression levels in MK as a function of ploidy classification determined by PI staining. Due to cytoplasmic expansion, RhoA expression increased as expected in larger, more mature MK, but this increase was repressed by RhoAsiLNA. In the right panel, nucleated (PI+) non-MK bone marrow cells showed no change in RhoA protein levels. Figure 3B ) RhoA siLNA regulates RhoA-dependent MK function. RhoA knockout mice and RhoA inhibitor-treated mice demonstrated that acute RhoA repression leads to increased MK ploidy. Compared with control siLNA, RhoA siLNA led to increased MK ploidy, indicating a specific functional role of in vivo RhoA repression in MK. Figure 3C ) Peripheral blood platelet counts increased with RhoAsiLNA, corresponding to the increase in MK ploidy Figure 3B ). Figure 3D ) Using RhoA siLNA, there was no change in mean platelet volume, confirming that increased MK ploidy drives elevated platelet counts but does not generate larger platelets.

[0044] Figures 4A - 4BThe administration of naked P2y12 (P2ry12) siLNA by infusion reduced the platelet ADP activation response and prevented injury-induced arterial thrombosis in WT mice, phenocopying currently available first-line antiplatelet drugs that target P2Y12.( Figure 4A )P2ry12 siLNA infusion reprogrammed platelets to have reduced ADP responsiveness. Platelet ADP responses in whole blood cell suspensions were assayed by FACS 24 h after P2ry12 (open circles) or control scrambled (CTL, closed circles) siLNA infusion to assess ADP-induced activation of the GpIIb / IIIa integrin (using the JonA antibody) (upper panel) and α-granule secretion (using the P-selectin antibody) (lower panel). p < 0.05. n = 3.( Figure 4B )At 96 h (day 5), anesthetized mice were subjected to arterial injury of the carotid artery (“injury”) by application of a filter paper soaked in 7.5% FeCl3 solution for 90 s. Blood flow was monitored with a Doppler probe downstream of the injury site until 30 min after injury. As expected, arterial occlusion and blockage of blood flow occurred in control mice between 5 and 12 min after injury. In contrast, the artery remained patent in mice treated with naked P2ry12 siLNA (targeting the P2y12 ADP receptor, the target of currently available first-line antiplatelet antithrombotic drugs), and no occlusion was observed within the experimental time frame. N = 3 for each experiment.

[0045] Figures 5A - 5D Single injection of naked miR-223-3p miRNA mimics transiently and potently increased platelet ADP reactivity and thrombotic response.( Figure 5A )miR-223-3p injection resulted in a ~4-fold mean increase in platelet ADP reactivity, measured by flow cytometry with the Jon / A antibody that recognizes the activated form of integrin GpIIb / IIIa, 24 h after injection, whereas control Cel-miR-67 or saline infusion had no effect on reactivity. The increased reactivity was transient, indicating depletion of the targeted miR and remodeling of the targeted protein at later times.( Figure 5B )The entire circulating platelet population showed a right shift in ADP reactivity. This result indicates a potent direct effect on circulating platelets, but an effect on MKs cannot be excluded.( Figure 5C )The effect of miR-223-3p infusion on injury-induced arterial thrombosis was tested by FeCl3 injury 24 h after injection, as Figure 4BThe same. miR-223-3p infusion enhances the thrombotic phenotype. Shown is the occlusion time of the carotid artery after injury and removal of FeCl3 insult. n = 9 controls, n = 6 miR-223-3p-treated mice. miR-223-3p infusion enhances the thrombotic phenotype, indicating a direct and potent effect of naked miR-NA on platelet physiological responses.( Figure 5D ) Figure 5C Summary data of middle carotid artery occlusion time.

[0046] Figures 6A - 6B Shows that single or repeated addition of naked ADAM17 siRNA to platelets in the form of stored concentrates protects against loss of GP1bα.( Figure 6A )Single direct addition of naked ADAM17 siRNA to platelet stored concentrates consistently maintains the total amount and surface expression of GP1bα in stored platelets up to 48 hours. After 48 hours, GP1bα levels drop to low levels similar to those of control siRNA, indicating turnover of ADAM17 and GP1bα.( Figure 6B )Multiple addition of ADAM17 siRNA to stored platelet concentrates (by precipitating and washing platelets, repeating transfection, and then reconstituting with fresh plasma) - given at 0 hours and again at 48 hours - prolongs the maintenance time of GP1bα in stored platelets.

[0047] Figures 7A - 7B Shows that single addition of naked ADAM17 siLNA to platelet stored concentrates protects against loss of GP1bα function.( Figure 7A )Shown is the change in the percentage of platelet aggregation (agglutination) over time after addition of the indicated concentration of ristocetin (Rs). Naked ADAM17 siLNA was directly added once to platelet stored concentrates on day 0, maintaining GP1bα functional reactivity within the 5-day experimental time frame. These data first demonstrate an improvement in the cellular physiological function of stored platelets by this method.( Figure 7B )GP1bα expression in stored platelets.

[0048] Figure 8 Shows platelet-specific miR-NA uptake: aggregates of platelets and platelet extracellular vesicles with leukocytes in peripheral blood, e.g., uptake by leukocytes was not detected.

[0049] Figure 9 Shows normal CBC and megakaryocyte size (ploidy class) using RhoA siLNA.

[0050] Figure 10Show normal CBC using miL-223-3p.

[0051] Figure 11 Show normal CBC using P2ry12 siLNA.

[0052] Figure 12 Is an illustration outlining a general method according to an embodiment of the present invention: Intravenous injection of a modeled infusion of a modified small RNA to selectively transfect megakaryocytes (as shown here) and circulating blood platelets.

[0053] Figure 13 Show si / miLNA sequences designed and used for the experiments presented herein; some experiments used miR-223-3p mimics without fluorophore labels. Sequences of the guide strand and the passenger strand are shown 5’→3’, and they were hybridized before use to produce a double-stranded oligonucleotide with overhangs at each end.

[0054] Figures 14A - 14C Show that miLNA uptake occurs only in MEG-01 cells during megakaryocyte differentiation. MEG-01 promegakaryocytes + / - PMA were cultured for different days as shown to induce megakaryocyte differentiation and then transfected in vitro with naked miL:AF488 for 1 hour. ( Figure 14A ) Show miLNA fluorescence and percentage of miLNA-positive (miL+) cells. miLNA uptake appears only starting from day 4 of differentiation. ( Figure 14B ) Initiation of CD41 expression (megakaryocyte / platelet marker) is consistent with the ability of miLNA uptake induced at day 4. ( Figure 14C ) miLNA uptake is evident in CD41+ cells.

[0055] Figure 15 Show the evaluation of the therapeutic window (TW) of P2ry12 siLNA and ticagrelor in mice. The TW was evaluated as the interval between the antithrombotic effect and the bleeding effect within a 3-log dose range of ticagrelor or P2ry12 siLNA after intravenous administration. The antithrombotic effect was also tested in mice treated with scrambled control siLNA, and the highest dose of P2ry12 siLNA (500 μg / kg; n = 5) was tested; the results are shown as large black squares. Control siLNA had no protective effect on thrombosis, while P2ry12 siLNA had a dose-dependent protective effect on thrombosis. Thrombosis – squares and dashed line; hemostasis inhibition – circles and dotted lines. n = 3 - 10.

[0056] Figure 16 Show a representative example from the FeCl3 arterial injury occlusive thrombosis assay.

[0057] Figure 17 Representative Doppler flow tracings 24 hours after IV administration of P2ry12 siLNA following carotid artery injury are shown. The right panel shows occlusion stability as a function of P2y12 inhibition measured by FACS. Lower left ( Figure 17 continued ) shows occlusion time as a function of siLNA IV dose. Experiments were stopped at 20 min (1200 s); any measurement at 1200 s represents restoration of blood flow at the end of the experimental time frame.

[0058] Figure 18 Diversity in reactivity within the circulating platelet population is shown.

[0059] Figure 19 F2RL3 siLNA-mediated platelet hPAR4 inhibition in hPAR4 mice is shown. Platelet lysates were extracted 24 hours after a single injection of F2RL3 (hPAR4) siLNA into hPAR4 mice and immunoblotted with a PAR4 antibody. hPAR4 TG / mPar4 KO indicates mice transgenic for human PAR4 and lacking the murine Par4 gene.

[0060] Figure 20 F2RL3 siLNA reprograms platelets to have reduced reactivity to PAR4 agonists is shown. hPAR4 mice were infused with 0.5 mg / kg F2RL3 or control siLNA, and 24 hours later platelet PAR4-activating peptide (PAR4-AP) responses in platelets from whole blood cell suspensions were tested by flow cytometry, where the Jon / A antibody indicates integrin activation (left) and the P-selectin antibody indicates granule secretion (right). n = 6.

[0061] Figure 21 F2RL3 siLNA provides protection against thrombosis while having minimal effects on bleeding is shown. hPAR4TG / mPar4 KO mice were injected with 0.5 mg / kg F2RL3 or control siLNA and subjected to FeCl3 thrombosis (left) or tail tip cut bleeding (right) at 24 hours. Blood loss is shown as the ratio of blood loss weight (g) to starting mouse weight (g). n = 3.

[0062] Figures 22A - 22D In vivo siLNA-mediated silencing of Gsα (mRNA, Gnas) in murine platelets at 24 hours, the prohemostatic effects of Gnas and Ptgir siLNA, and rescue of Gnas and Ptgir siLNA on acquired (ticagrelor-induced) bleeding are shown. Figure 22AShow the inhibition of Gsα by Gnas siLNA in mouse platelets 24 hours after tail vein injection of siLNA. n = 3. Figure 22B Show that in mice treated with Gnas (Gsα) or Ptgir (IP, prostacyclin receptor) siLNA, Figure 21 as measured in Figure 22C bleeding is reduced. All experiments show N. Figure 22D Show that hemostasis is accelerated in mice with Gnas siLNA compared to controls after tail tip transection at 1.5 mm diameter. The experiment was stopped at 20 minutes (1800 seconds). N = 6.

[0063] Figure 23 Show the dynamic Gsα expression during platelet lifespan. The ratio of Gsα expression to total platelets in new platelets (dashed line) and existing platelets (solid line) is shown as ± s.e.m. 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.

[0064] Figures 24A - 24B Show ( Figure 24A ) selective in - vivo miLNA uptake by ( Figure 24B ) platelets and (

[0065] Figure 25 ) bone marrow megakaryocytes. Cells were collected from un - infused WT and miR - 223KO mice (-), and miR - 223KO mice (+) 18 hours after miR - 223 - 3p infusion, which were labeled with surface markers and single - cell FAC sorted for each population as indicated. As in previous studies, the presence of miR - 223 - 3p was detected by PCR from poly(dA) - tailed cDNA in each population, directly indicating cell - type - specific in - vivo miLNA uptake in platelets / MK.

[0066] Figure 26Shows the targeting effect of subcutaneous administration of siLNA in mice using osmotic pumps. 3-day release pumps carrying 0.6 mg / kg P2ry12 (red open circles) or control (blue closed circles) siLNA were implanted subcutaneously in mice, and platelet ADP-induced integrin activation (left) and P-selectin exposure (right) were evaluated daily by flow cytometry. Data are shown as fold area under the ADP dose-response curve (as in Figure 4) ± s.e.m. n = 3. *, p < 0.03; **, p < 0.05. Detailed Description

[0067] In the present invention, the general approach is to introduce microRNA mimics (miRNA) and / or short interfering RNAs (siRNA) into megakaryocytes and / or platelets to regulate the expression of their cognate target genes, which contributes to specific cellular, physiological, and pathophysiological functions as outlined above. The mi / siRNA inhibits the translation of its target mRNA in a sequence-specific manner, thereby preventing the expression of the cognate protein. Thus, the mi / siRNA inhibits the protein expression of the target gene.

[0068] Definitions

[0069] As used herein, each of the following terms has an associated meaning 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 pertains. In general, the nomenclature and laboratory procedures used herein in animal pharmacology, pharmaceutical sciences, peptide chemistry, and organic chemistry are those well known and commonly employed in the art. It should be understood that the order of steps or the order of performing certain actions is not important so long as the present teachings remain operable. The use of any section headings is for the purpose of facilitating the reading of this document and should not be construed as limiting; information related to a section heading may appear within or outside of that particular section. All publications, patents, and patent documents mentioned in this document are incorporated herein by reference in their entirety as if individually incorporated by reference.

[0070] In this application, when an element or component is recited as being included in a list of recited elements or components and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and optionally a group consisting of two or more of the recited elements or components.

[0071] In the methods described herein, the acts can be performed in any order, unless a temporal or operational sequence is explicitly recited. Additionally, unless the claim language explicitly recites that the recited acts are to be performed separately, they can be performed concurrently. For example, the acts of making X as claimed and making Y as claimed can be performed concurrently in a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0072] In this document, unless the context clearly dictates otherwise, the terms "a", "an", or "the" are used to include one or more than one. Unless otherwise indicated, the term "or" is used to refer to a non-exclusive "or". The phrase "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B".

[0073] When referring to measurable values such as amounts, durations, etc., "about" as used herein means including variations of ±20% or ±10% from the specified value, in some embodiments ±5% from the specified value, in some embodiments ±1% from the specified value, and in some embodiments ±0.1% from the specified value, because such variations are suitable for performing the disclosed methods.

[0074] The terms "alteration" or "alter" or "modulate" 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 the methods described herein. In some embodiments, an alteration in the expression level includes a 10% change, 25% change, 40% change, and a 50% or greater change in the expression level.

[0075] As used herein, the term "antimiR" refers to a chemically modified single-stranded antisense oligonucleotide that inhibits miRNA function. AntimiRs range in length from 8-mer oligonucleotides that target the seed to antimiRs that are fully complementary to the mature miRNA. As synthetic reverse complements, they block miRNA activity by competing with the target 3'UTR mRNA site for miRNA binding.

[0076] As used herein, the term "antagomiR" refers to a 3'-cholesterol-conjugated, 2'-O-methyl-modified antisense oligonucleotide that inhibits miRNA function. AntagomiRs are fully complementary to the mature miRNA.

[0077] As used herein, the term cell refers to cells (such as, for example, megakaryocytes) and / or cell fragments (such as, for example, platelets).

[0078] A disease or disorder is "attenuated" if the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences the symptoms, or both, are reduced.

[0079] The term "cleavage" refers to the breaking of a covalent bond, such as the breaking of a covalent bond in the backbone of a nucleic acid molecule. Cleavage can be initiated by a variety of methods, including but not limited to enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-strand cleavage and double-strand cleavage are possible. Double-strand cleavage can be caused by two different single-strand cleavage events. RNA cleavage can result in the production of blunt ends or staggered ends.

[0080] In the present disclosure, terms such as "comprises", "comprising", "containing", and "having" can have the meanings given to them by U.S. patent law and can mean "includes", "including", etc.; "consisting essentially of" or "consists essentially" likewise have the meanings given by U.S. patent law, and the term is open-ended, allowing for features in addition to those recited, provided that the basic or novel features of the recited features are not changed by the presence of additional features, but rather exclude prior art embodiments.

[0081] A "disease" refers to the health state of an animal in which the animal is unable to maintain homeostasis and in which the health of the animal will continue to deteriorate if the disease is not improved.

[0082] A "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but the health state of the animal is less favorable than it would be in the absence of the disorder. A disorder does not necessarily lead to a further decline in the health state of the animal if left untreated.

[0083] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0084] As used herein, the term "regulatory gene" refers to regulatory genes and / or gene products and / or any genetic material. In certain embodiments, "regulatory gene" refers to, for example, regulatory mRNA, pre-mRNA, lncRNA, snRNA, snoRNA, tRNA, rRNA, YRNA, piRNA.

[0085] As used herein, "microRNA" or "miRNA" describes small non-coding RNA molecules, typically about 15 to about 50 nucleotides in length, preferably 17 - 23 nucleotides, which can play a role in regulating gene expression through a process such as that known as RNA interference (RNAi). RNAi describes a phenomenon whereby the presence of an RNA sequence that is complementary or antisense to a sequence in a target gene messenger RNA (mRNA) results in the suppression of expression of the target gene. miRNAs are processed from hairpin precursors (pre-miRNAs) of about 70 or more nucleotides, which are derived from primary transcripts (pri-miRNAs) by sequence cleavage by RNAse III enzymes. miRBase is a comprehensive microRNA database at the address www.mirbase.org, which is incorporated herein by reference in its entirety for all purposes.

[0086] As used herein, "homologous" refers to subunit sequence identity between two polymer molecules, such as between two nucleic acid molecules, e.g., between two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit positions in the two molecules are each occupied by the same monomer subunit; for example, if a position in each of 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 (e.g., five positions in a ten-subunit length polymer) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) match or are homologous, the two sequences are 90% homologous.

[0087] As used herein, "identity" refers to subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, such as between two polypeptide molecules. When two amino acid sequences have the same residue at the same position; for example, if a position in each of two polypeptide molecules is occupied by arginine, they have identity at that position. Identity or the degree to which two amino acid sequences have the same residue at the same position upon alignment is typically 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 in two sequences (e.g., five positions in a ten-amino acid length polymer) are the same, the two sequences have 50% identity; if 90% of the positions (e.g., 9 out of 10) match or are the same, the two amino acid sequences have 90% identity.

[0088] As used herein, the term "locked nucleic acid (LNA)" refers to a modified RNA nucleotide in which the ribose moiety is modified to have an additional bridge connecting the 2'-oxygen to the 4'-carbon.

[0089] As used herein, the term "modified" means that the state or structure of the molecule or cell of the present invention is altered. A molecule can be modified in a variety of ways, including chemically, structurally, and functionally. A cell can be modified by the introduction of nucleic acid.

[0090] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of response in a subject as compared to the level of response in a subject in the absence of treatment or compound, and / or as compared to the level of response in an otherwise identical but untreated subject. The term encompasses disrupting and / or affecting a natural signal or response such that a beneficial therapeutic response in a subject (preferably, a human) is mediated.

[0091] As used herein, the term "neo-generated platelets" refers to platelets produced by megakaryocytes between 0 and 48 hours relative to in vivo administration of siLNA or miLNA or transfection ex vivo or in vitro.

[0092] In the context of the present invention, the following abbreviations are used for the commonly occurring nucleic acid bases. "A" refers to adenosine, "C" refers to cytosine (cytidine), "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0093] As used herein, the term "naked RNA oligonucleotide" refers to an RNA oligonucleotide that is not encapsulated in any type of liposome or nanoparticle and is not anchored to any other molecule or adjuvant. Naked RNA oligonucleotides include, for example, microRNA mimics (miRNAs) or short interfering RNAs (siRNAs).

[0094] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection or infusion techniques.

[0095] The term "oligonucleotide" generally refers to short polynucleotides, usually not exceeding about 60 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T".

[0096] As used herein, "polynucleotide" includes cDNA, RNA, DNA / RNA hybrids, antisense RNA, siRNA, miRNA, snoRNA, tRNA, YRNA, genomic DNA, synthetic forms, and hybrid polymers, including both sense and antisense strands, and may be chemically or biochemically modified to include non-natural or derivatized, synthetic or semi-synthetic nucleobases. In addition, alterations of wild-type or synthetic genes are also included within the scope of the present invention, including but not limited to deletions, insertions, substitutions of one or more nucleotides, or fusions with other polynucleotide sequences. As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful in the present disclosure and a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates the administration of the compound to a patient. There are a variety of techniques for administering compounds 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.

[0097] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which 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 undesirable biological effects or interacting in a harmful manner with any of the components of the composition in which it is contained.

[0098] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle that participates in transporting or facilitating the transport of a compound useful in the present disclosure in a patient such that it can perform its intended function, such as a liquid or solid filler, stabilizer, dispersant, suspending agent, diluent, excipient, thickening agent, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful in the present disclosure, and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as 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, and absorption delaying agents, etc., that are compatible with the activity of the compound useful in the present disclosure and physiologically acceptable to the patient. "Pharmaceutically acceptable carrier" may further include pharmaceutically acceptable salts of the compound useful in the present disclosure. Other additional ingredients that can be included in the 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.

[0099] As used herein, the term "pharmaceutically acceptable salt" 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 or inclusion compounds thereof.

[0100] As used herein, a "pharmaceutically effective amount", "therapeutically effective amount" or "effective amount" of a compound refers to an amount of the compound sufficient to provide a beneficial effect to an object to which the compound is administered.

[0101] As used herein, the terms "prevent" or "prevention" mean, in the case where neither has occurred, not developing a disorder or disease, or, in the case where a disorder or disease has already developed, the disorder or disease not further developing. Also considered is the ability of an individual to prevent some or all of the symptoms associated with the disorder or disease.

[0102] "Small interfering RNA" or "siRNA" means double-stranded RNA. Optimally, the siRNA has a length of 18, 19, 20, 21, 22, 23 or 24 nucleotides and has a base overhang at its 3' end. These dsRNAs can be introduced into a single cell or an entire animal; for example, they can be introduced systemically through the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity.

[0103] The terms "siLNA" and "miLNA" refer to LNA-modified siRNA molecules and LNA-modified miRNA molecules, respectively.

[0104] As used herein, the terms "object", "individual" and "patient" are used interchangeably and can refer to a human or non-human mammal or bird. Non-human mammals include, for example, livestock and pets such as sheep, cattle, pigs, dogs, cats and murine mammals. In certain embodiments, the object is a human.

[0105] "Substantially identical" means that a polypeptide or nucleic acid molecule exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Such sequences have at least 60%, at least 80%, at least 85%, at least 90%, at least 95% or even at least 99% identity to the sequence being compared at the amino acid or nucleic acid level.

[0106] "Target site" or "target sequence" refers to a genomic nucleic acid sequence, a nucleic acid moiety to which a binding molecule can specifically bind under conditions sufficient to effect binding.

[0107] 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 a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0108] As used herein, the term “treatment” or “treating” is defined as the administration or application of a therapeutic agent, i.e., a compound useful in the present disclosure (alone or in combination with another agent), to a patient, or to an isolated tissue or cell line from a patient (e.g., for diagnostic or ex vivo applications), who has a disease or disorder and / or symptoms of a disease or disorder, with the purpose of curing, healing, alleviating, relieving, altering, remedying, improving, ameliorating, or affecting the disease or disorder and / or the symptoms of the disease or disorder. Such treatment can be specifically tailored or modified based on knowledge obtained from the field of pharmacogenomics.

[0109] Ranges: Throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the disclosure. Accordingly, the range description should be considered to have specifically disclosed all the 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 subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0110] Description

[0111] The object of the present invention is to provide a platform for regulating megakaryocyte development, platelet production, and / or platelet reactivity in vivo, ex vivo, and in vitro to improve all clinical conditions disclosed elsewhere herein, as well as to extend the lifespan and function of stored platelets while reducing the inflammatory response in stored platelet units.

[0112] Without wishing to be bound 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 direct ability to transfect megakaryocytes and platelets with regulatory small RNAs while minimizing off-target tissue transfection in vivo, ex vivo, and in vitro.

[0113] Importantly, although unassisted uptake of oligonucleotides (also known as "gymnosis") has been explored for therapeutic use, uptake efficiency and tissue targeting remain unresolved problems, and the present invention addresses these problems in megakaryocytes and platelets.

[0114] Composition

[0115] In one aspect, the present invention provides a composition for selectively modulating gene expression in megakaryocytes (MK) and / or platelets and / or platelets generated from transfected megakaryocytes. The composition comprises naked (e.g., unencapsulated) RNA oligonucleotides. In certain embodiments, the RNA oligonucleotides are suspended in an aqueous solution but are not encapsulated in any type of liposome or nanoparticle and are not anchored to any other molecule or adjuvant. In certain embodiments, the aqueous solution is, for example, a sterile saline solution.

[0116] In certain embodiments, the RNA oligonucleotides are selected from: double-stranded (ds) miRNA mimics, which comprise modified RNA base(s), wherein the guide strand represents the nucleotide sequence of a native miRNA; siRNA; and any combination thereof. In certain embodiments, the RNA oligonucleotides can be synthesized easily and inexpensively.

[0117] In certain embodiments, the RNA oligonucleotides selectively transfect megakaryocytes and platelets in vivo, ex vivo, or in vitro. In certain embodiments, in vivo transfection is used, for example, to modulate megakaryocyte development, function, and gene expression, platelet production, and platelet in vivo function, including controlling bleeding, thrombosis, and inflammation. In certain embodiments, ex vivo transfection is used, for example, to increase the functional lifespan of stored platelet concentrates and / or reduce their inflammatory state. In certain embodiments, in vitro transfection is used, for example, in stem cell therapy methods, such as modulating megakaryocytes derived from induced pluripotent stem cells.

[0118] In certain embodiments, the RNA oligonucleotides are designed to avoid degradation by plasma and tissue ribonucleases (RNAases). In certain embodiments, the RNA oligonucleotides are non-immunogenic. In certain embodiments, the RNA oligonucleotides are designed to avoid an immune response or elicit little to no immune response.

[0119] In certain other embodiments, the RNA oligonucleotides optionally comprise modified nucleotide(s), such as, for example, locked nucleic acid(s) (LNA) modified to include a methylene bridge bond between the 2'-oxygen and 4'-carbon of the pentose ring and 2'-OMe RNA base(s) methylated at the 2'-oxygen and / or 2'-fluorine (2'-F) base(s).

[0120] In certain embodiments, for RNA nucleotides where the 3' terminal residue in the guide strand overhang includes uracil(s) (one or more U), the U(s) is / are replaced by thymine(s) (one or more T) (since uracil cannot be modified to LNA due to its structure) to become LNA(T).

[0121] It is known that LNA-based RNA oligonucleotides confer strong resistance to RNAse cleavage of RNA. RNAse is rich 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. In addition, the precise placement of LNA and 2'-OMe RNA nucleotides within double-stranded miRNA mimics (siRNA or antimiR) confers several key properties; these properties include, for the guide strand: a 3' overhang consisting of 2 or 3 LNA bases facilitates the incorporation of the guide strand into the RNA-induced silencing complex (RISC), but avoids the loss of inhibitory activity; for the passenger strand: a truncated 5' end plus a 5' terminal LNA base inactivates the strand, a moderate number of LNA bases (5 - 6) evenly distributed in the strand confers RNase tolerance, a di-uridine at the 3' end provides an overhang and further inactivates the strand, and 2'-OMe modification of the 3' terminal uridine and most or all adenosine nucleotides greatly reduces immunogenicity. The guide strand sequence of the miRNA mimic is determined by the native sequence of the given mature miRNA.

[0122] In certain embodiments, instead of 2'-OMe nucleotides, 2'-F nucleotides will also be sufficient such that those bases in the passenger strand of the duplex si or miLNA complex will be replaced by 2'-OMe to produce a similar effect: protection from endonucleases and avoidance of inducing an immune response.

[0123] In certain embodiments, the siRNA is about 15 to about 30 nucleotides in length and is designed to target the 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 long.

[0124] In certain embodiments, the antimiR contains a reverse complementary sequence of the 5' end of the target miRNA, generally nucleotides 1 - 8, but not limited to this region. In certain embodiments, the antimiR consists entirely of LNA bases.

[0125] 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 the same as the guide strand sequence shown in Table 1. In certain embodiments, the passenger strand has a sequence substantially the same as 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.

[0126] In certain embodiments, the naked RNA oligonucleotide comprises only unmodified nucleotides.

[0127] In certain embodiments, the RNA oligonucleotide is thermostable, i.e., stable at freezing / deep freezing temperatures, refrigeration temperatures, room temperature, and body temperature.

[0128] In certain embodiments, the composition is administered to a subject to enter its bloodstream. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human subject.

[0129] On the other hand, the present invention provides a composition for selectively modulating the gene expression of one or more target genes in the cells of a subject, wherein the composition comprises cells transfected with a naked RNA oligonucleotide, and wherein the cells are at least one selected from megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide.

[0130] On yet another hand, the present invention provides a composition for selectively modulating the gene expression of one or more target genes in cells ex vivo or in vitro, wherein the composition comprises cells transfected with a naked RNA oligonucleotide, and wherein the cells are at least one selected from megakaryocytes and platelets.

[0131] In certain embodiments, the naked RNA oligonucleotide is as described elsewhere herein.

[0132] Method

[0133] On the other hand, the present invention provides a method for selectively modulating the gene expression of one or more target genes in the 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 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.

[0134] Although platelets do not contain a nucleus, they do have other forms of genetic material that regulate protein expression, and regulating gene expression involves interaction with downstream gene products (such as, for example, mRNA).

[0135] In certain embodiments, the term "regulatory gene" includes regulatory genes and / or gene products and / or any genetic material. In certain embodiments, "regulatory gene" includes, but is not limited to, regulatory mRNA, pre-mRNA, lncRNA, snRNA, snoRNA, tRNA, rRNA, YRNA, piRNA.

[0136] 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.

[0137] In certain embodiments, administration results in, for example, changes in megakaryocyte development, platelet production, megakaryocyte function, and / or platelet function.

[0138] In yet another aspect, the present invention provides methods of antiplatelet therapy that are improved relative to standard-of-care agents for antithrombotic use while reducing the clinical bleeding risk. In certain embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of the composition described elsewhere herein.

[0139] In certain embodiments, antiplatelet therapy targets include, but are not limited to, P2Y12 (ADP receptor, gene P2RY12). This protein product is the target of current first-line antiplatelets - including cangrelor, ticagrelor, and clopidogrel (Plavix), which are used as anticoagulants worldwide - but they incur variable clinical bleeding risks, ranging from moderate to severe, such as life-threatening intracranial hemorrhage. MK / platelet-specific knockout of P2Y12 via this platform provides improved antithrombotic efficacy and reduced bleeding risk compared to complete pharmacological blockade, thus impacting many patients in a variety of clinical scenarios of reduced and managed thrombosis risk.

[0140] In certain embodiments, antiplatelet therapy targets include, but are not limited to, PAR1 (thrombin receptor). This protein is the target of vorapaxar, which has withdrawn from most clinical applications due to bleeding. MK / platelet-specific PAR1 knockout via this platform provides an alternative to vorapaxar while improving antithrombotic efficacy and reducing bleeding risk, thus impacting many patients in a variety of clinical scenarios of reduced and managed thrombosis risk.

[0141] In yet other embodiments, antiplatelet therapy targets include, but are not limited to, FcγRIIa (gene, FCGR2A). This is an antibody receptor on platelets and other blood cells that mediates immunothrombosis, e.g., thrombosis in response to increased immune activity. MK / platelet-specific knockout of FcγRIIa via this platform can provide strong protection against immunothrombosis in various inflammatory clinical settings and for many patient cohorts, with increased specificity of action and thus reduced side effects compared to current standards of care.

[0142] In yet other embodiments, antiplatelet therapy targets include, but are not limited to, reactive platelet intracellular proteins. These targets include a long list of putative targets similar to those described above that can be modified by this platform technology in a personalized medicine approach based on the platelet expression profile of an individual patient. For example (one of many possibilities), a patient with overexpression of the cytoplasmic inhibitor of platelet receptor function, who has reduced platelet reactivity secondary to a bleeding diathesis, can be treated by targeting the overexpressed inhibitor protein(s).

[0143] In another aspect, the present invention provides a method for treating platelet-induced inflammation, thromboinflammation, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition described elsewhere herein.

[0144] In certain embodiments, targets for treating thromboinflammation include, but are not limited to, platelet-derived inflammatory cytokines and their upstream regulators. These targets include, but are not limited to, interleukins and their upstream inducers such as NLRP3, CCL lymphocyte activators, etc. Platelets contribute to inflammation by releasing inflammatory cytokines; e.g., contributing to an excessive inflammatory response in sepsis, which is a major cause of death. However, in many cases, completely blocking inflammation would impair the necessary patient immune response. MK / platelet-specific knockout of inflammatory cytokines via this platform can provide improved anti-inflammatory efficacy while maintaining necessary immune function.

[0145] In other embodiments, targets for treating thromboinflammation include, but are not limited to, PAR4 (protease-activated receptor 4). PAR4 antagonists are also used as antiplatelet and anti-inflammatory drugs. PAR4 plays a major role in platelet production of inflammatory substances. However, in many cases, completely blocking PAR4 may not be desirable. MK / platelet-specific knockout of PAR4 via this platform can provide improved anti-inflammatory efficacy while maintaining necessary immune function.

[0146] In yet other embodiments, targets for treating thromboinflammation include, but are not limited to, intracellular platelet proteins that mediate the generation of platelet-derived microvesicles (there are multiple targets in this category), which is an active process driven by the action of multiple proteins and occurs due to platelet stimulation (such as platelet stimulation caused by vascular injury, trauma), due to thrombolytic or thrombectomy treatment for ischemia, or in the context of atherosclerosis and atherothrombosis. These microvesicles are both pro-inflammatory and pro-coagulant. The present platform technology will reduce the generation of platelet microvesicles and / or reduce the expression of pro-inflammatory or pro-coagulant components, thereby producing anti-inflammatory and anticoagulant results superior to the current standard of care.

[0147] In yet another aspect, the present invention provides treatments for acute bleeding and bleeding disorders, including acquired and inherited bleeding disorders. These treatments can be considered anti-bleeding or pro-hemostatic therapies. The bleeding disorders treated with the present invention include, but are not limited to, platelet reactivity disorders and coagulation disorders. Acquired bleeding disorders include, but are not limited to, bleeding disorders induced by antiplatelet or anticoagulant therapies. Targets for anti-bleeding / pro-hemostatic therapies 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 platelet reactivity regulators.

[0148] In yet another aspect, the present invention provides treatments for thrombocytopenia, thrombocytosis, and other pathological manifestations of disrupted megakaryocyte development, wherein the method comprises administering a therapeutically effective amount of the composition described elsewhere herein to a subject in need thereof.

[0149] These conditions are driven by changes in gene expression in developing megakaryocytes. For example, upregulation of transcription factors inhibits MK development, favoring the erythroid lineage, leading to thrombocytopenia, or conversely, factors drive platelet production, leading to thrombocytosis. Targeting these upregulated mediators that alter cell fate can be used to restore normal MK development and normal platelet production.

[0150] On the other hand, the present invention provides methods for treating platelet storage damage as outlined above. In certain embodiments, the methods comprise administering to a subject in need thereof a therapeutically effective amount of the compositions described elsewhere herein. In certain embodiments, targets for treating platelet storage damage include, but are not limited to, ADAM17 metalloprotease, which is responsible for the cleavage of platelet vWF receptor GP1bα. In certain embodiments, targets for treating platelet storage damage include, but are not limited to, ADAM10 metalloprotease, which is responsible for the cleavage of platelet collagen receptor GPVI. In certain embodiments, targets for treating platelet storage damage include, but are not limited to, miR-326 that de-represses the anti-apoptotic master regulator BCLxL (using antmiR-326). In certain embodiments, targets for treating platelet storage damage include, but are not limited to, neuraminidase I, which is responsible for the desialylation of platelet surface proteins, resulting in accelerated clearance of stored platelets after transfusion. In certain embodiments, targets for treating platelet storage damage include, but are not limited to, inflammatory cytokines as described elsewhere herein and proteins involved in microvesicles as described elsewhere herein, each of which is released by platelets over time during storage, resulting in a pro-inflammatory state of platelet storage concentrates. In yet other embodiments, other mediators of platelet death, dysfunction, and inflammatory state during storage are also targets of this platform technology.

[0151] On the other hand, the present invention provides improved treatment of thrombosis risk associated with hormonal effects, including gender differences in thrombosis risk, increasing thrombosis risk with age and menopause, estrogen therapy, hormone replacement therapy, etc. In certain embodiments, the methods comprise administering to a subject in need thereof a therapeutically effective amount of the compositions described elsewhere herein. In certain embodiments, targets for treating thrombosis include, but are not limited to, estrogen receptor β (ERβ, gene ESR2), which is the major estrogen receptor in MK / platelets, has established non-nuclear functions that alter platelet reactivity and is associated with thrombosis.

[0152] In yet other embodiments, targets for treating thrombosis include, but are not limited to, genes involved in hormonal responses, such as adrenergic receptors, androgen receptors, and other hormone receptors expressed in platelets. In certain embodiments, these genes are targeted by specifically targeting MK / platelets, reducing platelet reactivity without compromising hormonal body functions. This set of targets together represents a major area of improvement over current standards of care.

[0153] On the other hand, the present invention provides methods for selectively modulating the gene expression of one or more target genes in megakaryocytes or platelets in vitro or ex vivo, where the megakaryocytes or platelets are derived from at least one selected from cell culture systems, concentrates, cell suspensions, tissue homogenates, organoids, tissues, and organs, and the method includes transfecting the megakaryocytes or platelets with naked RNA oligonucleotides. In certain embodiments, the target gene or target protein is at least one selected from those listed in Table 1.

[0154] In certain embodiments, platelets transfected (in vivo or in vitro) with the naked RNA oligonucleotides of the present invention can be used as delivery vehicles to deliver those naked RNA oligonucleotides to other target cells and / or target tissues in the body, including, for example, tumor cells, white blood cells, inflammatory cells, endothelial cells, etc. The delivery mechanism from transfected platelets to heterologous cells and tissues can include extracellular vesicles released by the transfected platelets, and these extracellular vesicles can include microparticles (also known as microvesicles), exosomes, ectosomes, or apoptotic bodies, or other platelet releasates carrying the transfected naked RNA oligonucleotides. In the example of tumor cells, solid tumor cells allow platelet microparticles carrying naked RNA oligonucleotides to reach the tumor due to their leaky vasculature. Thus, compared to cells in normal tissues, the transfer of mi / siRNA from platelets may be selective for tumor cells, and platelet microparticles are less accessible to these cells in normal tissues.

[0155] In certain embodiments, the present invention provides methods for improved cell culture therapy for in vitro MK (such as directly isolated or derived from stem cell culture MK) for bone marrow reconstitution and for industrial platelet production. In certain embodiments, the method includes transfecting the MK with at least one RNA oligonucleotide described elsewhere herein.

[0156] In certain embodiments, the cell culture is, for example, stem cell culture.

[0157] In certain embodiments, the concentrate is, for example, a platelet storage concentrate containing autologous plasma. In certain embodiments, gene expression in platelets is regulated to extend the lifespan and function of stored platelets while reducing the inflammatory response in stored platelet units.

[0158] In certain embodiments, transfection is achieved ex vivo by, for example, direct addition to a platelet storage unit containing autologous plasma, or addition 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 addition to megakaryocytes or platelets cultured in vitro. mi / siRNA / antimiR selectively transfects megakaryocytes and platelets and modulates their function driven by changes in the expression of the target gene(s).

[0159] In certain embodiments, transfection is carried out without the use of synthetic carriers or adjuvants.

[0160] The compositions and methods described herein are improvements over lentivirus or other methods that carry risks associated with gene therapy methods using live viruses, and are in a manner of increasing platelet production.

[0161] In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human subject. In certain embodiments, the subject is in need of at least one of the following: antiplatelet therapy, anti-inflammatory therapy for treating thromboinflammation, treatment of thrombocytopenia, thrombocytosis, and other pathological manifestations of disrupted megakaryocyte development, platelet transfusion with platelets free of platelet storage damage, treatment of thrombosis, treatment of acquired bleeding disorders, and treatment of hereditary hemorrhagic disorders.

[0162] Kit

[0163] In another aspect, the present invention provides a kit that includes a composition comprising a naked RNA oligonucleotide for selectively regulating the expression of at least one gene in megakaryocytes and / or platelets and instructional materials for its use. In certain embodiments, the composition is for in vivo and / or ex vivo and / or in vitro regulation of gene expression.

[0164] In yet another aspect, the present invention provides a kit that includes a composition comprising cells transfected with a naked RNA oligonucleotide for selectively regulating the expression of at least one gene in megakaryocytes and / or platelets and instructional materials for its use. In certain embodiments, the composition is for in vivo and / or ex vivo and / or in vitro regulation of gene expression. In certain embodiments, the composition is as described elsewhere herein. In certain embodiments, the naked RNA oligonucleotide is as described elsewhere herein.

[0165] Pharmaceutical composition

[0166] The compositions or pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount 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 appropriate dosages can be determined through clinical trials.

[0167] The pharmaceutical compositions of the present invention can be administered in solid or liquid forms such as tablets, capsules, powders, solutions, suspensions, emulsions, etc. The pharmaceutical compositions of the present invention can be administered by the following means: oral, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, by nasal instillation, by implantation, by intracavitary or intravesical instillation, intravitreal, intraarterial, intralesional, transdermal, or by application to mucous membranes. In some embodiments, the composition can be applied to the nose, pharynx, or bronchi by inhalation, for example.

[0168] The dosage of the above-mentioned treatment to be administered to a patient will vary with the exact nature of the condition being treated and the recipient of the treatment, and can be determined by physical and physiological factors such as body weight, severity of the condition, prior or concurrent treatment interventions, and route of administration. The dosage scaling of the administration can be carried out according to practices accepted in the art. For example, for adult patients, the dosage of miR mimics will generally be in the range of 1 to about 100 mg, usually administered once a month for a period between 1 and 12 months. The preferred monthly dosage is 1 to 10 mg per month, but in some cases, larger dosages exceeding 10 mg per month may be used.

[0169] The amount of the human dose can initially be determined by extrapolation from the amount of the compound used in mice, as one of ordinary skill in the art recognizes that modifying the human dose is routine in the art compared to animal models. In certain embodiments, it is contemplated that the dose can include the following effective amounts per administration: 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 up to 1000 mg / kg / body weight or more, and any range derivable therefrom. In other embodiments, the 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 body weight. In other embodiments, it is contemplated that the effective amount can be in the range of about 1 microgram of the compound to about 100 mg of the compound. In other embodiments, the effective amount can 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 another embodiment, the 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, 95 mg per day. In an exemplary embodiment, the effective amount includes less than about 50 mg per day. Of course, the amount of the single dose or the amount of the daily dose can be adjusted up or down based on the results of the initial clinical trials and the needs of the particular subject, as is routine in such treatment regimens. One of ordinary skill in the art will recognize the need to modify the conditions and circumstances of administration.

[0170] The precise determination of what will be considered an effective dose is based on the individual factors of each subject, including their body size, age, gender, weight, and condition of the particular subject. One of ordinary skill in the art can readily determine the dose based on the present disclosure and knowledge in the art.

[0171] Optionally, the methods of the present invention provide for administering the compositions of the present invention to a suitable animal model to determine the dosage of the composition(s), the concentration of the components therein, and the timing of administering the composition(s), which elicits tissue repair, reduces cell death, or induces another desired biological response. Such determination does not require undue experimentation, but is routine and can be determined without undue experimentation.

[0172] The bioactive agent can be conveniently provided to a subject as a sterile liquid preparation (e.g., an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition), which can 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, liquid formulations are desirable because they are convenient to administer, especially by injection. Viscous compositions may be preferred in situations where prolonged contact with tissue is desired. Such compositions are formulated within an appropriate viscosity range. The liquid or viscous composition can contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0173] A sterile injectable solution is prepared by suspending talampanel and / or perampanel in a required amount of a suitable solvent and, if desired, in association with various amounts of other ingredients. Such compositions can be mixed with a suitable carrier, diluent, or excipient (such as sterile water, physiological saline, dextrose, dextran, or the like). The composition can also be lyophilized. Depending on the route of administration and the desired formulation, the composition can contain auxiliary substances such as wetting agents, dispersing agents, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, etc. Suitable formulations can be prepared by referring to standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th Edition, 1985 (incorporated herein by reference) without undue experimentation.

[0174] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. The action of microorganisms can be ensured against by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). Prolonged absorption of injectable drug forms can be produced by using agents that delay absorption, such as aluminum monostearate and gelatin. However, according to the present invention, any vehicle, diluent, or additive used must be compatible with the cells or agents present in their conditioned medium.

[0175] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and tears. The desired isotonicity of the compositions of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is particularly preferred for buffers containing sodium ions.

[0176] If desired, a pharmaceutically acceptable thickening agent (such as methylcellulose) can be used to maintain the viscosity of the composition at a selected level. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, such as a liquid dosage form (e.g., whether the composition is to be formulated as a solution, suspension, gel or another liquid form, such as a timed release form or a liquid-filled form). Those skilled in the art will recognize that the components of the composition should be selected to be chemically inert.

[0177] It should be understood that the methods and compositions to be used in the present invention are not limited to the specific formulations set forth in the examples. The following examples are presented to provide a complete disclosure and description to those of ordinary skill in the art of how to prepare and use the cells, expansion and culture methods, and treatment methods of the present invention, and are not intended to limit the scope that the inventors regard as their invention.

[0178] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the purview of those skilled in the art. Such techniques are well explained in the following documents: "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); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and can thus be taken into account in the preparation and practice of the present invention. Particularly useful techniques for specific embodiments will be discussed in the following sections.

[0179] It should be understood that, no matter how large the values and ranges provided herein are, the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the present invention. Therefore, all values and ranges encompassed by these values and ranges are included within the scope of the present invention. In addition, all values falling within these ranges and the upper or lower limits of the value ranges are also contemplated by this application. The description of a range should be considered to have specifically disclosed all possible sub-ranges as well as the individual numerical values within that range, and, where appropriate, the fractional integers of the numerical values within the range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of how broad the range is.

[0180] The following examples further illustrate aspects of the present invention. However, they are in no way a limitation on the teachings or disclosure of the present invention set forth herein.

[0181] Experimental Examples

[0182] The present invention will be further described in detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to cover any and all variations that become apparent from the teachings provided herein.

[0183] Without further elaboration, 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. Accordingly, the following working examples specifically point out preferred embodiments of the present invention and should not be construed in any way as limiting the remainder of the disclosure.

[0184] Materials and Methods

[0185] Double - stranded RNA design and synthesis and preparation of "naked" dsRNA.

[0186] The ~19-29 nucleotide (nt) guide and passenger strand RNAs of double-stranded siRNA duplexes were designed from the full-length target mature (spliced) mRNA sequences using siDesign (sidirect2.rnai.jp / ), following 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, and the primary candidates were selected from the top-scoring candidates using the scoring method in Fakhr, E. et al. Cancer gene therapy 23, 73-82, (2016). PMID: 26987292, with a minimum score of 14. Candidate sequences were designed by this software, incorporating 2-nt overhangs at each 3’ end of the duplex. Double-stranded miRNA mimics were designed as the full-length sequence of the mature miRNA as the guide RNA strand, and the passenger strand was the exactly reverse complementary sequence, truncated at the first 2 or 3 5’ nt to generate a 3’ overhang in the guide strand and appended with diuridine (UU) at the 3’ end, generating a UU overhang in the duplex. Dithymidine (TT) can also be substituted for UU. The sequences were then modified by the following rules: 1) The 2-nt 3’ overhang sequence in the siRNA passenger strand 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 in 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 need not be converted, although this is preferred. In addition, U residues generally cannot participate in LNA, so wherever U appears in the overhang, it will not become LNA. Also, the nt added at the 3’ end can never be U because the design rules for sequence selection enforce G or C at the terminal position); 3) The first 5’ terminal nt in the passenger strand was converted to the corresponding LNA; 4) Adenosine (A) in the passenger strand was converted to the corresponding 2’-O-methyl (2’-OMe) A nt (at least 5 2’-OMeA nt) and distributed as evenly as possible throughout the passenger strand sequence.If Ant is limited, Unt is converted to 2'-OMe nt, resulting in a total of at least five 2'-OMe A or Unt; 5) The second U in the di-uridine (UU) overhang at the 3'-end of the passenger strand is also converted to 2'-OMe U; 6) Except for the 5'-terminal LNA nt (which cannot be U) in the passenger strand, five C, G, or Ant in the passenger strand are converted to the corresponding LNA nt, distributed as evenly as possible in the passenger strand sequence. siRNAs and miRNA mimics with the above modifications are synthesized by standard oligonucleotide synthesis, annealed into duplexes, purified, and verified. The purified RNA duplexes are suspended in a buffer made of sterile deoxy / deionized water, aliquoted, and stored at -20 °C or -80 °C for up to one year. Freeze / thaw cycles are not recommended, but if kept to a minimum, efficiency may not be significantly reduced. RNA duplexes can be combined by mixing these suspensions for multiplexing as needed for specific indications.

[0187] In vivo transfection.

[0188] Naked dsRNA duplexes in a sterile liquid suspension are administered to the bloodstream of a subject at empirically determined concentrations. Administration can reach the peripheral blood circulation by any route, including but not limited to intravenous or intra-arterial, via a needle, catheter, or any other direct administration route to the blood vasculature, or by an indirect route, such as intramuscular, subcutaneous, direct transdermal administration or absorption into a patch, intraocular, intranasal, or intraperitoneal. Oral administration may be feasible. The dosage and frequency of administration depend on the target gene and the specific indication. Generally, naked dsRNA is administered at approximately 0.2 mg / kg per day, but this can vary. The total duration of administration also varies depending on the indication.

[0189] Ex vivo and in vitro transfection.

[0190] Naked dsRNA duplexes in a sterile liquid suspension are directly added to stored platelet concentrates or megakaryocytes or platelets cultured in vitro at empirically determined concentrations suitable for each desired target mRNA or mRNA. Generally, the concentration is between approximately 100 - 1000 nM. Transfection generally begins at the time of collection of the platelet concentrate from a donor or megakaryocyte or platelet culture or shortly after collection, but can begin at any time depending on the indication and can be repeated daily during storage prior to infusion or throughout the duration of cell culture as needed for the indication.

[0191] Table 1: Shows target genes and RNA oligonucleotide sequences for targeting these genes.

[0192]

[0193]

[0194]

[0195]

[0196]

[0197] Sequence key points:

[0198] Bold italic = locked nucleic acid (LNA) modification

[0199] Bold = 2'-OMe nucleotide or 2'-F modification.

[0200] All sequences are 5' --> 3'

[0201] Regarding the evaluation of the therapeutic window of P2ry12 siLNA and ticagrelor in mice

[0202] In age-matched wild-type C57Bl6 / J mice, the therapeutic window (TW) was evaluated as the interval between antithrombotic and bleeding effects within a 3-log dose range of ticagrelor or P2ry12 siLNA (gene name, P2y12; protein name, P2y12) after intravenous administration. A scrambled siLNA without predicted human mRNA targets was used as a control. Thrombosis was tested using FeCl3 carotid artery injury and Doppler monitoring of blood flow downstream of the injury site, and bleeding was tested by tail clip amputation, blood collection, and body weight measurement to determine blood loss. The tests were performed in anesthetized mice 5 minutes after ticagrelor administration and 24 hours after siLNA administration.

[0203] Thrombosis: The left carotid artery was exposed, excess tissue was removed, and it was placed on a Doppler probe connected to a blood flow monitor. The artery was elevated (temporarily interrupting blood flow), and a 2 x 2 mm square filter paper soaked with 4 μL of 7.5% FeCl3 was placed on the exposed carotid artery for 90 seconds to induce injury. After removing the filter paper, blood flow was monitored for 20 min. In a typical experimental setting, without antithrombotic treatment, an occlusive blood clot (thrombosis) would form within approximately 9 - 11 minutes after vascular injury, resulting in downstream blood flow blockade. In some cases, treatment that reduced blood clot growth to a moderate level would be observed as an incomplete recovery of blood flow through the narrowed blood vessel. Thrombosis is represented by a square and is expressed as the percentage of blood flow blockade at the 20-min endpoint after injury compared to the starting flow rate, calculated from the average flow rates at the start and the final 100 seconds.

[0204] Bleeding: Cut the tip of the mouse tail with a diameter of 1 mm (measured with a caliper) using a new blade, and immerse the tail in warm saline for 20 min. The increase in the weight of the saline tube after measurement provides the blood loss within the measurement time range. Hemostatic inhibition is shown as circles and calculated as blood loss / mouse initial body weight (wt, g / g), expressed as the fold change in the average blood loss / mouse body weight observed compared to the vehicle control (n = 18).

[0205] The therapeutic window (TW) was calculated as the ratio of the dose that caused a 3-fold increase in blood loss compared to the control (hemostatic inhibition, blue dotted line) to the dose that caused a 50% restoration of blood flow (thrombosis, red short dashed line). Results are shown as ±s.e.m. n = 3 - 8 for each experiment. n.s., not significantly different from the control.

[0206] Example 1: Selective in vitro transfection of bone marrow megakaryocytes by naked double - stranded miRNA mimics.

[0207] Suspend the freshly isolated murine bone marrow cell suspension from femurs without washing (e.g., using the existing plasma and ribonuclease to mimic the in vivo state) and incubate it with the indicated concentration of AF488 (green)-conjugated miL-223-3p ds mimics for 1 hour, then perform real-time imaging or fix and process. Perform real-time imaging of AF488 on the whole unfixed bone marrow cell suspension captured on poly- L -lysine (PLL)-coated slides ( Figure 1A ). Only megakaryocytes (MK) show green fluorescence, indicating MK-specific uptake. Counterstain the fixed bone marrow cells to examine the MK / platelet marker Cd41, and the nuclear dye is DAPI ( Figure 1B ). Only MK shows miLNA uptake, while other bone marrow cells do not. In this experiment, the bone marrow cells were co-incubated with both miLNA-AF488 and the unmodified form of miR-223-3p conjugated to AlexaFluor647. No fluorescence is shown in the figure below, indicating degradation of the unmodified miRNA mimic. Counterstain the cells in ( Figure 1B ) to examine the two MK / platelet markers Cd42d and Cd41, and it is shown that MK uptake occurs at different stages including the immature growth stage (small MK) ( Figure 1C ).

[0208] Example 2: Selective in vivo transfection of bone marrow megakaryocytes and platelets by infused naked double - stranded miRNA mimics.

[0209] Infuse the miL-223-3p conjugated to AlexaFluor488 in ( Figure 1A ) into the tail vein of WT mice. The extraction and processing of the bone marrow cell suspension are the same as in ( Figures 1A - 1C)。The whole bone marrow cell suspension extracted 2 hours after infusion, which was captured on poly-L-lysine (PLL)-coated slides and imaged in real time with respect to AF488( Figure 2A )。Only megakaryocytes (MKs) showed green fluorescence, indicating MK-specific in vivo uptake. Fixed bone marrow cells were counterstained to examine MK / platelet markers Cd42d and Cd41, and the nuclear dye was DAPI( Figure 2B )。At 18 and 72 hours after infusion, only MKs and platelets showed miLNA uptake, while other bone marrow cells did not. Analysis of miL uptake (by fluorescence) of cells in ( Figure 2B ) by flow cytometry, using Cd41 / Cd42d and size (forward scatter FSC) to identify MKs( Figure 2C )。In this experiment, the spleen was also extracted and the splenocyte suspension was analyzed at 72 hours, jointly showing robust MK uptake and miLNA retention in bone marrow and splenic MKs. Peripheral blood cells from the same animals as above were fixed and captured on cover slips, and platelets were identified with Cd42d antibody. The in vivo uptake efficiency of platelet miLNA was close to 100%, as previously observed for washed platelets in vitro( Figure 2D )。

[0210] Example 3: Infused naked RhoA siLNA selectively inhibits RhoA protein expression in bone marrow megakaryocytes and regulates megakaryocyte development and platelet production in vivo.

[0211] These experiments demonstrated the effects of infused naked siLNA on megakaryocyte (MK) target gene expression and cellular function. As detailed previously, RhoA or Cel-miL-67 siLNA was infused into WT mice. Peripheral blood was sampled daily to evaluate blood cells, and bone marrow was extracted 96 hours after the start of the experiment. Cel-miL-67 was used as a negative control. RhoA siLNA selectively repressed RhoA protein expression in bone marrow MKs after infusion( Figure 3A )。Using RhoA antibody, MKs were labeled in combination with Cd42d antibody, and propidium iodide (PI) was used to label nuclear DNA. RhoA levels were measured by flow cytometry in permeabilized whole bone marrow cell suspensions. (Left panel) RhoA expression levels in MKs as a function of ploidy classification determined by PI staining. Due to cytoplasmic expansion, RhoA expression increased as expected in larger and more mature MKs, but this increase was repressed by RhoA siLNA. (Right panel) Nucleated (PI+) non-MK bone marrow cells showed no change in RhoA protein levels. RhoA siLNA regulates RhoA-dependent MK function( Figure 3B) RhoA knockout mice and mice treated with RhoA inhibitors showed that acute RhoA inhibition led to an increase in MK ploidy. Compared with control siLNA, RhoA siLNA led to an increase in MK ploidy, indicating a specific functional role of RhoA inhibition in MK in vivo. Figure 3C showed that peripheral blood platelet counts increased with RhoA siLNA, corresponding to the increase in MK ploidy ( Figure 3B ). Using RhoA siLNA, the mean platelet volume did not change, confirming that the increase in MK ploidy drives an increase in platelet counts but does not generate larger platelets ( Figure 3D ).

[0212] Example 4: Infused naked P2y12 (P2ry12) siLNA reduces platelet ADP reactivity and prevents injury - induced arterial thrombosis in WT mice, phenocopying current first - line P2Y12 - targeted anti - platelet drugs (e.g., Plavix, cangrelor, ticagrelor).

[0213] Figures 4A - 4B showed that the infusion of naked P2y12 (P2ry12) siLNA reduced platelet ADP activation responses and prevented injury-induced arterial thrombosis in WT mice, phenocopying current first-line antiplatelet drugs targeting P2Y12. ( Figure 4A ) P2ry12 siLNA infusion reprogrammed platelets to have reduced ADP reactivity. At 24 hours after P2ry12 or scrambled control (CTL) siLNA infusion, platelet ADP responses in whole blood cell suspensions were tested by flow cytometry (FACS) to assess ADP-induced GpIIb / IIIa integrin activation (using JonA antibody) (upper panel) and α-granule secretion (using P-selectin antibody) (lower panel). p < 0.05. n = 3. Figure 4B ) WT mice were infused via the tail vein with 0.2 mg / kg of saline (control) or naked P2ry12 siLNA once daily for 4 days. At 96 hours (day 5), anesthetized mice were subjected to arterial injury of the carotid artery ("injury") by applying a filter paper soaked in 7.5% FeCl3 solution for 30 seconds, then the filter paper was removed and blood flow was restored. Blood flow was monitored with a Doppler probe downstream of the injury site until 30 minutes after injury.

[0214] As Figure 4B shown, as expected, between 5 - 12 minutes after injury, the arteries in control mice occluded, blocking blood flow. In contrast, the arteries in mice treated with naked P2ry12 siLNA (targeting the P2y12 ADP receptor, the target of current first-line antiplatelet antithrombotic drugs) remained patent, and no occlusion was observed within the experimental time frame. n = 3 for each experiment.

[0215] Example 5: Single injection of naked miR - 223 - 3p miRNA mimic transiently and potently increases platelet ADP reactivity. Figure 5A

[0216] These experiments demonstrated the effect of infused naked miLNA on circulating platelets. miR-223-3p (miL-223-3p) was used because this miRNA was found to target a key negative regulator of platelet response to ADP agonist; importantly, these targets turn over rapidly in platelets and must be translated constitutively. It was hypothesized that overexpression of miR-223-3p and miL-223-3p would result in direct platelet effects by repressing the target proteins in platelets - independent of the MK effect.

[0217] As Figure 5B shown, injection of miL-223-3p resulted in a mean 4-fold increase in platelet ADP reactivity evaluated 24 h after injection - measured by flow cytometry using the Jon / A antibody that recognizes the activated form of integrin GpIIb / IIIa, while control Cel-miL-67 or saline infusion had no effect on reactivity. The increased reactivity was transient, indicating depletion of the targeted miLNA and reconstitution of the targeted protein at later times. As Example 6: shown, the entire circulating platelet population showed a rightward shift in ADP reactivity. This result demonstrated a potent direct effect on circulating platelets, but an effect on MK could not be excluded. Single or repeated addition of naked ADAM17 siRNA to platelets in the form of storage concentrate protects against GP1bα loss. Figure 6A

[0218] For initial testing, ADAM17 or control (Cel-miR-67) siRNA (unmodified; the Cel-miR-67 sequence was identical to the Cel-miL-67 sequence used elsewhere) was obtained from Dharmacon and added to freshly isolated, washed human platelets for 1 h (as previously reported), after which the platelets were reconstituted with autologous plasma to generate platelet-rich plasma, which was then maintained at room temperature with gentle agitation for up to 96 h, mimicking the storage conditions commonly used for platelet concentrates in transfusion recipients. The ADAM17 metalloprotease is known to cause progressive proteolytic cleavage of the GP1bαvWF receptor on platelets, resulting in reduced hemostatic function of stored platelets - a key component of platelet storage lesion. It was assumed that ADAM17 siRNA would be protected from such degradation. Note: Platelet counts remained equal within the experimental time frame (results not shown). Figure 6B Shown, single direct addition of naked ADAM17 siRNA to platelet storage concentrates consistently maintained total and surface expression of GP1bα in stored platelets for up to 48 h. After 48 h, GP1bα levels decreased to low levels similar to those of control siRNA, indicating turnover of ADAM17 and GP1bα. Figure 13showed that multiple additions of ADAM17 siRNA to stored platelet concentrates (by pelleting and washing the platelets, repeating the transfection, and then reconstituting with autologous plasma) - given at 0 hours and again at 48 hours - prolonged the maintenance of GP1bα in stored platelets. Based on these data, ADAM17 siLNA( Example 7: Single addition of naked ADAM17 siLNA to platelet storage concentrate protects against GP1bα functional loss. ) was designed and generated.

[0219] Figure 13

[0220] ADAM17 or control (Cel-miL-67) siLNA (modified as shown in Figure 7A ) was added directly to freshly isolated platelet-rich human plasma and then maintained for up to 96 hours at room temperature with gentle agitation, mimicking the storage conditions common for transfused recipient platelet concentrates.

[0221] At the indicated times, aliquots of platelet-rich plasma were transferred to an aggregometer at 37 °C and given 0.5, 1.0, or 1.5 mg / mL of the GP1bα cofactor ristocetin (Rs), which triggered GP1b-dependent platelet aggregation by cross-linking plasma vWF, and this was monitored over time by light transmission. Another aliquot was isolated for western blotting to assess the GP1bα expression level in platelets.

[0222] Figure 7B showed the change in the percentage of platelet aggregation over time after addition of the indicated concentration of ristocetin (Rs). Naked ADAM17 siLNA was added directly as a single dose to platelet storage concentrates on day 0, maintaining GP1bα functional responsiveness over the 5-day experimental time frame. These data demonstrate for the first time that, by this method, improvement of the cellular physiological functions of stored platelets. Example 8: Therapeutic window (TW) of P2ry12 siLNA and ticagrelor in mice. , and GP1bα expression in stored platelets. These results support the use of naked siLNA to modulate antiplatelet storage lesion. Precise timed addition of key targeted siLNA is expected to generate platelets with reactivity profiles similar to those of freshly isolated platelets, achieving maximal hemostatic efficacy in transfused recipients in a timed manner.

[0223] Figure 15

[0224] In a first series of experiments, siLNA was tested against murine P2y12 (mRNA, P2ry12; P2Y12 in humans) - the main ADP receptor on platelets (also expressed in some other cells) which, following secretion of ADP by partially stimulated platelets in response to other stimuli, is responsible for the so-called "secondary" feedback stimulation via autocrine and paracrine signalling, the ADP then stimulating a secondary response via P2Y12. The ADP-driven P2Y12 secondary activation response is a key tipping point for thrombus formation due to overgrowth of the blood clot. Current antiplatelet SOC non-aspirin monotherapies mainly focus on P2Y12 inhibitors (clopidogrel, prasugrel, cangrelor or ticagrelor). Dual antiplatelet therapy typically includes a P2Y12 inhibitor and aspirin. Aspirin, while beneficial and generally safe, has not solved the clinical thrombosis problem and is currently used less, especially in elderly patients who often experience adverse effects. The unmet need addressed here is the well-documented and unresolved induction of clinical bleeding in patients on P2Y12 monotherapy or dual therapy.

[0225] Figure 15 The data in represent testing of the therapeutic window of P2y12 siLNA versus the P2Y12 antagonist ticagrelor in a murine model - the safe dose range that provides an antithrombotic effect (therapeutic benefit) without causing excessive bleeding. As with all siLNA, P2y12 siLNA is designed to act as siRNA once internalized into platelets and / or megakaryocytes. The siRNA function is to specifically anchor to the target mRNA (P2y12 in this case) and prevent translation of the homologous protein (P2y12) via the cell's native RNA-induced silencing machinery.

[0226] From Example 9: P2y12 siLNA destabilizes thrombi and prevents thrombus formation in a P2y12 surface density - dependent manner. As can be seen, the TW of ticagrelor was determined to be 6.8, while the TW of P2ry12 siLNA was determined to be >100. Thus, the ratio of TWs, P2y12 siLNA / ticagrelor >14-fold. The TW of ticagrelor observed in mice was very consistent with the TW established previously in rats using a similar method and reflected the clinical observations in humans. The actual upper limit of the TW of P2ry12 siLNA remains unknown as increased bleeding was not observed at the highest test dose. In addition, low doses of 1 to 10 μg / kg also provided therapeutic benefit (blocking 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, scrambled control siLNA had no protective effect on thrombosis as occlusion was observed in all mice receiving scrambled siLNA, similar to untreated mice suffering from thrombotic injury.

[0227] Figure 17 Example 10: P2y12 siLNA selectively normalizes high ADP reactivity and P2y12 levels in newly generated platelets.

[0228] Twenty-four hours after a single IV injection of a range of P2y12 or scrambled siLNA, P2y12 surface expression was evaluated by flow cytometry using a fluorophore-conjugated P2y12 antibody as a function of testing thrombosis blockade. As Figure 18 shown, a threshold level of siLNA-mediated P2y12 inhibition was observed to destabilize thrombi and produce an antithrombotic effect. Approximately 20% inhibition of P2y12 was sufficient to destabilize thrombi and restore blood flow, while approximately 35 - 40% inhibition appeared sufficient to prevent the formation of any occlusion despite vascular injury.

[0229] Figure 18 Figure 18

[0230] Platelet reactivity was monitored as a function of platelet lifespan in vivo. As Figure 18 shown, newly generated platelets were less responsive to ADP (the physiological ligand for the P2y12 receptor) and thromboxane (another "sub" agonist blocked by aspirin) compared to the mean reactivity of the total population, while thrombin and convulxin reactivity (the major "prime" activating agonists) were similar throughout the lifespan. The P2y12 surface density was also found to be highest in new platelets relative to the total population ( Figure 18, right), and this high level of P2y12 decreases over time, indicating that P2y12 synthesis occurs mainly in megakaryocytes and / or newly formed platelets. P2y12 siLNA normalizes this increase in the highly expressed subset but has no effect on existing P2y12 levels, as predicted for siRNA-mediated gene silencing. Thus, P2ry12 siLNA reduces thrombosis by preferentially suppressing elevated P2y12 levels in younger platelets to normalize ADP responsiveness, while older platelets with reduced P2y12 synthesis are resistant to siLNA-mediated silencing because existing P2y12 protein is not targeted, and hemostasis is therefore maintained.

[0231] Using pulse-chase labeling with a single infusion of the Gp1bβ-X488 antibody, the reactivity of ( Example 11: PAR4 as a second (new) siLNA target in platelets. , left) and the ( Figure 20 , right) P2y12 surface expression of ex vivo mouse platelets were monitored throughout their lifespan. The Gp1bβ antibody conjugated to a fluorophore for tracking by flow cytometry was injected into the bloodstream, where they specifically label platelets without labeling other cells, but these antibodies are inert and do not activate platelets or cause them to be destroyed. In vivo antibody labeling reaches saturation within 1 hour. Thus, when tracked by flow cytometry from blood samples, Gp1bβ+ platelets (fluorophore positive, shown as solid lines) represent the entire platelet population at the time of pulse labeling. When platelets are extracted at later time points - in the case above, at 24-hour intervals after pulse labeling - Gp1bβ- platelets (fluorophore negative, shown as dashed lines) represent newly formed platelets that did not undergo pulse labeling at the "0" hour time point. Over time, the percentage of these Gp1bβ- platelets in the total platelet pool becomes increasingly large because pre-existing platelets are cleared over time (the lifespan of murine platelets is approximately 5 days). This is evident from the convergence of the red and blue lines at later time points.

[0232] In Example 12: Targeting of non - receptor cytoplasmic proteins by siLNA and anti - bleeding / pro - hemostatic effects. , the results are shown as relative integrin activation (top row) and α-granule secretion (bottom row) - two independent measures of platelet activation - or P2y12 levels compared to the total population in newly formed platelets (Gp1bβ-, dotted line) and existing platelets (Gp1bβ+, solid line). Upper right, control; lower right, 24 hours after P2ry12 siLNA injection. All results are ± s.e.m. n = 3 - 6.

[0233] ​

[0234] PAR4 is another platelet GPCR that is important for platelet activation. It is one of the two major thrombin receptors on human platelets, and thrombin is the main agonist for so-called "primary" platelet activation that initiates blood clot formation. Only Par4 is the sole thrombin receptor in mouse platelets. Small molecule antagonists of PAR4 have been developed as putative antithrombotic drugs. It is to be considered whether PAR4 knockout, as an alternative antithrombotic target to P2Y12, as a single target, and possibly in combination with P2Y12 antagonists or P2Y12 siLNA, can produce benefits. Unlike P2y12 / P2Y12, the mouse and human Par4 / PAR4 genes are so different that functional studies of clinical significance are best focused on the human gene (if possible). Therefore, humanized PAR4 mice were developed that carry the human PAR4 transgene (including all surrounding regulatory sequences) and lack mouse Par4, and were backcrossed for multiple generations on the WTC57Bl / 6J background. Platelet PAR4 has been studied previously, and studies have shown that the human PAR4 transgene expressed at similar levels on mouse platelets as on human platelets supports PAR4 functions mapped to human platelet physiology in these mice. Therefore, these mice (hereinafter referred to as "hPAR4" mice) provide a unique resource to test the inhibition of human PAR4 with designed and created human-targeted siLNA (gene and siLNA name, F2RL3). Intravenous injection of F2RL3 siLNAIV into these mice resulted in substantial inhibition of human PAR4 in their platelets within 24 hours.

[0235] Silencing of platelet hPAR4 resulted in a modest decrease in platelet responsiveness to a receptor-specific peptide agonist, which does not stimulate other PARs or receptors, called PAR4 activation peptide or PAR4-AP.

[0236] Since PAR4 is the only responsive thrombin receptor on these platelets, it is predicted that modest PAR4 inhibition will be sufficient to achieve an antithrombotic effect. As ​ shown, in the FeCl3 carotid artery injury model, intravenous injection of naked F2RL3siLNA - at the same dose that produced a modest decrease in platelet responsiveness to PAR4 agonist stimulation - was sufficient to block thrombus formation at 24 hours. However, although F2RL3 siLNA had an antithrombotic effect - achieved at the highest dose used so far to test P2y12 siLNA, which is 500 μg / kg - no increase in bleeding was observed in these mice compared to control hPAR4 or WT mice. The full therapeutic window of F2RL3 siLNA remains to be determined. These PAR4 data indicate that multiple siLNAs targeting different genes (mRNAs) are functional, as indicated by reduced target protein expression and expected functional effects.

[0237] ​

[0238] Gsα is a cytoplasmic small G protein involved in platelet signal transduction. Although P2y12 and PAR4 are different genes, their protein products are both members of the G protein-coupled receptor family. These data on Gsα in Figure 22 indicate that siLNA silences the expression of various types of protein targets. Notably, in circulating platelets, Gsα protein also exhibits different expression kinetics compared to P2y12. Gsα appears to be most highly expressed in older platelets. Thus, siLNA normalizes the expression of target proteins in the entire platelet population in a target-specific manner. In addition, the data in Figure 22 also demonstrate the anti-bleeding / pro-hemostatic in vivo effect of siLNA-mediated suppression of endogenous negative regulatory proteins in platelets, including Gsα (siLNA, Gnas) and IP (siLNA, Ptgir). The data in Figure 22 further demonstrate the rescue of acquired bleeding diathesis in mice by combined Gnas / Ptgir siLNA, which were acutely treated with a moderate dose of ticagrelor, resulting in increased bleeding and rescued by siLNA.

[0239] Example 13: miR-126 / miR-126* platelet and megakaryocyte specific uptake demonstrated by PCR

[0240] As an orthogonal method to study the selective in vivo uptake of naked mi / siLNA by megakaryocytes / platelets, miR-223 knockout (KO) mice were used to monitor the internalization of ectopically infused naked miL-223-3p in isolated cell populations sorted by FAC using specific markers by PCR. miR-223-3p was detected in all tested BM and blood cells of WT mice, but as expected, was absent in cells of miR-223 KO mice. After infusion of miL-223-3p into miR-223 KO mice, miR-223-3p was observed in blood platelets and bone marrow megakaryocytes, but the infused miLNA was not detected in blood or bone marrow white cells / leukocytes (WBC, including Cd45+ WBC progenitors). Since all these populations were single-cell sorted based on surface markers (and size, for megakaryocytes), heterotypic cell aggregates were eliminated. These results indicate that only megakaryocytes and platelets support the unique ability to internalize infused naked double-stranded miLNA, while other cells in hematopoietic tissues or blood do not.

[0241] Example 14: Anti-miR broad-spectrum hematopoietic cell targeting

[0242] Compared with platelet / megakaryocyte-specific in vivo targeting of double-stranded miLNA and siLNA, it was found that single-stranded 8-nucleotide anti-miR (anti-miR-223-3p) - composed of locked nucleic acid (LNA), mimicking McKenzie / Tsygankov anti-miR-148a and carrying a 3'-FAM fluorophore (anti-miL) - was internalized by blood platelets and, within 1 hour of intravenous administration in WT mice, was internalized in vivo by red blood cells (RBCs) and white blood cells / leukocytes (WBCs), as Figure 25 shown. Notably, RBCs are much larger in volume than platelets, and WBCs are much larger in volume than RBCs; these relatively larger volumes compared to platelets explain the significantly lower relative mean fluorescence intensity (per cell) observed. Thus, all blood cells exhibit similar ability to internalize anti-miRs modeled on anti-miR-148a. Therefore, anti-miRs / antagomiRs do not possess the uniqueness of platelet / megakaryocyte-specific internalization and utilization that miLNA and siLNA possess.

[0243] Example 15: Long-term modulation of platelet reactivity by siLNA using a subcutaneous osmotic pump

[0244] As Figure 26 shown, platelet reactivity regulation can be achieved by subcutaneous administration, thus providing an alternative route to parental administration. In mice carrying a subcutaneous osmotic pump with stable release of P2ry12 siLNA, platelet ADP responses were inhibited, indicating that the siLNA payload was delivered to the bloodstream and platelets, and platelet function was regulated by subcutaneous administration of siLNA. Enumerated Embodiments

[0245] The following exemplary embodiments are provided, and their numbers should not be construed as indicating a level.

[0246] Embodiment 1 provides a method for selectively regulating the gene expression of one or more target genes in the cells of a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a naked RNA oligonucleotide,

[0247] wherein the cells are at least one selected from megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide.

[0248] Embodiment 2 provides the method of Embodiment 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.

[0249] Embodiment 3 provides the method of Embodiments 1-2, wherein the naked RNA oligonucleotide is selected from miRNA, siRNA, and any combination thereof.

[0250] Embodiment 4 provides the method of Embodiments 1-3, wherein the naked RNA oligonucleotide does not contain any modified nucleotides / bases.

[0251] Embodiment 5 provides the method of Embodiments 1-4, wherein the transfection is carried out without using a synthetic carrier or adjuvant.

[0252] Embodiment 6 provides the method of Embodiments 1-5, wherein the naked RNA oligonucleotide optionally contains at least one modification selected from locked nucleic acid (LNA), 2'-fluorobase, and 2'-O-methylated base.

[0253] Embodiment 7 provides the method of Embodiments 1-6, wherein the naked RNA oligonucleotide is heat-stable.

[0254] Embodiment 8 provides the method of Embodiments 1-7, wherein the naked RNA oligonucleotide is non-immunogenic.

[0255] Embodiment 9 provides the method of Embodiments 1-8, wherein the naked RNA oligonucleotide is resistant to ribonuclease (RNase) cleavage.

[0256] Embodiment 10 provides the method of Embodiments 1-9, wherein the composition is administered intravenously.

[0257] Embodiment 11 provides the method of Embodiments 1-10, wherein the administration alters megakaryocyte development, platelet production, megakaryocyte function, and / or platelet function.

[0258] Embodiment 12 provides the method of Embodiments 1-11, wherein the subject requires at least one of the following:

[0259] i. Antiplatelet therapy,

[0260] ii. Anti-inflammatory therapy for treating thromboinflammation,

[0261] iii. Treatment of thrombocytopenia, thrombocytosis, and other pathological manifestations of disrupted megakaryocyte development,

[0262] iv. Platelet transfusion with platelets free from platelet storage damage,

[0263] v. Treatment of thrombosis,

[0264] vi. Treatment of acquired bleeding disorders, and

[0265] vii. Treatment of hereditary bleeding disorders.

[0266] Embodiment 13 provides the method of Embodiments 1-12, wherein the composition comprises a saline solution.

[0267] Embodiment 14 provides the method of Embodiments 1-13, wherein the subject is a mammal.

[0268] Embodiment 15 provides the method of Embodiments 1-14, wherein the mammal is a human.

[0269] Embodiment 16 provides a method for selectively regulating the gene expression of one or more target genes in cells in vitro or ex vivo, the method comprising transfecting the cells with a naked RNA oligonucleotide, wherein the cells are at least one selected from megakaryocytes and platelets.

[0270] Embodiment 17 provides the method of Embodiment 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.

[0271] Embodiment 18 provides the method of Embodiments 16-17, wherein the cells are derived from at least one selected from cell culture systems, concentrates, cell suspensions, tissue homogenates, organoids, tissues, and organs.

[0272] Embodiment 19 provides the method of Embodiments 16-18, wherein the cell culture is a stem cell culture.

[0273] Embodiment 20 provides the method of Embodiments 16-19, wherein the concentrate is a platelet-rich plasma storage concentrate containing autologous plasma.

[0274] Embodiment 21 provides the method of Embodiments 16-20, wherein the transfection is carried out without using synthetic carriers or adjuvants.

[0275] Embodiment 22 provides the method of Embodiments 16-21, wherein the naked RNA oligonucleotide is thermostable.

[0276] Embodiment 23 provides the method of Embodiments 16-22, wherein the naked RNA oligonucleotide is non-immunogenic.

[0277] Embodiment 24 provides the method of Embodiments 16-23, wherein the naked RNA oligonucleotide is resistant to cleavage by ribonuclease (RNase).

[0278] Embodiment 25 provides a method for delivering a naked RNA oligonucleotide to a target tissue and / or cell of a subject, wherein the method comprises using platelets transfected with the naked RNA oligonucleotide as a carrier to deliver the naked RNA oligonucleotide to the target tissue and / or target cell.

[0279] Embodiment 26 provides the method of Embodiment 25, 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.

[0280] Embodiment 27 provides the method of Embodiments 25-26, wherein the target cell is one selected from tumor cells, white blood cells, or inflammatory cells.

[0281] Embodiment 28 provides the method of Embodiments 25-27, wherein the target tissue comprises endothelial cells.

[0282] Embodiment 29 provides a composition for selectively regulating the gene expression of one or more target genes in the cells of a subject, wherein the composition comprises cells transfected with a naked RNA oligonucleotide, and the cells are at least one selected from megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide.

[0283] Embodiment 30 provides the composition of Embodiment 29, wherein the naked RNA oligonucleotide is suspended in a sterile saline solution.

[0284] Embodiment 31 provides a composition for selectively regulating the gene expression of one or more target genes in cells ex vivo or in vitro, wherein the composition comprises cells transfected with a naked RNA oligonucleotide, and the cells are at least one selected from megakaryocytes and platelets.

[0285] Embodiment 32 provides the composition of Embodiment 31, wherein the cells are derived from at least one selected from cell culture systems, concentrates, cell suspensions, tissue homogenates, organoids, tissues, and organs.

[0286] Embodiment 33 provides a kit, which includes a composition having a naked RNA oligonucleotide for selectively regulating the expression of at least one gene in cells and instruction materials for its use, wherein the cells are at least one selected from megakaryocytes, platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide.

[0287] Other embodiments

[0288] The recitation of a list of elements in any definition of a variable herein includes defining the variable as any single element or combination (or sub-combination) of the listed elements. The recitation of an embodiment herein includes the embodiment as any single embodiment, or in combination with any other embodiment or part thereof.

[0289] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

[0290] Although the invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and variations of the invention can be devised by other persons skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A method for selectively regulating the 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 megakaryocytes, circulating platelets, and platelets generated from megakaryocytes transfected with the naked RNA oligonucleotide.

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 miRNA, siRNA, and any combination thereof.

4. The method according to claim 1, wherein the naked RNA oligonucleotide does not contain any modified nucleotides / bases.

5. The method according to claim 1, wherein the transfection is carried out without using a synthetic carrier or adjuvant.

6. The method according to claim 1, wherein the naked RNA oligonucleotide optionally comprises at least one modification selected from locked nucleic acid (LNA), 2'-fluorobase, and 2'-O-methylated base.

7. The method according to claim 6, wherein the naked RNA oligonucleotide is heat-stable.

8. The method according to claim 6, wherein the naked RNA oligonucleotide is non-immunogenic.

9. The method according to claim 6, wherein the naked RNA oligonucleotide is resistant to cleavage by ribonuclease (RNase).

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

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

12. The method according to claim 1, wherein the subject requires at least one of the following: i. Antiplatelet therapy, ii. Anti-inflammatory therapy for treating thromboinflammation, iii. Treatment of other pathological manifestations of thrombocytopenia, thrombocytosis, and disrupted megakaryocyte development, iv. Platelet transfusion that requires protection from platelet storage damage or transfusion due to the subject's platelet disorder, v. Treatment of thrombosis, vi. Treatment of acquired bleeding disorders, and vii. Treatment of hereditary bleeding disorders.

13. The method according to claim 1, wherein the composition comprises a saline solution.

14. The method according to claim 1, wherein the subject is a mammal.

15. The method according to claim 14, wherein the mammal is a human.

16. A method for selectively regulating the gene expression of one or more target genes in cells in vitro or ex vivo, the method comprising transfecting the cells with a naked RNA oligonucleotide, wherein the cells are at least one selected from megakaryocytes and platelets.

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 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.

19. The method according to claim 18, wherein the cell culture is a stem cell culture.

20. The method according to claim 18, wherein the concentrate is a platelet-rich plasma concentrate containing autologous plasma.

21. The method according to claim 16, wherein the transfection comprises transfection without using a synthetic carrier or adjuvant.

22. The method according to claim 16, wherein the naked RNA oligonucleotide is heat-stable.

23. The method according to claim 16, wherein the naked RNA oligonucleotide is non-immunogenic.

24. The method according to claim 16, wherein the naked RNA oligonucleotide is resistant to cleavage by ribonuclease (RNase).

25. A method for delivering a naked RNA oligonucleotide to a target tissue and / or cell of a subject, wherein the method comprises using platelets transfected with the naked RNA oligonucleotide as a carrier to deliver the naked RNA oligonucleotide to the target tissue and / or target cell.

26. The method according to claim 25, 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.

27. The method according to claim 25, wherein the target cells are selected from tumor cells, white blood cells, or inflammatory cells.

28. The method according to claim 25, wherein the target tissue comprises endothelial cells.

29. A composition for selectively regulating the gene expression of one or more target genes in cells of a subject, wherein the composition comprises cells transfected with a naked RNA oligonucleotide, and 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.

30. The composition according to claim 29, wherein the naked RNA oligonucleotide is suspended in a sterile saline solution.

31. A composition for selectively regulating the gene expression of one or more target genes in cells ex vivo or in vitro, wherein the composition comprises cells transfected with a naked RNA oligonucleotide, and wherein the cells are at least one selected from the group consisting of megakaryocytes and platelets.

32. The composition according to claim 31, 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.

33. A kit comprising a composition having a naked RNA oligonucleotide for selectively regulating the expression of at least one gene in cells and instructional materials for its use, wherein 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.

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