Pharmaceutical composition for use in treatment of venous thromboembolism

Abstract

The present invention relates generally to human medicine, and more specifically microRNA (miR)-replacement therapy for management of Venous Thromboembolism (VTE). In particular, the present invention provides pharmaceutical compositions comprising a miR mimic and a pharmaceutically acceptable carrier. The pharmaceutical compositions disclosed herein may be administered systemically, in particular by intravenous administration (IV).

Description

[0001] PHARMACEUTICAL COMPOSITION FOR USE IN TREATMENT OF VENOUS THROMBOEMBOLISM

[0002] Field of the invention

[0003] The present invention relates generally to human medicine, and more specifically microRNA (miR)-replacement therapy for management of Venous Thromboembolism (VTE). In particular, the present invention provides pharmaceutical compositions comprising a miR mimic and a pharmaceutically acceptable carrier. The pharmaceutical compositions disclosed herein may be administered systemically, in particular by intravenous administration (IV). Further, the present invention also relates to a kit comprising a pharmaceutical composition, comprising a miR mimic and a pharmaceutically acceptable carrier, and an instructional material for use thereof.

[0004] Background of the invention

[0005] A venous thrombus is a blood clot (thrombus) that forms within a vein. A common type of venous thrombosis is deep vein thrombosis (DVT), which is caused by a blood clot in a deep vein, usually in the leg or in the pelvic veins. If the thrombus breaks off (embolizes) and flows in the blood towards the lungs, it can become a pulmonary embolism (PE), a blood clot in the lungs. DVT and PE are both considered to represent medical conditions within the definition of VTE.

[0006] VTE, in the form of deep vein thrombosis (DVT) and pulmonary embolism (PE), is a common disease, with an annual incidence of 1-2 per 1000 individuals. It is associated with serious short- and long-term complications, including post thrombotic syndrome, recurrence, and death. VTE is most common in adults older than 60, but it may occur at any age. People who are obese, have cancer or are suffering from autoimmune disorders and cardiovascular diseases seem to be at higher risk of developing VTE than others.

[0007] VTE events typically occur after an individual has been subjected to one or more provoking factors (e.g. surgery, trauma, acute myocardial infarction, acute ischemic stroke or immobilization such as bed rest for more than 3 days) but may also occur even though a patient has not been subjected to any of the known provoking factors. These two sub-groups of VTE are typically referred to as provoked VTE and unprovoked VTE respectively.

[0008] Today’s prophylactic treatment of VTE is limited to administration of various kinds of anticoagulants such as heparin, warfarin, apixaban, rivaroxaban and dabigatran. Patients may experience serious side effects such as excessive or spontaneous bleeding of anticoagulant drugs. This, in addition to the need for following meticulous dosage regimes is a clear disadvantage for patients in need of VTE treatment. A non-pharmaceutically approach for prophylactic treatment of VTE is the use of compression stockings.

[0009] Despite advances in thromboprophylaxis, time trend studies have shown a slight increase in the incidence of VTE during the past two decades. To reduce the health burden associated with VTE, there is an urgent need in the art to provide improved treatment strategies for VTE with as few side effects as possible. miRs (micro-RNAs) are small non-coding RNAs that post-transcriptionally downregulate gene expression through mRNA degradation or inhibition of translation. Since the discovery of miRs as a class of regulatory molecules, their critical role in many biological and pathological processes has become apparent.

[0010] In addition to their common intracellular localization, a substantial number of miRs can be detected extracellularly in various body fluids. Circulating miRNAs are stably present in plasma and have emerged as a promising class of biomarkers for many diseases, including cancer and cardiovascular diseases (CVDs). Given their potential contribution to the pathogenesis of a number of diseases, miRs have also been explored as therapeutic targets.

[0011] US 10034902 discloses methods for treatment of nerve disease / disorder using genetically modified stem cells overexpressing certain miRs and / or where the expression of certain miRs are inhibited. This document also mentions treatment of traumatic lesions of the nervous system due to thrombosis or embolisms, to induce neurogenesis and provide survival factors to minimize insult to damaged neurons.

[0012] WO2017 / 163132 discloses that miR previously has been utilized in a method for promoting wound healing in patients, where the method comprises contacting the wound with a composition comprising one or more miRs. Use of exosomes containing miRs is demonstrated.

[0013] W02012 / 020308 discloses compositions and methods for treating acute tissue damage based on “pathfinder calls” and / or associated miR. The acute tissue damage may be related to ischemic conditions resulting from i.e. blood clots, thrombosis, atherosclerotic plaque or other obstructions of vasculature.

[0014] WO2017 / 207855 discloses miRs (miR-124, miR-145, miR-20a, miR-296, miR-34a, miR-374, miR-19b, and miR-15a) for use in diagnosis of primary antiphospholipid syndrome. International Journal of molecular medicine, 18 Dec 2019, XP55729116, GR ISSN: 1107-3756, DOI: 10.3892 / ijmm.2019.4434 demonstrates an in-vitro antiinflammatory effect of an anti-miR-15a-5p mimic on pulmonary artery smooth muscle cells.

[0015] WO2013 / 192576 discloses treatment of cardiovascular conditions by using an inhibitor of miR-15a.

[0016] W02019 / 028094 discloses compositions comprising an isolated cortical bone stem cell-derived exosome and at least one RNA molecule. It is suggested that said compositions may be useful for treating a cardiovascular disease or disorder in a subj ect.

[0017] CN1 11184734 suggests that hsa-miR-15a-5p or hsa-miR-16-5p may be useful for treating cardiac hypertrophy.

[0018] Despite a noteworthy progress of miR research in CVDs, there is still a lack of knowledge on the distinctive miR patterns specific for VTE in the general population.

[0019] Previous studies have described differential expression patterns of certain miRs in patients with acute PE, such as miR-28-3p as a potential plasma marker for diagnosis of pulmonary embolism (Thromb Res, 2016. 138: p. 91-95).

[0020] Another study has been done on patients with suspected first episode of DVT, discussing the diagnostic potential of miR signatures (Thromb Haemost, 2016. 116(2): p. 328-36). This scientific paper has also been cited elsewhere (Thromb Haemost, 2023. 21 :7-17).

[0021] However, findings regarding miR expression in VTE patients observed in the aforementioned studies were inconsistent. Moreover, the studies were limited by relatively small sample sizes and were conducted using a case-control design. Therefore, the expression profile of these miRs could be merely a consequence rather than a mediator of the thrombotic disease. Whether miR levels are associated with future risk of VTE remains unknown.

[0022] Summary of the invention

[0023] The present inventors have solved the above-mentioned needs by providing a pharmaceutical composition comprising a miR mimic which counteracts some of molecular mechanisms involved in the pathogenesis of VTE. A first aspect of the present invention relates to a pharmaceutical composition for use in the treatment of venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic, such as miR-15a-5p and in particular human miR-15a-5p, and a pharmaceutically acceptable carrier.

[0024] A second aspect of the present invention relates to a method of treating venous thromboembolism (VTE) in a subject in need thereof, comprising administering to the subject a composition comprising a pharmaceutically effective amount of a miR- 15a-5p mimic and a pharmaceutically acceptable carrier.

[0025] A third aspect of the present invention relates to a kit comprising a pharmaceutical composition and an instructional material for use thereof; wherein the pharmaceutical composition comprises a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier; and the instructions material comprises instructions for treating VTE in a subject, wherein the treating includes administering the pharmaceutical composition to the subject. miR15a-5p mimic

[0026] It is preferred that the miR-15a-5p mimic is human.

[0027] In one embodiment the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a seed sequence represented by nucleotides 2 to 9 from the 5’ end of SEQ ID NO:3 (AGCAGCAC). It is preferred that the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.

[0028] In another embodiment the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:3 by 0 to 5 nucleotide substitutions, preferably by 0 to 4 nucleotide substitutions, more preferably by 0 to 3 nucleotide substitutions, even more preferably by 0 to 2 nucleotide substitutions, and most preferably by 0 to 1 nucleotide substitutions or by 0 nucleotide substitutions; with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:3. It is preferred that the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.

[0029] In another embodiment the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:4 by 0 to 4 nucleotide substitutions, preferably by 0 to 3 nucleotide substitutions, more preferably by 0 to 2 nucleotide substitutions, even more preferably by 0 to 1 nucleotide substitutions, and most preferably by 0 nucleotide substitutions; with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:4. It is preferred that the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.

[0030] In another embodiment the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO: 5 by 0 to 3 nucleotide substitutions, preferably by 0 to 2 nucleotide substitutions, more preferably by 0 to 1 nucleotide substitutions and most preferably by 0 nucleotide substitutions; with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:5. It is preferred that the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.

[0031] In another embodiment the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:6 by 0 to 2 nucleotide substitutions, more preferably by 0 to 1 nucleotide substitutions and most preferably by 0 nucleotide substitutions; with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:6. It is preferred that the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.

[0032] In another embodiment the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:7 by 0 to 4 nucleotide substitutions, preferably by 0 to 3 nucleotide substitutions, more preferably by 0 to 2 nucleotide substitutions, even more preferably by 0 to 1 nucleotide substitutions, and most preferably by 0 nucleotide substitutions; with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 1 to 8 from the 5’ end of SEQ ID NO:7. It is preferred that the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.

[0033] In another embodiment the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO: 8 by 0 to 3 nucleotide substitutions, preferably by 0 to 2 nucleotide substitutions, more preferably by 0 to 1 nucleotide substitutions and most preferably by 0 nucleotide substitutions; with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 1 to 8 from the 5’ end of SEQ ID NO:8. It is preferred that the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.

[0034] In another embodiment the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:9 by 0 to 2 nucleotide substitutions, more preferably by 0 to 1 nucleotide substitutions and most preferably by 0 nucleotide substitutions; with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 1 to 8 from the 5’ end of SEQ ID NO:9. It is preferred that the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.

[0035] It is preferred that the oligonucleotide consists of from 15 to 40 linked nucleobases, such as 15 to 35 linked nucleobases, 15 to 30 linked nucleobases, 15 to 25 linked nucleobases, 15 to 22 linked nucleobases, 17 to 35 linked nucleobases, 17 to 30 linked nucleobases, 17 to 25 linked nucleobases, 15 to 22 linked nucleobases, 19 to 35 linked nucleobases, 19 to 30 linked nucleobases, 19 to 25 linked nucleobases, 19 to 22 linked nucleobases, 21 to 35 linked nucleobases, 21 to 30 linked nucleobases, 21 to 25 linked nucleobases or 21 to 22 linked nucleobases.

[0036] In a more preferred embodiment the oligonucleotide consists of from 15 to 29 linked nucleobases, 17 to 27 linked nucleobases, 19 to 25 linked nucleobases, 21 to 23 linked nucleobases, or 22 linked nucleobases.

[0037] In an even more preferred embodiment the oligonucleotide consists of from 19 to 25 linked nucleobases, such as 21 to 23 linked nucleobases.

[0038] In the most preferred embodiment the oligonucleotide consists of 22 linked nucleobases.

[0039] In another embodiment

[0040] - the miR15a-5p mimic is an oligonucleotide;

[0041] - the oligonucleotide comprising a first nucleotide sequence derived from any one of SEQ ID NO:3 to 9 by 0 to 2 nucleotide substitutions, preferably by 0 to 1 nucleotide substitutions, and even more preferably by 0 nucleotide substitutions; with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:3; and

[0042] - the oligonucleotide consists of from 15 to 40 linked nucleobases.

[0043] In a preferred embodiment according to the present invention, the miR15a-5p mimic is an oligonucleotide; the oligonucleotide comprising a nucleotide sequence of any one of SEQ ID NO:3 to 9; and the oligonucleotide consists of from 15 to 40 linked nucleobases.

[0044] In an even more preferred embodiment according to the present invention, the miR15a-5p mimic is an oligonucleotide; and the oligonucleotide is any one of SEQ ID NO:3-9. In the most preferred embodiment according to the present invention, the miR15a- 5p mimic is an oligonucleotide; and the oligonucleotide is SEQ ID NO:3.

[0045] In one embodiment, the oligonucleotide is double-stranded.

[0046] In one embodiment, the oligonucleotide is a RNA oligonucleotide.

[0047] Carrier

[0048] In one embodiment according to the present invention the pharmaceutically acceptable carrier is a liposome, the liposome preferably being suitable for in vivo delivery of miRs in general, in particular the miR15a-5p mimic.

[0049] In another embodiment the pharmaceutically acceptable carrier is a lipid nanoparticle, the lipid nanoparticle preferably being suitable for in vivo delivery of miRs in general, in particular the miR15a-5p mimic.

[0050] In a preferred embodiment the pharmaceutically acceptable carrier is Invivofectamine 3.0 Reagent kit provided by Thermo Fisher Lot: 2435463.

[0051] In one embodiment the miR15a-5p mimic is associated to, encapsulated within and / or embedded within the pharmaceutically acceptable carrier.

[0052] Venous thromboembolism

[0053] In one embodiment according to the present invention, venous thromboembolism (VTE) is deep vein thrombosis (DVT) and / or pulmonary embolism (PE).

[0054] In another embodiment venous thromboembolism (VTE) is deep vein thrombosis (DVT).

[0055] In another embodiment venous thromboembolism (VTE) is pulmonary embolism (PE).

[0056] In another embodiment venous thromboembolism (VTE) is provoked venous thromboembolism (VTE).

[0057] In another embodiment venous thromboembolism (VTE) is unprovoked venous thromboembolism (VTE).

[0058] Administration

[0059] In one embodiment according to the present invention, the pharmaceutical composition is to be administered by intravenous administration (IV). In another embodiment according to the present invention, the pharmaceutical composition is to be administered systemically.

[0060] Treatment

[0061] One embodiment according to the present invention relates to a pharmaceutical composition for use in the prophylactic and therapeutic treatment of venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier.

[0062] Another embodiment according to the present invention relates to a pharmaceutical composition for use in the prophylactic treatment of venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier.

[0063] Another embodiment according to the present invention relates to a pharmaceutical composition for use in the therapeutic treatment of venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier.

[0064] One embodiment according to the present invention relates to a pharmaceutical composition for use in the prophylactic and therapeutic treatment of provoked venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier.

[0065] Another embodiment according to the present invention relates to a pharmaceutical composition for use in the prophylactic treatment of provoked venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier.

[0066] Another embodiment according to the present invention relates to a pharmaceutical composition for use in the therapeutic treatment of provoked venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier.

[0067] One embodiment according to the present invention relates to a pharmaceutical composition for use in the prophylactic and therapeutic treatment of unprovoked venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier. Another embodiment according to the present invention relates to a pharmaceutical composition for use in the prophylactic treatment of unprovoked venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier.

[0068] Another embodiment according to the present invention relates to a pharmaceutical composition for use in the therapeutic treatment of unprovoked venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier.

[0069] Chemical modification

[0070] In one embodiment according to the present invention, the miR15a-5p mimic is an oligonucleotide, wherein the oligonucleotide comprises at least one phosphorothioate linkage.

[0071] In one embodiment according to the present invention, the miR15a-5p mimic is an oligonucleotide, wherein the oligonucleotide comprises a plurality of phosphorothioate linkages.

[0072] In another embodiment the miR15a-5p mimic is an oligonucleotide, wherein the oligonucleotide comprises at least one 2’ -fluoro base.

[0073] In another embodiment the miR15a-5p mimic is an oligonucleotide, wherein the oligonucleotide comprises a plurality of 2’ -fluorobases.

[0074] In another embodiment the miR15a-5p mimic is an oligonucleotide, wherein the oligonucleotide comprises at least one 2’ -methoxy base.

[0075] In another embodiment the miR15a-5p mimic is an oligonucleotide, wherein the oligonucleotide comprises a plurality of 2’ -methoxy bases.

[0076] In one embodiment the miR15a-5p mimic is an oligonucleotide, wherein the oligonucleotide comprises at least one phosphorothioate linkage and at least one 2’- fluoro base.

[0077] In one embodiment the miR15a-5p mimic is an oligonucleotide, wherein the oligonucleotide comprises at least one phosphorothioate linkage and at least one 2’- methoxy base. In one embodiment the miR15a-5p mimic is an oligonucleotide, wherein the oligonucleotide comprises at least one phosphorothioate linkage, at least one 2’- fluoro base and at least one 2’-methoxy base.

[0078] Brief description of the figures

[0079] Figure la-b illustrates incidence and size of venous thrombi over time in mice treated with miR-15a-5p and controls.

[0080] Control miR-15a-5p

[0081] Figure la shows the number of mice who had developed thrombi at 6h, 24h, and 48h. Figure lb shows mean thrombus volume (mm3) assessed by ultrasound at 6h, 24h, and 48h in the mice who had developed thrombi. Error bars represent standard error of the mean (SEM).

[0082] Figure 2a-c illustrates thrombosis and bleeding at 48h in mice treated with miR- 15a-5p and controls. Figure 2a shows the proportion (percent) of mice who had developed thrombosis at 48h (light grey=no thrombus; dark grey=thrombus). Figure 2b shows the thrombus weight in mg at 48h. Figure 2c shows the bleeding time in seconds at 48h.

[0083] Figure 3a-b illustrates incidence and size of venous thrombi over time in mice treated with antagomiR-15a and controls.

[0084] . O Control

[0085] M AntagomiR- 15a

[0086] Figure 3a shows the number of mice who had developed thrombi at 6h, 24h, and 48h. Figure 3b shows mean thrombus volume (mm3) assessed by ultrasound at 6h, 24h, and 48h in the mice who had developed thrombi. Error bars represent standard error of the mean (SEM).

[0087] Figure 4a-c illustrates thrombosis and bleeding at 48h in mice treated with antagomiR-15a and controls. Figure 4a shows the proportion (percent) of mice who had developed thrombosis at 48h (light grey=no thrombus; dark grey=thrombus). Figure 4b shows the thrombus weight in mg at 48h. Figure 4c shows the bleeding time in seconds at 48h.

[0088] Figure 5a-b illustrates the human mir-15a stem-loop structure. Figure 5a highlights the mature sequences of miR-15a-5p (nucleotide 14-35) and miR-15a-3p (nucleotide 51-72) respectively. Figure 5b highlights the seed sequences of miR-15a-5p (nucleotide 15-22) and miR-15a-3p (nucleotide 51-58) respectively. Figure 6 illustrates the biogenesis pathway of canonical miRNAs (Int. J. Mol.

[0089] Sci. 2020, 21(1), 132). miRNA biogenesis is mediated by multiple steps, including the transcription of primary miRNA transcripts, nuclear processing by Drosha, nucleocytoplasmic export by XPO5, cytoplasmic processing by Dicer, and formation of the RISC with Ago proteins.

[0090] Definitions

[0091] The term “thrombus” refers to the final product of the blood coagulation cascade in hemostasis. The term “thrombus” may be used interchangeably with “blood clot” or “clot” herein.

[0092] The term “embolism” refers to the lodging of a material inside a blood vessel which blocks or restricts blood flow. The material may be a thrombus.

[0093] The term “deep vein thrombosis” (DVT) refers to a blood clot in a deep vein, usually in the leg. DVT can sometimes also affect the arm or other veins.

[0094] The term “pulmonary embolism” (PE) refers to when a DVT clot breaks free from a vein wall, travels to the lungs and then blocks some or all of the blood supply of a pulmonary artery.

[0095] The term “provoked VTE” refers to VTE events occurring after an individual has been subject to provoking factors, e.g. surgery, trauma, or acute medical conditions, acute myocardial infarction, acute ischemic stroke, acute infections within 12 weeks before the event; cancer at the time of the VTE event; immobilization such as bed rest > 3 days or confinement to wheelchair within the last 8 weeks, or other factors described as provoking by a physician in the medical record e.g. intravascular catheter.

[0096] The term “unprovoked VTE” refers to a VTE event occurring when the patient has not been subject to any of the afore-mentioned provoking factors within the preceding 12 weeks.

[0097] The terms “miRNA” and “miR” are used interchangeably herein and relates to small, non-coding RNAs that are capable of modulating gene expression, including gene silencing, by one or more mechanisms.

[0098] The term “seed sequence” as used herein refers to a continuous string of 8 nucleotides. The seed region is a contributing factor to miR-mRNA binding. The term “miR mimics” as used herein refer to RNA molecules designed to mimic endogenous miRs.

[0099] The term “antagomiRs” as used herein refers to RNA molecules designed to silence endogenous miRs. Antagomirs are a kind of antisense oligonucleotide, as their sequence is complementary to their specific miR target.

[0100] Sequence Listing

[0101] “ RNA sequence;bAnatagomiR — 15a-5p; miR — 15a-5p mature sequence: bold; miR — 15a-5p seed sequence: bold and underlined.

[0102] Detailed description of the invention

[0103] Throughout the present disclosure relevant terms are to be understood consistently with their typical meanings established in the relevant art, i.e. the art of medicine, pharmacology, pharmaceutical chemistry, biology, biochemistry and physiology.

[0104] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of medicine, pharmacology, pharmaceutical chemistry, biology, biochemistry and physiology.

[0105] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.

[0106] Despite advances in thromboprophylaxis, time trend studies have shown a slight increase in the incidence of VTE during the past two decades. To reduce the health burden associated with VTE, there is an urgent need in the art to provide improved treatment strategies for VTE with as few side effects as possible.

[0107] In the search for a solution to the above-mentioned problems, the inventors of the present invention initiated an epidemiologic study looking for associations between miR plasma levels and events of VTE. Surprisingly, a clear association between low plasma levels of miR-15a-5p and events of VTE was found. Based on those results miR-15a-5p mimics emerged as promising candidates for prophylactic and / or therapeutic treatment of VTE.

[0108] In order to investigate whether the expression profile of miR-15a-5p was a mediator rather than merely a consequence of the thrombotic disease, the inventors decided to initiate an in-vivo study looking at protective impact of miR-15a-5p on VTE risk (example 1).

[0109] Mice were subjected to surgical treatment which promotes development of thrombi in the inferior vena cava (IVC). Prior to this treatment, vehicle (control) or miR- 15a-5p was administrated into the tail vein of the mice, and thrombus formation and size were monitored by ultrasound imaging at 6-, 24- and 48-hours post-surgery.

[0110] At 48 hrs, a bleeding test was conducted and thereafter the inferior vena cava (IVC) inspected for presence of thrombi and thrombus size / weight after death.

[0111] The incidence and size of venous thrombi over time in mice treated with miR-15a- 5p and vehicle are shown in figure la-b. Both the incidence (58% vs. 83%) and the thrombus volume among those that developed thrombi (12 vs 15 mm3) were lower in mice treated with miR-15a-5p compared to mice treated with vehicle. Similar findings occurred for the proportion of thrombus and thrombus weight after necropsy at 48 hours (figure 2a-b). Bleeding time conducted 48 hours after surgery displayed greater variation and on average was modestly prolonged among those treated with miR-15a-5p compared to vehicle treated mice.

[0112] In view of the above, it became clear that miR-15a-5p in fact prevents thrombosis formation in-vivo. To further support a protective role for miR-15a-5p on VTE risk, the inventors decided to conduct a study investigating whether inhibition of miR- 15a-5p would have an opposite effect to miR-15a-5p on thrombus formation in mice. Mice were subjected to surgical treatment which promotes development of thrombi in the inferior vena cava (IVC). Prior to this treatment, vehicle (control) or antagomiR-15a-5p was administrated into the tail vein of the mice, and thrombus formation and size were monitored by ultrasound imaging at 6-, 24- and 48-hours post-surgery. At 48 hrs, a bleeding test was conducted and thereafter the inferior vena cava (IVC) inspected for presence of thrombi and thrombus size / weight after death.

[0113] The surgeons noticed increased bleeding tendency already at surgery and at necropsy of all antagomir-15a-5p treated animals. Among the mice treated with antagomiR-15a-5p, 44% of the animals died due to PE, confirmed by a veterinarian, within 4 hours post-surgery. The incidence and size of venous thrombi over time in mice treated with antagomiR-15a-5p and vehicle are shown in figure 3. The number of mice with a venous thromboembolic event were similar between groups for antagomir-15a-5p treated mice and for vehicle treated mice (figure 3a), whereas the thrombus volume was slightly higher in antagomir-treated animals (figure 3b). Accordingly, few antagomir-treated animals were available for measurement of thrombus weight at necropsy (figure 4b), but thrombus weights appeared comparable between groups. The bleeding time were slightly prolonged among mice treated with antagomiR-15a-5p compared to vehicle treated mice (figure 4c).

[0114] Even though treatment with antagomiR-15a-5p had no obvious adverse effect on the incidence of thromboembolic events, the severity of the events with 44% of the antagomir-treated animals dying from PE combined with sever bleeding tendency during surgery and necropsy support that treatment with antagomiR-15a-5p provided an opposing phenotype compared to the miR-15-5p treated animals which further supports a protective role for miR-15a-5p on VTE risk.

[0115] In short, the present inventors have demonstrated a protective role for miR-15a-5p on VTE risk and that antagomiR-15a-5p provides an opposing phenotype compared to the miR-15-5p treated animals. Based on these results, the inventors have provided a new pharmaceutical composition, the composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier, for use in the treatment of VTE. miRs are small non-coding RNAs that span between 18-24 nucleotides. miRs regulate gene expression on a post-transcriptional level through base-pairing with complementary sequences of the 3 'untranslated region (UTR) of messenger RNAs (mRNA). This interaction results in gene silencing by cleavage of the mRNA strand or destabilization of the mRNA through shortening of its polyA tail or inhibiting the translation of the mRNA into proteins. miRs were first identified in the organism C. elegans in the early 1990s. Currently, more than 2500 mature miRs have been identified in the human genome. A significant fraction appears to be highly conserved in other animals. miR genes are transcribed by RNA polymerase II in the cell nucleus as long singlestranded RNAs (ssRNA) referred to as the “primary miR”, or “pri-miR” (figure 6). The ssRNA transcript forms a hairpin loop structure, which signals for RNA nuclease cleavage by a nuclear protein complex called Drosha / Dgcr8. The resulting short hairpin RNA is termed “precursor-miR” or “pre-miR”. Pre-miRs are released into the cytoplasm by the nuclear export protein exportin 5 and undergo cleavage by the enzyme Dicer into ~22 nucleotide-long mature miRs (figure 5a). After strand separation, the single-stranded mature miRs become incorporated into the miR induced silencing complex (miRISC).

[0116] Within the miRISC, the ~22 nucleotide miR recruits Argonaute (AGO) to specific target sites via base-pairing interactions. Perfect base-pairing of the miR with its target site, which is common in plants but rare in animals, results in endonucleolytic cleavage by AGO of the target RNA. Animal miRs typically form a partial duplex with their target site, which prevents cleavage and instead relies on AGO co-factors to regulate target expression through translational repression and mRNA destabilization.

[0117] Pairing of nucleotides 2-9 of the miR, commonly referred to as the seed sequence or seed region (figure 5b), to its target site has generally been considered the minimal element needed to engage a target mRNA (Bartel DP (2018) Metazoan MicroRNAs. Cell 173, 20-51}. Indeed, structural studies have shown that only sequences within the seed sequence of the AGO-bound miR are available for initial pairing to a target site (Cell. 2012 Jul 6; 150(l):233; Nature. 2012 Jun 21; 486(7403): 368-374; Science. 2012 May 25; 336(6084): 1037-1040}. Additionally, single molecule studies have demonstrated the importance of the seed in stable target site engagement (Cell. 2015 Jul 2; 162(1): 96-107; Molecular Cell 59, 117- 124 July 2, 2015}. However, it has previously been suggested that AGO may undergo a conformational change once miRISC binds target RNA, and that this conformational change may allow for extended seed pairing and exposes part of the miR 3’ region for additional interactions with the target. Hence, it may be that pairing to miR 3 ’-end sequences also may have an impact on the specificity of targeting and the regulatory mechanisms.

[0118] The present inventors have demonstrated a protective role for a miR-15a-5p mimic (SEQ ID NO:3) on VTE risk. Based on the prior art discussed above, it is believed that pairing of nucleotides 2-9 from the 5’ end of SEQ ID NO:3 (seed sequence, figure 5a) to its target site represents the minimal element needed to engage a target mRNA. Hence, in one embodiment according to the present invention the miR15a- 5p mimic is an oligonucleotide, the oligonucleotide comprising a seed sequence represented by nucleotides 2 to 9 from the 5’ end of SEQ ID NO: 3.

[0119] The seed sequence of the miR-15a-5p mimic (SEQ ID NO:3) used in example 1 is situated at positions 2 to 9 from the 5’ end of the nucleotide sequence. Hence, it is believed to represent a preferred embodiment if the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.

[0120] As previously discussed it seems that the seed sequence represents the minimal element needed to engage their target mRNA. However, it has also previously been suggested that pairing to the 3 ’-end of miR sequences may have an impact on target specificity. Having this in mind, it seems reasonable to believe that perfect basepairing of the 3’-end of the miR sequence with its target site is not essential in order to achieve the asserted technical effect, but that perfect base-pairing may represent a preferred embodiment. miRs with some nucleotide substitutions at the 3 ’end of the miR sequence should therefore be expected to provide the asserted technical effect.

[0121] Hence, in one embodiment according to the present invention the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:3 by 0 to 5 nucleotide substitutions (i.e. some substitutions are allowed); with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:3 (i.e. the seed sequence is intact). As previously discussed, it is believed to represent a preferred embodiment if the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.

[0122] As previously discussed it seems reasonable to believe that perfect base-pairing of the 3 ’-end of the miR sequence with its target site is not essential in order to achieve the asserted technical effect. miRs with some nucleotide deletions at the 3 ’end of the miR sequence should therefore be expected to provide the asserted technical effect.

[0123] Hence,

[0124] - in one embodiment according to the present invention the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:4 by 0 to 4 nucleotide substitutions (i.e. some substitutions and deletions are allowed); with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:4 (i.e. the seed sequence is intact); in another embodiment according to the present invention the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:5 by 0 to 3 nucleotide substitutions (i.e. some substitutions and deletions are allowed); with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:5 (i.e. the seed sequence is intact);

[0125] - in another embodiment according to the present invention the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:6 by 0 to 2 nucleotide substitutions (i.e. some substitutions and deletions are allowed); with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:6 (i.e. the seed sequence is intact).

[0126] With reference to previous discussions it seems that the seed sequence represents the minimal element needed to engage their target mRNA. Further, it has also previously been suggested that pairing to the 3 ’-end of miR sequences may have an impact on target specificity. However, the prior art seems to be completely silent as to the importance of the nucleotide(s) positioned on the 5’ end of the seeding sequence.

[0127] Hence,

[0128] - in one embodiment according to the present invention the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:7 by 0 to 4 nucleotide substitutions (i.e. some substitutions and deletions are allowed); with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 1 to 8 from the 5’ end of SEQ ID NO:7 (i.e. the seed sequence is intact);

[0129] - in another embodiment according to the present invention the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:8 by 0 to 3 nucleotide substitutions (i.e. some substitutions and deletions are allowed); with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 1 to 8 from the 5’ end of SEQ ID NO: 8 (i.e. the seed sequence is intact);

[0130] - in another embodiment according to the present invention the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a first nucleotide sequence derived from SEQ ID NO:9 by 0 to 2 nucleotide substitutions (i.e. some substitutions and deletions are allowed); with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 1 to 8 from the 5’ end of SEQ ID NO:9 (i.e. the seed sequence is intact). miR mimics that restore miRNA expression and / or function is highly sought-after therapeutic strategies for effective manipulation of miR levels. In this regard, carrier vehicles that facilitate proficient and safe delivery of miR-based therapeutics are important to the clinical success of these pharmaceuticals (Methods

[0131] Protoc. 2021 Mar; 4(1): 10). Suitable carrier vehicles are well known to the skilled person.

[0132] Hence, in one embodiment the pharmaceutically acceptable carrier is a liposome or a lipid nanoparticle which is suitable for in vivo delivery of miR. The miR15a-5p mimic is preferably associated to, encapsulated within and / or embedded within the pharmaceutically acceptable carrier.

[0133] Some of the limitations associated with miR delivery are susceptibility to degradation by nucleases, rapid clearance from blood, immunotoxicity, and low tissue permeability. Chemical modifications of miRNAs have significantly improved their stability and provided protection against nucleases (J. Pharmacol. Exp. Ther. 1996;277:923-937; EMBO Mol. Med. 2014;6:851-864. doi: 10.15252 / emmm.201100899; Proc. Natl. Acad. Sci. USA. 2000;97:5633-5638. doi: 10.1073 / pnas.97.10.5633; Int. J. Endocrinol. 2019;2019:6782653. doi: 10.1155 / 2019 / 6782653).

[0134] A non-limiting list of chemical modifications that may improve function and potency of miR mimics are provided in the following:

[0135] - Phosphonothioate (PS) modification substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone, which blocks, or at least reduces, the ability of many nucleases to degrade this bond. PS modifications can be placed uniformly throughout a sequence or can be selectively placed at critical locations, leaving some unmodified phosphodiester (PO) linkages.

[0136] - Use of 2’-O-methyl (2'0me) RNA impedes the ability of many nucleases to degrade single-stranded oligonucleotides.

[0137] - Locked nucleic acids (LNAs) have become a prominent modification employed in anti-miRs both in vitro and in vivo. LNAs are bicyclic nucleic acids that tether the 2'-0 to the 4'-C via a methylene bridge, effectively locking the structure into a 3'-endo sugar conformation. This modification provides stabilization against nucleases and also offers the greatest increase in binding affinity of any of nucleic acid modifications in common use today.

[0138] Thus, chemical modifications suitable to improve tissue permeability, reduce degradation by nucleases, decrease immunotoxicity and reduce clearance from blood are well known to the skilled person. How to introduce such chemical modifications in a miR sequence is part of the common general knowledge. In one embodiment the miR15a-5p mimic is an oligonucleotide, wherein the oligonucleotide comprises at least one phosphorothioate linkage, at least one 2’- fluoro base, at least one 2’-methoxy base or any combination thereof.

[0139] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

[0140] Examples

[0141] Example 1: “Protective impact of miR-15a on VTE risk”

[0142] Animal model and housing conditions

[0143] For all experiments, 8-week-old C57BL / 6J female mice were used; mice were purchased from Janvier Labs at the age of 7 weeks and were kept for an acclimation period of one week prior experiments in the SPF (specific pathogen free) animal facility of Synovo GmbH. All experimental animals had ad libitum access to standard rodent chow and tap water. Further details for animal housing are given in Table 1 below.

[0144] Table 1 :

[0145] “Temperature, humidity and room conditions are continuously monitored by Monnit sensors and automatically recorded.bThe food is analysed by the manufacturer.

[0146] “The water used in the study is the water used for human consumption in the area. Experimental formulations

[0147] Vehicle and miRNA15a-5p formulations were done similarly. Briefly, 250 nmol miRNA15a-5p mimic 2.0, HPLC / IVR-grade were purchased from Thermo Fisher (product number 13486696). A 48 mg / mL stock solution was produced from lyophilized miRNA15a-5p and UltraPure Water RNAse free (Invitrogen, Lot: 2242301). The 48 mg / mL stock solution was kept frozen at -20 °C until experimental use. On day -1 of venous stenosis interventions, both vehicle and miRNA15a-5p formulations were prepared freshly. Therefore, miRNA15a-5p was pre-diluted to a 9.6 mg / mL stock with Ultra Pure Water RNAse free. To achieve a 0.4 mg / mL final treatment concentration solution using the Invivofectamine 3.0 Reagent kit (Thermo Fisher Lot: 2435463) and an application volume of 5 mL / kg, an appropriate volume of the 9.6 mg / mL miRNA15a-5p stock was mixed 1 : 1 with complexation buffer contained in the Invivofectamine 3.0 Reagent kit. Subsequently, the mixture was mixed 1 : 1 with Invivofectamine 3.0 Reagent and immediately vortexed thoroughly to ensure complex formation. The mixture of miRNA15a-5p stock, complexation buffer and Invivofectamine 3.0 Reagent was placed for 30 min in a heating block at 50 °C. Finally, the miRNA complex solution was diluted 6-fold with sterile, RNAse free, physiological saline. This procedure was done according to the manufacturers protocol with slight deviations (Invivofectamine 3.0 Reagent kit (Thermo Fisher Lot: 2435463)). The formulation was performed using RNAse free pipette tips and RNAse free 1.5 mL reaction tubes. In case of vehicle preparation, the miRNA containing solution was replaced by sterile, RNAse free, physiological saline solution of equal volume.

[0148] Inferior vena cava stenosis model

[0149] All experiments were approved by local authorities (Regierungsprasidium Tubingen) under approval number SYN 01 / 20. The body weight was assessed daily starting on experimental day -1. All mice were shaved and depilated at the abdomen 24 h before surgical intervention. Subsequently, 12 h prior venous thrombosis intervention surgery (study day -1), awake, restrained mice were intravenously injected with 5 mL / kg vehicle, or with 2 mg / kg miRNA15a-5p mimics in a volume of 5 mL / kg, prepared as described in experimental formulations. Intravenous injections were done in the lateral tail vein with 30G 1 mL insulin syringes. After the injections, mice were placed back into their home cages. On the next day (study day 0), animals were prepared for surgeries. 20 min prior surgical interventions, mice were subcutaneously injected with 0.1 mg / kg Buprenorphin in 5 mL / kg physiological saline solution. 10 min later, anesthesia was induced with Medetomidin (0.5 mg / kg) / Midazolam (5 mg / kg) in saline solution injected intramuscularly in an application volume of 1.5 mL / kg body weight. Anesthesia depth was assessed by testing the toe pinching reflex (gentle compression of the mid digit of a hind paw with a pair of forceps). Upon complete anesthesia and analgesia, mice were placed on heating mats to maintain 37°C body temperature, eyes were covered with eye ointment and an iodine containing anti-inflammatory solution (Betaisadona) was applied on the shaved and depilated abdominal skin. For surgical procedures, Zeiss surgical microscopes were used. With scissors, a laparotomy was performed by incision of the abdominal skin and peritoneum lateral to the midline with a length of ~l-2 cm. Cotton swabs were pre-soaked in warmed physiological saline solution and used to move duodenumjejunum and ileum out of the abdominal cavity and besides the mouse. Guts were placed and also covered with pre-soaked medical gauze, again using warmed physiological saline solution. Subsequently, the abdominal aorta and the inferior vena cava were gently separated with a fine surgical grade forceps. The ligation of the inferior vena cava was done next to the left renal vein branch. For ligation, a 7-0 vicryl suture was used together with a spacer (5-0 prolene suture). The spacer suture was placed between the surgical grade 7-0 suture and the inferior vena cava. Subsequently, Ixlxl surgical knots were made using the same suture to achieve 90% ligation of the inferior vena cava, afterwards the spacer was removed and the remaining blood flow through the inferior vena cava was visually assessed. The largest side branch of the vena cava was, if appropriate and if accessible, ligated accordingly omitting the spacer suture. After successful ligations, duodenum, jejunum and ileum were placed back in the abdominal cavity, the peritoneum was sutured first, followed by the abdominal skin (abdominal skin was closed with an intracutaneous suturing technique using 6-0 vicryl suture). After completion of the surgical process, animals were injected with 2.5 mg / kg Atipamezol and 0.5 mg / kg Flumazenil in an application volume of 8.5 mL / kg of physiological saline to antagonize anesthesia, but not analgesia. The abdominal skin was again treated with Betaisadona to avoid inflammation of the suture site. Subsequently, mice were placed in pre-warmed cages that were placed on heating mats (37 °C), containing tissues, agar gel, water-soaked food pellets and mouse housings. Mice were observed by the animal caretakers till animals recovered from anesthesia. Animals were kept in these cages for 6 h until the first abdominal ultrasound measurement was performed. After the 6 h ultrasound scan, animals were placed back in their home cages supplied with pre-wetted mouse chow pellets, agar and plenty of nesting material. The surgical procedure was adapted and modified from Grover et al (PMID: 33094904).

[0150] Ultrasound imaging

[0151] To non-invasively assess thrombus volumes in vivo after inferior vena cava stenosis surgeries, animals were scanned 6 h, 24 h and 48 h after the intervention surgery at the abdomen along the midline using a VEVO 3100 ultrasound imaging system for small animals (FujiFilm, Visual Sonics) with a MX550D transducer (transmit frequency range 25-55 MHz, centre transmit frequency 40 MHz). Scans were performed with an axial resolution of 40 pm. Axial, two-dimensional scans were performed in 1 mm increments starting at the occlusion site, likewise, trans axial scans were done. Thereby, the trans axial scans were done around the center largest diameter of the inferior vena cava, respectively. For ultrasound imaging, animals were placed on a heated mouse imaging platform to maintain body temperature. Animals were covered with cotton cloth to further ensure maintaining body temperature at 36.5 ± 0.5 °C. All scans were done under isoflurane anesthesia (1.5 % in 98.5 % room air), therefore, animals were first placed in a chamber to induce isoflurane anesthesia at a concentration of 2.5 % isoflurane in 97.5 % room air. Subsequently, mice were placed in the supine position with the nose in a custom- made nose cone supplying isoflurane anesthesia for the scanning procedure (1.5 % in 98.5 % room air). Using Vevo LAB 5.6.1 software for data analysis, regions of interest were drawn on the basis of B-mode anatomical scans that were recorded as described in this paragraph. To calculate thrombus volumes, assessed radii were used together with the assessed thrombus length assuming that it is cylindrical in shape. Final ultrasound volumes were cross-correlated to ex vivo assessed thrombus weights to ensure validity of the calculation method and of the imaging approach.

[0152] Bleeding test

[0153] Finally, 48 h after the intervention surgery, a tail bleeding test was performed to assess blood coagulation times in vehicle and miRNA15a-5p mimics treated animals, respectively. The method was adopted and modified from Rossaint et al.(PMID: 23231375). Briefly, mice were anesthetised with intramuscularly injected 0.5 mg / kg Medetomidin / 5 mg / kg Midazolam / 0.05 mg / kg Fentanyl in application volume of 1.5 mL / kg body weight of saline solution. Anesthesia depth was assessed by testing the toe pinching reflex (gentle compression of the mid digit of a hind paw with a pair of forceps). Upon complete anesthesia and analgesia, mice were placed on a heating pad above a 250 mL glas beaker containing physiological saline solution with a temperature of 37 °C. The temperature of the saline solution was feed-back monitored with a heating module and a temperature probe inserted in the beaker. Subsequently, the tail was capped with a sharp scissor approximately 3 mm distal from the tail tip and immersed in the pre-heated saline solution. The tail tip bleeding time was assessed by two independent observers. The time was stopped when bleeding ceased for 5 s.

[0154] Necropsy

[0155] 48 h after the intervention surgery, and directly following the terminal tail bleeding test, animals were euthanized with CO2. After death of the animal was confirmed by a veterinarian, heart blood was taken from the left ventricle, immediately mixed with 3.8 % sodium citrate buffer 1 : 10 and stored on ice. Subsequently, the inferior vena cava was gently removed with a sharp micro scissor and surgical grade forceps at the site of ligation, stretching down to the external iliac vein branching of the inferior vena cava. The thrombus was gently removed from the excised part of the vena cava under a surgical microscope. Then, two liver samples were taken from the liver being still attached in the carcass of the mouse with a maximum dimension of 5 x 5 x 5 mm and a maximum weight of -100 mg. The piece of inferior vena cava was stored in a minimum of 250 pL RNAlater at -20 °C until analysis by qPCR. The thrombi weights were measured using microscales, following length measurements and thrombi photographs on millimeter-scaled laminated papers together with the respective animal study ID. Then, the thrombi were transferred to 4 % paraformaldehyde solution for 24 h and kept in 70 % ethanol until paraffin embedding. The collected liver pieces were stored in five volumes RNAlater (Thermo Fisher) at -20 °C until preparation for qPCR. Finally, platelet-poor plasma was prepared from collected heart blood samples by centrifugation at 2500 g for 15 min at RT. The platelet-poor plasma was then centrifuged a second time at 2500 g for 15 min at RT to yield the final platelet poor plasma used for analysis of coagulation factors.

[0156] Results

[0157] In order to investigate whether miR-15a-5p has a protective impact on VTE risk, i.e. prevents thrombosis formation in vivo, the inventors performed an intervention study in a mouse venous thrombosis model using the inferior vena cava stensosis model. Female mice (8-week-old C57BL / 6J) mice were used for all experiments.

[0158] Twelve hours prior to surgery vehicle or miR-15a-5p (2 mg / kg) (n=12 in each group) were administrated into the tail vein of the mice, and thrombus formation (incidence) and size (thrombus volume) were monitored by ultrasound imaging at 6- , 24- and 48-hours post-surgery. At 48 hrs, a bleeding test was conducted before the mice were euthanized and the inferior vena cava (IVC) inspected for presence of thrombi and thrombus size / weight after death.

[0159] The incidence and size of venous thrombi over time in mice treated with miR-15a- 5p and vehicle are shown in figure 1. Both the incidence (58% vs. 83%) and the thrombus volume among those that developed thrombi (12 vs 15 mm3) were lower in mice treated with miR-15a-5p compared to mice treated with vehicle. The protective effect of miR-15a-5p appeared to be more pronounced during the first 24 hours after the surgery. Similar findings occurred for the proportion of thrombus and thrombus weight after necropsy at 48 hours (figure 2a-b). Bleeding time conducted 48 hours after surgery displayed greater variation and on average was modestly prolonged among those treated with miR-15a-5p compared to vehicle treated mice.

[0160] In order to investigate whether inhibition of miR-15a-5p would have an opposite effect to miR-15a-5p on thrombus formation in mice, the inventors treated mice with vehicle (n=8) or antagomiR-15a-5p (n=9). Twelve hours prior to surgery vehicle or antagomiR-15a-5p (lOmg / kg) (has-miR-15a-5p miRCURY LNA miRNA Inhibitor from QIAGEN) was administrated into the tail vein of the mice, and thrombus formation (incidence) and size (thrombus volume) were monitored by ultrasound imaging at 6-, 24- and 48-hours post-surgery. At 48 hrs, a bleeding test was conducted before the mice were euthanized and the IVC inspected (presence of thrombi and thrombus size (thrombus weight) after death.

[0161] The surgeons noticed increased bleeding tendency already at surgery and at necropsy of all antagomir-15a-5p treated animals. Among the mice treated with antagomiR-15a-5p (lOmg / kg), 4 (44%) animals died due to PE, confirmed by a veterinarian, within 4 hours post-surgery. The incidence and size of venous thrombi over time in mice treated with antagomiR-15a-5p and vehicle are shown in figure 3. The number of mice with a venous thromboembolic event were similar between groups (6 (67%) for antagomir-15a-5p treated mice and 6 (75%) for vehicle treated mice (figure 3 a), whereas the thrombus volume were slightly higher in antagomir- treated animals (figure 3b). Accordingly, few antagomir-treated animals were available for measurement of thrombus weight at necropsy (figure 4b), but thrombus weights appeared comparable between groups. The bleeding time were slightly prolonged among mice treated with antagomiR-15a-5p compared to vehicle treated mice (figure 4c).

[0162] Even though treatment with antagomiR-15a-5p had no obvious adverse effect on the incidence of thromboembolic events, the severity of the events with 44% of the antagomir-treated animals dying from PE combined with sever bleeding tendency during surgery and necropsy support that treatment with antagomiR-15a-5p provided an opposing phenotype compared to the miR-15-5p treated animals.

Claims

CLAIMS1.A pharmaceutical composition for use in the treatment of venous thromboembolism (VTE), the pharmaceutical composition comprising a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier.2.The pharmaceutical composition for use according to claim 1, wherein the miR-15a- 5p mimic is human miR-15a-5p mimic.3.The pharmaceutical composition for use according to any one of the preceding claims, wherein the miR15a-5p mimic is an oligonucleotide, the oligonucleotide comprising a seed sequence represented by nucleotides 2 to 9 from the 5’ end of SEQ ID NO:3.4.The pharmaceutical composition for use according to claim 3, wherein the seed sequence is situated at positions 2 to 9 from the 5’ end of the oligonucleotide.5.The pharmaceutical composition for use according to any one of the preceding claims, wherein- the miR15a-5p mimic is an oligonucleotide;- the oligonucleotide comprising i) a first nucleotide sequence derived from SEQ ID NO: 3 by 0 to 5 nucleotide substitutions; with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:3; ii) a first nucleotide sequence derived from SEQ ID NO:4 by 0 to 4 nucleotide substitutions; with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:4;iii) a first nucleotide sequence derived from SEQ ID NO:5 by 0 to 3 nucleotide substitutions; with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:5; iv) a first nucleotide sequence derived from SEQ ID NO: 6 by 0 to 2 nucleotide substitutions; with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:6; v) a first nucleotide sequence derived from SEQ ID NO: 7 by 0 to 4 nucleotide substitutions; with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 1 to 8 from the 5’ end of SEQ ID NO:7; vi) a first nucleotide sequence derived from SEQ ID NO: 8 by 0 to 3 nucleotide substitutions; with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 1 to 8 from the 5’ end of SEQ ID NO:8; or vii) a first nucleotide sequence derived from SEQ ID NO: 9 by 0 to 2 nucleotide substitutions; with the proviso that nucleotides 1 to 8 from the 5’ end of the first nucleotide sequence is identical with nucleotides 1 to 8 from the 5’ end of SEQ ID NO:9.6.The pharmaceutical composition for use according to any one of the preceding claims, wherein the oligonucleotide consists of from 15 to 40 linked nucleobases, such as 15 to 30 linked nucleobases, 20 to 25 linked nucleobases or 20 to 22 linked nucleobases.7.The pharmaceutical composition for use according to any one of the preceding claims, wherein- the miR15a-5p mimic is an oligonucleotide;- the oligonucleotide comprising a first nucleotide sequence derived from any one of SEQ ID NO:3 to 9 by 0 to 2 nucleotide substitutions; with the proviso that nucleotides 2 to 9 from the 5’ end of the first nucleotide sequence is identical with nucleotides 2 to 9 from the 5’ end of SEQ ID NO:3; and the oligonucleotide consists of from 15 to 40 linked nucleobases.8.The pharmaceutical composition for use according to any one of the preceding claims, wherein- the miR15a-5p mimic is an oligonucleotide;- the oligonucleotide comprising a nucleotide sequence of any one of SEQ ID NO:3 to 9; and- the oligonucleotide consists of from 15 to 40 linked nucleobases.9.The pharmaceutical composition for use according to any one of the preceding claims, wherein- the miR15a-5p mimic is an oligonucleotide; and- the oligonucleotide is selected from the group consisting of any one of SEQ ID NO:3-9.10.The pharmaceutical composition for use according to any one of the preceding claims, wherein the miR15a-5p mimic is miR15a-5p.11.The pharmaceutical composition for use according to any one of the preceding claims, wherein the miR15a-5p mimic is human miR15a-5p.12.The pharmaceutical composition for use according to any one of the preceding claims, wherein- the miR15a-5p mimic is an oligonucleotide; and- the oligonucleotide is SEQ ID NO:3.13.The pharmaceutical composition for use according to any one of the preceding claims, wherein the pharmaceutically acceptable carrier is a liposome or a lipid nanoparticle which is suitable for in vivo delivery of miR.The pharmaceutical composition for use according to any one of the preceding claims, wherein the miR15a-5p mimic is associated to, encapsulated within and / or embedded within the pharmaceutically acceptable carrier.15.The pharmaceutical composition for use according to any one of the preceding claims, wherein venous thromboembolism (VTE) is pulmonary embolism (PE) and / or deep vein thrombosis (DVT).16.The pharmaceutical composition for use according to any one of the preceding claims, wherein venous thromboembolism (VTE) is pulmonary embolism (PE).17.The pharmaceutical composition for use according to any one of the preceding claims, wherein venous thromboembolism (VTE) is deep vein thrombosis (DVT).18.The pharmaceutical composition for use according to any one of the preceding claims, wherein the pharmaceutical composition is to be administered by intravenous administration (IV).19.A kit comprising a pharmaceutical composition; and an instructional material for use thereof; wherein- the pharmaceutical composition comprises a pharmaceutically effective amount of a miR-15a-5p mimic and a pharmaceutically acceptable carrier;- the instructions material comprises instructions for treating VTE in a subject, wherein the treating includes administering the pharmaceutical composition to the subject.

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