Treatment of factor x deficiency with fitusiran

Abstract

The present disclosure provides methods of treating factor X deficiency with fitusiran in a mammal in need thereof.

Description

Attorney Docket: 15979-20206.40 TREATMENT OF FACTOR X DEFICIENCY WITH FITUSIRAN CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 572,854, filed April 1, 2024, and U.S. Provisional Application No. 63 / 631,193, filed April 8, 2024, the contents of each of which are incorporated herein by reference in their entireties. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (159792020640SEQLIST.xml; Size: 31,471 bytes; and Date of Creation: March 26, 2025) is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Coagulation factor X (FX) is a vitamin K-dependent protein that circulates in plasma at a concentration of about 10 μg / ml (Versteeg et al., Physiol Rev. (2013) 93(1):327- 58). Upon activation by the tissue factor / factor VIIa (TF / FVIIa)-complex or the factor IXa / factor VIIIa (FIXa / FVIIIa)-complex, FXa constitutes the enzymatic component of the prothrombinase-complex that converts prothrombin into thrombin. Thrombin, in turn, is responsible for the formation of a stable fibrin-rich blood clot.

[0004] Hereditary FX deficiency is a rare autosomal recessive disorder that occurs in about 1 in every 1,000,000 individuals (Uprichard et al., Blood Rev. (2002) 16(2):97-110; Menegatti et al., Semin Thromb Hemost. (2009) 35(4):407-15). FX deficiency can be classified into type I (low FX antigen and low FX activity) or type II (near-normal FX antigen, low FX activity). Clinically, FX deficiency produces a variable bleeding tendency, with a strong inverse association between plasma activity levels and frequency and severity of bleeding. Whereas patients with residual FX activity between 10 and 40% usually suffer minor spontaneous bleeding or bleeding after trauma, patients with activity levels below 10% are at high risk for major spontaneous bleeding (Peyvandi et al., J Thromb Haemost. (2012) 10(4):615-21). Patients with <1% FX activity are the most severely affected, with the occurrence of intracranial hemorrhages, gastrointestinal bleeding and hemophilia-like bleeding episodes (Peyvandi, supra; Brown et al., Haemophilia (2008) 14(6):1176-82).

[0005] Current treatment options have long been restricted to non-specific FX replacement therapy (e.g., fresh frozen plasma, prothrombin-complex concentrates, and FIXAttorney Docket: 15979-20206.40 concentrates with a specified FX content), and only one high-purity human plasma-derived FX concentrate (Coagadex®; Bio Products Laboratory, UK) has become available for routine prophylaxis and on-demand treatment of bleeding episodes (Menegatti, supra; Payne et al., Haemophilia (2022) 28(4):523-31).

[0006] Thus, there remains a need for methods of treating factor X deficiency in mammals. SUMMARY OF THE INVENTION

[0007] The present disclosure provides a method of treating factor X deficiency in a mammal, comprising administering fitusiran to the mammal in need thereof. Also provided are a method of prophylaxis to prevent or reduce bleeding in a mammal having factor X deficiency, a method of reducing use of factor X replacement factor to treat a bleeding event in a mammal with factor X deficiency, a method of increasing thrombin generation in a mammal having factor X deficiency, and a method of improving hemostatic activity in a mammal having factor X deficiency, all comprising administering fitusiran to the mammal in need thereof.

[0008] In some embodiments, the factor X deficiency is type I factor X deficiency. The factor X deficiency may be severe. In some embodiments, the factor X activity in the mammal is less than 1% of a normal level. In some embodiments, the factor X deficiency is type II factor X deficiency.

[0009] In some embodiments, the mammal is a mouse. In other embodiments, the mammal is a human.

[0010] In some embodiments, fitusiran is administered to the mammal by subcutaneous injection. Fitusiran may be administered in a pharmaceutical composition comprising phosphate-buffered saline.

[0011] Also provided are fitusiran for use in treating factor X deficiency, in prophylaxis to prevent or reduce bleeding in a mammal having factor X deficiency, in reducing use of factor X replacement factor to treat a bleeding event in a mammal with factor X deficiency, in increasing thrombin generation in a mammal having factor X deficiency, or in improving hemostatic activity in a mammal having factor X deficiency. This application also provides use of fitusiran in the manufacture of a medicament for treating factor X deficiency, in prophylaxis to prevent or reduce bleeding in a mammal having factor X deficiency, in reducing use of factor X replacement factor to treat a bleeding event in a mammal with factorAttorney Docket: 15979-20206.40 X deficiency, in increasing thrombin generation in a mammal having factor X deficiency, or in improving hemostatic activity in a mammal having factor X deficiency.

[0012] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description. BRIEF DESRIPTION OF THE FIGURES

[0013] FIGS. 1A-1B show the expanded structural formula, chemical formula, and molecular mass of fitusiran (sodium form; “fitusiran sodium”); (A-116858 = fitusiran sense strand (SEQ ID NO:1); A-116861 = fitusiran antisense strand (SEQ ID NO:2)).

[0014] FIG. 2 shows a schematic of an engineered f10 gene with loxP-sequences surrounding exons 4 & 5 engineered to be expressed in mice. Upon activation of Cre recombinase, both exons, which are flanked by loxP sites, are excised. P1 and P2 indicate location of primers used for post-induction analysis.

[0015] FIG. 3A shows PCR-analysis of genomic DNA isolated from liver of poly- Inosinic-poly-Cytidylic (pI:pC)-treated Cre+- and Cre--mice. A virtually complete recombined F10-locus was observed in Cre+-mice.

[0016] FIG. 3B shows a plot of Russell’s viper venom factor X activator (RVV-X)- induced FXa amidolytic activity over time in a f10WT-mouse and f10low-mice.

[0017] FIG. 3C shows a plot of Residual FX activity measured in RVV-X-induced activity assay. Limit of Detection (LOD) of this assay is 0.5 % FX. All mice having <1% FX activity were designated as f10low-mice.

[0018] FIG. 3D shows the survival curve of pI:pC-treated f10WTmice and f10low-mice (both n=7 at beginning of survival assay). No differences in survival after 12 months were observed. Y-axis measures percent survival. X-axis measures time after pI:pC in months.

[0019] FIGS. 4A-4B show the clotting assays Prothrombin time (PT; FIG. 4A) and activated Partial Prothrombin Time (aPTT; FIG. 4B) performed using plasma of pI:pC- treated f10WT-mice (n=8) and f10low-mice (n=8). Horizontal lines represent mean values. Statistical analysis was performed using unpaired Student’s t-test.

[0020] FIGS. 4C-4D show plots of thrombin generation (TG) performed using tissue factor (TF; FIG. 4C) or FXIa (FIG. 4D) as initiator. Solid lines represent mean values, andAttorney Docket: 15979-20206.40 shaded areas standard deviation (SD). Number of measurements are indicated for each condition. Detailed values of TG parameters (lag-time, endogenous thrombin potential, thrombin peak and time to peak) are summarized in Table 3.

[0021] FIG. 5A shows representative digital composite fluorescence and bright-field images of platelet accumulation and fibrin deposition in f10WT(n=46 clots) and f10low(n=44 clots) mice before (0 s) and 20, 80, 120, 200 and 300 s after laser injury.

[0022] FIGS. 5B and 5D show kinetic curves displaying platelet accumulation and fibrin deposition in f10WT(46 clots) and f10low(44 clots) mice. Plotted is the median integrated fluorescence intensity (IFI) for platelets (FIG. 5B) and fibrin (FIG. 5D) fluorescence versus time after injury.

[0023] FIGS. 5C and 5E show the distribution of the integrated platelet (FIG. 5C) or fibrin (FIG. 5E) fluorescence intensity for each of the 46 clots in f10WTmice and 44 clots in f10lowmice at maximal size. Bars represent median IFI values for each group. Statistical analysis was performed using the Mann-Whitney test.

[0024] FIG. 6A shows a plot of blood loss for f10WT, f10low, and FVIII-knock out mice. The terminal 3-mm of the tail was amputated from the tails of f10WT-mice, f10low-mice, and f10lowmice that received FX (50 U / kg) five min before injury and FVIII-deficient mice (f8KO). Blood loss was monitored for 30 min, and the amount of shed blood was measured. Statistical analysis was performed using one-way Anova with Tukey’s correction for multiple comparisons.

[0025] FIG. 6B shows a plot of the number of mice that had a primary bleeding stop following tail amputation and the number of mice that did not have a primary bleeding stop. The number of mice with a primary stop was significantly reduced in f10low-mice compared to f10WTand FVIII-deficient mice. Statistical analysis was performed using a Chi-square test for multiple groups.

[0026] FIG. 6C shows representative bleeding profiles of f10WT-mice, f10low-mice, and f10low-mice that received FX (50 U / kg) five minutes before injury. Bleeding profiles from FVIII-deficient mice that received FX (50 U / kg) five minutes before injury were also obtained.

[0027] FIG. 7 shows the number of clots over a 30-min period observed for f10WT-mice, f10low-mice, FVIII deficient mice (f8KO), and f10low-mice. f10low-mice received purified FX at 1 U / kg or 5 U / kg. Each dot on the graph represents an individual mouse. Horizontal bars represent median values. Statistical analysis was performed via one-way Anova with Tukey’s correction for multiple comparisons.Attorney Docket: 15979-20206.40

[0028] FIG. 8A shows a plot of antithrombin activity (%) for untreated f10lowmice and f10lowmice treated with fitusiran. Treated f10low-mice received two doses of fitusiran at a one-week interval. Seven days after the last dose, plasma was analyzed for antithrombin activity in non-treated and fitusiran-treated f10low-mice. Each dot on the graph represents an individual mouse, and horizontal bars represent mean values. Statistical analysis was performed using an unpaired Student’s t-test.

[0029] FIGS. 8B and 8C depict plots of TF-induced TG in non-treated and fitusiran- treated f10low-mice. Plasma levels of FX were <1% (FIG. 8B) or plasmas were spiked with FX (2 % of normal plasma) before measuring TG. Solid lines represent mean values, and shaded areas SD. Detailed values of thrombin generation parameters (lag-time, endogenous thrombin potential, thrombin peak and time to peak) are summarized in Table 3.

[0030] FIGS. 8D and 8E depict plots showing the number of clots over a 30-min period observed for f10low-mice and fitusiran-treated f10low-mice. FX activity levels were <1% (FIG. 8D). Mice received a low-dose FX (1 U / kg) five min before injury (FIG. 8E). Each dot on the graph represents an individual mouse. Horizontal bars represent median values. Statistical analysis was performed using an unpaired Student’s t-test. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present disclosure provides in vivo data demonstrating that fitusiran may be used to increase clot formation in a bleeding event in mammals (e.g., subjects) with factor X deficiency, such as severe deficiency in which the subjects have less than 1% of factor X activity and antigen of normal levels. Accordingly, the present disclosure provides methods of treating factor X deficiency; methods of prophylaxis (e.g., routine prophylaxis) to prevent or reduce bleeding and / or to prevent, or reduce the frequency of, bleeding episodes in subjects with factor X deficiency; methods of reducing amounts of factor X replacement factor needed to treat bleeding episodes in subjects with factor X deficiency in reducing use of factor X replacement factor to treat a bleeding event in a mammal with factor X deficiency, methods of increasing thrombin generation in a mammal having factor X deficiency; and methods of improving hemostatic activity in a mammal having factor X deficiency.

[0032] In some embodiments, the factor X deficiency is severe factor X deficiency or Type I factor X deficiency. In some embodiments, the mammal has severe factor X deficiency, characterized by factor X antigen levels <1% of normal and factor X activity <1%Attorney Docket: 15979-20206.40 of normal. In some embodiments, the mammal has type I factor X deficiency, characterized as having low factor X antigen levels and low factor X activity. In some embodiments, the mammal has type II factor X deficiency, characterized as having near-normal factor X antigen levels and low factor X activity. I. Composition for Treatment of Factor X Deficiency

[0033] Without being bound by theory, it is believed that fitusiran-mediated lowering of antithrombin (AT) levels will increase thrombin generation and thus improve hemostasis in patients with factor X deficiency. Antithrombin is encoded by the SERPINC1 gene.

[0034] Fitusiran, whose structure is described herein, is a synthetic, chemically modified double-stranded small interfering RNA (siRNA) oligonucleotide covalently linked to a tri- antennary N-acetyl-galactosamine ligand targeting the AT3 mRNA in the liver, thereby suppressing the synthesis of antithrombin. The nucleosides in each strand of fitusiran are connected through either 3’-5’ phosphodiester or phosphorothioate linkages, thus forming the sugar-phosphate backbone of the oligonucleotide.

[0035] The sense strand and the antisense strand of fitusiran contain 21 and 23 nucleotides, respectively. The 3’-end of the sense strand is conjugated to the N-acetyl- galactosamine containing moiety (referred to as L96) through a phosphodiester linkage. The sense strand contains two consecutive phosphorothioate linkages at its 5’ end. The antisense strand contains four phosphorothioate linkages, two at the 3’ end and two at the 5’ end. The 21 nucleotides of the sense strand hybridize with the complementary 21 nucleotides of the antisense strand, thus forming 21 nucleotide base pairs and a two-base overhang at the 3’-end of the antisense strand. See also U.S. Pat. 9,127,274, U.S. Pat. 11,091,759, and WO 2019 / 014187.

[0036] The two nucleotide strands of fitusiran are shown below: sense strand: 5’Gf-ps-Gm-ps-Uf-Um-Af-Am-Cf-Am-Cf-Cf-Af-Um-Uf-Um-Af-Cm-Uf- Um-Cf-Am-Af-L963’ (SEQ ID NO: 1), and antisense strand: 5’ Um-ps-Uf-ps-Gm-Af-Am-Gf-Um-Af-Am-Af-Um-Gm-Gm-Uf-Gm- Uf-Um-Af-Am-Cf-Cm-ps-Am-ps-Gm 3’ (SEQ ID NO: 2), wherein Af = 2’-fluoroadenosine (i.e., 2’-deoxy-2’-fluoroadenosine) Cf = 2’-fluorocytidine (i.e., 2’-deoxy-2’-fluorocytidine) Gf = 2’-fluoroguanosine (i.e., 2’-deoxy-2’-fluoroguanosine) Uf = 2’-fluorouridine (i.e., 2’-deoxy-2’-fluorouridine)Attorney Docket: 15979-20206.40 Am = 2’-O-methyladenosine Cm = 2’-O-methylcytidine Gm = 2’-O-methylguanosine Um = 2’-O-methyluridine “-” (hyphen) = 3’-5’ phosphodiester linkage sodium salt “-ps-” = 3’-5’ phosphorothioate linkage sodium salt and wherein L96 has the following formula:(I).

[0037] The expanded structural formula, molecular formula, and molecular weight of fitusiran (sodium form) are shown in FIGS. 1A-1B. As used herein, the term 2’- fluoroadenosine is used interchangeably with the term 2’-deoxy-2’-fluoroadenosine; the term 2’-fluorocytidine is used interchangeably with the term 2’-deoxy-2’-fluorocytidine; the term 2’-fluoroguanosine is used interchangeably with the term 2’-deoxy-2’-fluoroguanosine, and the term 2’-fluorouridine is used interchangeably with the term 2’-deoxy-2’-fluorouridine.

[0038] The structure of fitusiran can also be described using the following diagram, wherein the X is O:.Attorney Docket: 15979-20206.40

[0039] For use in the present treatment methods, fitusiran may be provided in a pharmaceutical composition comprising it and a pharmaceutically acceptable excipient. In certain embodiments, fitusiran is in sodium salt form.

[0040] In some embodiments, fitusiran is provided in an aqueous solution at a concentration of about 1 to about 200 mg / mL (e.g., about 50 to about 150 mg / mL, about 80 to about 110 mg / mL, or about 90 to about 110 mg / mL). As used herein, values intermediate to recited ranges and values are also intended to be part of this disclosure. In addition, ranges of values using a combination of any of recited values as upper and / or lower limits are intended to be included. In further embodiments, the pharmaceutical composition comprises fitusiran at a concentration of about 6.25, about 12.5, about 25, about 50, about 75, about 100, about 125, about 150, or about 200 mg / mL.

[0041] Unless otherwise indicated, a fitusiran weight recited in the detailed description of the present invention is the weight of fitusiran free acid (the active moiety), even though fitusiran is injected to patients subcutaneously in its sodium form (in an aqueous solution). For example, 100 mg / mL fitusiran means 100 mg of fitusiran free acid (equivalent to 106 mg fitusiran sodium, the drug substance) per mL.

[0042] In some embodiments, the pharmaceutical compositions comprise fitusiran in a phosphate-buffered saline. The phosphate concentration in the solution may be about 1 to 10 mM (e.g., 2, 3, 4, 5, 6, 7, 8, or 9 mM), with a pH of 6.0-8.0. The pharmaceutical compositions herein may include a preservative such as EDTA. Alternatively, the pharmaceutical compositions are preservative-free. In particular embodiments, the fitusiran pharmaceutical composition is preservative-free and comprises, consists of, or consists essentially of about 100 mg of fitusiran per mL of a 5 mM phosphate buffered saline (PBS) solution. The PBS solution is composed of sodium chloride, dibasic sodium phosphate (heptahydrate), and monobasic sodium phosphate (monohydrate). Sodium hydroxide solution and diluted phosphoric acid may be used to adjust the pH of the composition to 7.0.

[0043] The pharmaceutical composition may be provided in a container (e.g., a vial or a syringe). The container may contain single or multiple doses. In some embodiments, the container is a single-use container (e.g., a single-use ampule or a single-use syringe such as a single-use pre-filled syringe), with each container containing about 10 to about 100 mg fitusiran (e.g., about 10 mg, about 20 mg, about 25 mg, about 40 mg, about 50 mg, or about 80 mg). The fitusiran may be provided in a solid form in the container and reconstituted in an aqueous solution (e.g., PBS) prior to use, with the reconstituted solution containing about 1 to about 150 mg / mL (e.g., 100 mg / mL) fitusiran. In some embodiments, fitusiran is provided inAttorney Docket: 15979-20206.40 sodium salt form in a single-use glass vial or a single-use prefilled syringe (e.g., one with a safety system). In further embodiments, each vial or syringe contains about 80 mg of fitusiran in about 0.8 mL (or about 50 mg of fitusiran in about 0.5 mL, about 20 mg of fitusiran in about 0.2 mL, or about 10 mg of fitusiran in about 0.1 mL) of 5 mM phosphate buffered saline solution (pH 7.0); and the solution is administered to patients through subcutaneous injection. The solution can be stored at 2 to 30oC (e.g., 2 to 8oC).

[0044] In particular embodiments, the fitusiran composition for subcutaneous injection contains fitusiran in a 5 mM phosphate buffered saline having 0.64 mM NaH2PO4, 4.36 mM Na2HPO4, and 84 mM NaCl at pH 7.0. In certain embodiments, the composition of fitusiran solution for subcutaneous injection is shown in Table 1A: Table 1A. Exemplary Formulationq.s.: quantum satis.

[0045] In some embodiments, fitusiran is packaged in a pre-filled pen containing 50 mg of fitusiran (equivalent to 53 mg fitusiran sodium). In some embodiments, each single-use pre-filled pen contains 50 mg of fitusiran in 0.5 mL solution at a concentration of 100 mg / mL. In some embodiments, each pre-filled pen is designed to deliver 50 mg fitusiran in 0.5 mL which also contains: sodium chloride (2.455 mg), dibasic sodium phosphate (0.585 mg), monobasic sodium phosphate (0.044 mg), phosphoric acid (q.s. pH 7.0), sodium hydroxide (q.s. pH 7.0), and water for injection (q.s. 0.5 mL). Fitusiran may be supplied as a sterile, clear solution for subcutaneous injection in the 50 mg PFP. In some embodiments, the PFP contains a single dose of fitusiran and comprises a prefilled syringe (PFS) assembled with an automated injection system (pen). The PFS is a 1 mL colorless type 1 glass syringeAttorney Docket: 15979-20206.40 with a 29 gauge, (½ inch) stainless steel staked needle covered with a rigid needle shield. The syringe is closed with a rubber coated (bromobutyl gray) plunger stopper.

[0046] In some embodiments, fitusiran is packaged in a vial containing 20 mg of fitusiran (equivalent to 21 mg fitusiran sodium). In these embodiments, tach single-use vial contains 20 mg of fitusiran in 0.2 mL solution at a concentration of 100 mg / mL. In some embodiments, each vial contains 20 mg fitusiran in 0.2 mL which also contains: sodium chloride (0.982 mg), dibasic sodium phosphate (0.234 mg), monobasic sodium phosphate (0.018 mg), phosphoric acid (q.s. pH 7), sodium hydroxide (q.s. pH 7.0), and water for injection (q.s. 0.2 mL). Fitusiran may be supplied as a sterile, clear solution for subcutaneous injection in the vial. The vial comprises a single dose of fitusiran in a 2 mL colorless Type-1 glass vial with a bromobutyl rubber stopper and an aluminum polypropylene colored crimping cap.

[0047] In certain embodiments, the composition of fitusiran solution for subcutaneous injection is shown in Table 1B below.Attorney Docket: 15979-20206.40 Table 1B. Fitusiran Formulations

[0048] While the fitusiran dosage weight described herein refers to the weight of fitusiran free acid (active moiety), administration of fitusiran to patients herein refers to administration of fitusiran sodium (drug substance) provided in a pharmaceutically suitable aqueous solution (e.g., a phosphate-buffered saline at a physiological pH). II. Methods of Treatment

[0049] The present disclosure provides methods of treating factor X deficiency, prophylaxis (e.g., routine prophylaxis) to prevent or reduce bleeding or the frequency of bleeding episodes, and reducing factor X replacement factor use, in mammals (e.g., humans, non-human primates, pets, mice, or rats). The present disclosure also provides methods of increasing hemostatic (e.g., clotting) activity.

[0050] In some embodiments, the mammal is a mouse. In such embodiments, fitusiran may be administered at a dose of about 10-15 mg / kg (e.g., about 11, 12, 13, 14, or 15 mg / kg) about once per week. In some embodiments, fitusiran is administered more than one (e.g., two) times. In some embodiments, in f10lowmice treated with fitusiran, the residual antithrombin activity is 14 ±2 %.

[0051] In some embodiments, the mammal is a human. In human subjects, factor X deficiency may also be referred to as Stuart-Prower factor deficiency. Factor X may also be referred to as Stuart-Prower factor. In some embodiments, the human is a factor X deficiency patient.

[0052] Fitusiran may be administered to the mammal in need thereof through injection, e.g., subcutaneous injection, optionally in a pharmaceutical composition comprising PBS. InAttorney Docket: 15979-20206.40 certain embodiments, the pharmaceutical composition is administered by subcutaneous injection at a dose strength of, for example, about 10 to about 100 mg (e.g., about 10 to about 95 mg, about 40 to about 90 mg, about 50 to about 100 mg, about 50 to about 90 mg, about 50 to about 85 mg, or about 50 to about 80 mg) per dose. In particular embodiments, fitusiran is administered subcutaneously at about 10, about 20, about 50, or about 80 mg (weight of active moiety) per dose in a PBS solution as described above. Fitusiran may be administered in an amount sufficient to achieve clinical needs (e.g., alleviating symptoms of factor X deficiency such as bleeding) without causing thrombotic events.

[0053] A plurality of fitusiran doses may be administered to a subject at an interval of about one, about two, about three, about four, about five, about six, about seven, or about eight weeks, or of about one, about two, or about three months. In particular embodiments, a fixed dose of fitusiran (e.g., about 50 or about 80 mg subcutaneous injection) is administered to a subject (e.g., a patient with factor X deficiency) about once every four weeks or about once a month or about once every eight weeks or about once every other month.

[0054] In some embodiments, a human patient with factor X deficiency is treated with a subcutaneous dose of fitusiran at about 50 mg per dose about every two months (or about every eight weeks). In other embodiments, a human patient with factor X deficiency is treated with a subcutaneous dose of fitusiran at about 50 mg about every month (or about every four weeks). In yet other embodiments, a human patient with factor X deficiency is treated with a subcutaneous dose of fitusiran at about 80 mg about every two months (or about every eight weeks). In yet other embodiments, a human patient with factor X deficiency is treated with a subcutaneous dose of fitusiran at about 80 mg about every month (or about every four weeks). In yet other embodiments, a human patient with factor X deficiency is treated with a subcutaneous dose of fitusiran at about 20 mg about every two months (or about every eight weeks). In yet other embodiments, a human patient with factor X deficiency is treated with a subcutaneous dose of fitusiran at about 20 mg about every month (or about every four weeks). In yet other embodiments, a human patient with factor X deficiency is treated with a subcutaneous dose of fitusiran at about 10 mg about every month (or about every four weeks). III. Articles of Manufacture and Kits

[0055] The present invention also provides kits comprising a pharmaceutical composition for use in the present treatment methods. Such kits include one or more vials or one or moreAttorney Docket: 15979-20206.40 pre-filled syringes or injectors (e.g., pens) comprising a pharmaceutical composition of the invention and instructions for use, e.g., instructions for administering a therapeutically effective amount of fitusiran.

[0056] In some embodiments, fitusiran is packaged in a drug delivery device. In some embodiments, a drug delivery device may involve a needle-based injection system as described in Table 1 of section 5.2 of ISO 11608-1:2014(E). As described in ISO 11608- 1:2014(E), needle-based injection systems may be broadly distinguished into multi-dose container systems and single-dose (with partial or full evacuation) container systems. The container may be a replaceable container or an integrated non-replaceable container.

[0057] As further described in ISO 11608-1:2014(E), a multi-dose container system may involve a needle-based injection device with a replaceable container. In such a system, each container holds multiple doses, the size of which may be fixed or variable (pre-set by the user). Another multi-dose container system may involve a needle-based injection device with an integrated non-replaceable container. In such a system, each container holds multiple doses, the size of which may be fixed or variable (pre-set by the user).

[0058] As further described in ISO 11608-1:2014(E), a single-dose container system may involve a needle-based injection device with a replaceable container. As also described in ISO 11608-1:2014(E), a single-dose container system may involve a needle-based injection device with an integrated non-replaceable container. In some embodiments, each container holds a single dose, whereby the entire deliverable volume is expelled (full evacuation). In other embodiments, each container holds a single dose, whereby a portion of the deliverable volume is expelled (partial evacuation).

[0059] An exemplary sleeve-triggered auto-injector with manual needle insertion is described in WO2015 / 004052. Exemplary audible end-of-dose feedback mechanisms are described in WO2016 / 193346 and WO2016 / 193348. An exemplary needle-safety mechanism after using an auto-injector is described in WO2016 / 193352. An exemplary needle sheath remover mechanism for a syringe auto-injector is described in WO2016 / 193353. An exemplary support mechanism for supporting an axial position of a syringe is described in WO2016 / 193355.

[0060] Fitusiran may be supplied in a single-use vial containing 20 mg fitusiran (equivalent to 21 mg fitusiran sodium) in 0.2 mL of solution at a concentration of 100 mg / mL. Fitusiran may also be supplied in a pre-filled pen containing 50 mg of fitusiran (equivalent to 53 mg fitusiran sodium) in 0.5 mL solution at a concentration of 100 mg / mL.Attorney Docket: 15979-20206.40

[0061] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0062] According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any manner.

[0063] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. EXAMPLES Example 1: Fitusiran Reduces Bleeding in Factor X-Deficient Mice

[0064] The possibility that fitusiran could be used to ameliorate the bleeding tendency in FX-deficiency was explored. To this end, a mouse model was developed using the MX1-Attorney Docket: 15979-20206.40 Cre / loxP recombination system to induce targeted disruption of the murine f10 gene. This approach generated mice with a severe deficiency of FX antigen and activity (<1% of normal, which we refer to as f10low-mice), which was associated with a dramatic decrease in in vitro clotting activity and thrombin generation. These mice were viable, exhibited impaired thrombus formation in a laser-induced vessel wall injury model, and displayed a bleeding tendency in two different bleeding models. Bleeding could be reversed by the infusion of FX. Moreover, treatment with fitusiran strongly reduced bleeding, suggesting that this agent could be explored for its use in patients with a FX-deficiency. Methods Generation of mice

[0065] MX1Cre+f10flox / floxmice were prepared by Genoway (Lyon, France). Mice expressing a floxed f10-gene (F10tm1a(EUCOMM)Hmgu) were obtained from the European Mouse Mutant Archive (06365; HEPD0602_2_G07) (Graf et al., Sci Immunol. (2019) 4(39):eaaw8405) and crossed with flippase-expressing C57Bl / 6 mice to generate a conditional f10 allele (f10flox / flox). Subsequently, these mice were crossed with the MX1-Cre transgenic line (B6.Cg-Tg(Mx1-Cre)1CgnJ; Jackson Laboratories, Bar Harbor, USA). As a control, MX1Cre--littermates were used. Factor X (FX) deficiency was induced by three subsequent intraperitoneal injections of pI:pC (10 mg / kg; Invivogen, San Diego, CA, USA) over a two-week period. Genotyping

[0066] Genotyping of homogenous f10flox / flox-mice for MX1-Cre recombinase positivity was performed using DNA extracted by ear biopsies using a classic NaOH-based protocol and PCR amplification (KAPA2G Fast Genotyping Mix, Kapa Biosystems, Wilmington, MA) using the primer combination: 5’-CTGGAAAATGCTTCTGTCCGTTTGC-3’ (SEQ ID NO: 3) and 5’-AATCCATCGCTCGACCAGTTTAGTTACC-3’ (SEQ ID NO: 4).

[0067] PCR analysis of recombination of the f10-locus was performed using the primers: 5’-ATGCTGGAGTTCTTCGCCCACC-3’ (SEQ ID NO: 5) and 5‘-CCATGCTTGAACTGTAGGACCAGAGC-3’ (SEQ ID NO: 6). Activity Assays

[0068] FXa amidolytic activity was measured by incubating plasma with the FX- activating enzyme from Russell’s viper venom (RVV-X; 5 nM), and progress of FXa formation was monitored from the hydrolysis rate of 200 μM synthetic substrate S-2765 (Benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginineparanitroaniline-dihydrochloride;Attorney Docket: 15979-20206.40 Chromogenix, Milano, Italy). To determine Prothrombin Time (PT), mouse plasma samples were diluted in commercially available Owren-Koller buffer (Stago, Asnières, France) and 50 μl was mixed with 50 μl human FX-deficient plasma (Stago) and 100 μl neoplastine (Stago). To determine activated Partial Prothrombin Time (aPTT), plasma samples were diluted in commercially available Owren-Koller buffer, and 50 μl was mixed with 50 μl human FX- deficient plasma. Upon the addition of 25 μl aPTT-reagent (Stago), the clotting reaction was started by the addition of 50 μl CaCl2solution. All clotting assays were performed in a Diagnostica Stago ST4 coagulation analyzer. Thrombin Generation Assay

[0069] Thrombin generation in platelet-poor murine plasma was measured in a microtiter-plate fluorometer (Fluoroskan Ascent, ThermoLabssystems, Helsinki, Finland) according to the method using either tissue factor (1 pM) and phospholipids (4 μM) or FXIa (175 nM) and phospholipids (4 μM) (Hemker et al., Pathophysiology of Haemostasis and Thrombosis (2002) 32(5-6):249-53). After incubation for five minutes at 37°C, thrombin generation was initiated by the addition of 20 μl pre-warmed Fluo-substrate (20 mM Hepes (pH7.4) / 0.1 M Z-Gly-Gly-Arg-AMC / 2.5 mM CaCl2 / 60 mg / ml BSA). Thrombin generation curves were recorded continuously with a microtiter-plate fluorometer (Fluoroskan Ascent, Thermo Labsystems, Helsinki, Finland) under control of Thrombinoscope software (Thrombinoscope BV, Maastricht, The Netherlands), filtered for excitation at 390 nm and emission at 460 nm. Laser-Induced Vessel Injury

[0070] Intravital videomicroscopy of the cremaster muscle microcirculation was performed as previously described (Dubois, supra). Following cremaster preparation, microvessel data were obtained using an Olympus BX61WI microscope with a 60 × 0.9 NA water immersion objective. The fluorescence microscopy system has previously been described (Dubois, supra). Digital images were captured with a Hamamatsu C9300 CCD camera in 640×480–pixel format. Widefield images were captured every 174 ms. The entire system was controlled using Slidebook (Intelligent Imaging Innovations, Denver, CO) and this software was also used for image and fluorescent data analysis as previously described (Dubois, supra). Platelets were labeled using Alexa647-labeled rat anti-mouse CD41 Fab fragments (250 ng / g bodyweight; clone MWReg30, Emfret, Eibelstadt, Germany). Vessel wall injury was induced with a nitrogen dye laser (Micropoint; Photonics Instruments) focused through the microscope objective, parfocal with the focal plane and aimed at the vessel wall. Typically, one or two pulses were required to induce vessel wall injury. Ten toAttorney Docket: 15979-20206.40 15 thrombi were generated per mouse over a time course of about 120 minutes. New thrombi were formed upstream of earlier thrombi in all experiments. There were no characteristic trends in thrombus size or thrombus composition in sequential thrombi generated in a single mouse during an experiment. Tail-Clip Assay

[0071] A classic tail-clip assay was performed (Muczynski, supra). Briefly, 3 mm of the distal tip of the tail of anesthetized mice (ketamine and xylazine, 100 and 10mg / kg, respectively) was amputated using a razor blade. Blood shed by the injury was collected in isotonic saline solution constantly warmed at 37°C for 30 min and quantified measuring hemoglobin optical density. Saphenous Vein Puncture Model

[0072] The saphenous vein puncture model was also performed. Under observation with an operating microscope, the saphenous vein was exposed and first partially transected with microscissors followed by a small longitudinal cut. When the bleeding ceased, the formed clot was physically dislodged with the sharp tip of a 30-gauge needle, and dislodgement was repeated after each incidence of hemostasis over the course of 30 min. The total number of clots formed (hemostatic events) within a 30-min period was recorded. In this model, higher numbers of reforming clots correspond to better hemostatic control / capacity. Statistical Analysis

[0073] All data are presented as mean ± standard deviation (mean ± SD) unless indicated otherwise. Number (n) refer to the number of independent experiments or animals. The statistical analysis was performed using GraphPad Prism 9 software for Mac (La Jolla, California, USA). One-way analysis of variance (1-way ANOVA) followed by Tukey’s or Dunnett’s multiple comparison test was performed when comparing multiple groups. Pairwise analysis was performed using the unpaired Student’s t-test, with Welch-correction where appropriate. Multiple Chi-square test was performed online. P<0.05 was considered as statistically significant. Results Induction of Factor X Deficiency

[0074] Transgenic mice homogenous for a loxP-flanked f10 gene and expressing cre- recombinase under the control of the interferon-inducible MX1-promotor (ie. MX1Cre+f10flox / flox) were used to inactivate the f10 gene by administration of pI:pC (FIG. 2). Age- and sex-matched MX1Cre--littermates that were treated with pI:pC served as controls, and are referred to as f10WT-mice. PCR-analysis of genomic DNA isolated from the liverAttorney Docket: 15979-20206.40 indicated that cre-recombinase activation efficiently induces elimination of exons 4 and 5 of the f10 gene in MX1Cre+f10flox / flox-mice but not in f10WT-mice (FIG. 3A). Consequently, f10WT-mice displayed efficient RVV-X-induced FX amidolytic activity, whereas residual FX amidolytic activity was virtually undetectable in plasma from pI:pC-treated MX1Cre+f10flox / floxmice (FIG. 3B). In terms of residual activity, the majority of the latter group displayed activity levels below the detection limit of 0.5% (FIG. 3C). Moreover, no FX antigen could be detected in the plasma of these mice. In the remainder of the study, only mice exhibiting <1% FX activity were used, and we refer to these mice as f10low-mice. Importantly, besides strongly reduced FX activity, no reduction in red cell, white blood cell or platelet counts were detected, nor were levels of FVII, FVIII or FIX affected (Table 2). It was further observed that no differences in survival between unchallenged f10low-mice and f10WT-control mice over a one-year period (6 / 7 and 7 / 7; p=0.32; FIG. 3D). Inspection of mice sacrificed at three months (n=5) or at 12 months (n=6) revealed no signs of overt bleeding. These data indicate that a severe FX-deficiency can be induced, resulting in mice with less than 1% FX activity, which are viable for at least 1 year. Table 2. Hematological Parameters of f10WTand f10lowMice*: n=15-19 for each group;#: n=5-8 for each group; NMP: normal mouse plasma collected from non-pI:pC-treated f10loxP / loxPmice; FVIII activity and FIX activity were measured using chromogenic activity assays; VWF: Von Willebrand factor. Data represent mean±SD. Loss of Circulatory FX Results in Severely Impaired in vitro Coagulation

[0075] Plasma obtained from f10WT-mice and f10low-mice was then analyzed in a number of in vitro hemostasis assays. First, in both the PT (19±1 sec versus 112±39 sec; p=0.0002; FIG. 4A) and the aPTT (42±3 sec versus 170±42 sec; p=0.0002; FIG. 4B) the clotting time was significantly prolonged in plasma from f10low-mice compared to f10WT-littermates. Next, aplasmas of these mice for thrombin generation was analyzed, triggered by TF or FXIa.Attorney Docket: 15979-20206.40 Regardless of the trigger used, thrombin generation was strikingly reduced in plasma of f10low-mice (FIGS. 4C and 4D; Table 3). All of the relevant parameters (lag-time, endogenous thrombin potential, thrombin peak and time to peak) were significantly different between both groups. Together, these data are compatible with FX activity being severely reduced to levels below 1%. Table 3. Thrombin GenerationETP: endogenous thrombin potential; Data represent mean ± SD. Statistical analysis was performed using unpaired Student’s t-test, and Welch-correction was applied where necessary. Thrombus Formation in a Laser-Induced Vessel Injury Model

[0076] To study FX-deficiency in vivo, the kinetics of arteriolar thrombus formation in the cremaster microcirculation following laser-injury was evaluated. Both f10WTand f10lowmice were infused with fluorescently-labeled antibodies against fibrin and the platelet-surface marker CD41 to monitor thrombus formation in real-time (FIG. 5A). Compared to f10WTmice, f10lowmice were characterized by a reduced accumulation of platelets, illustrated by a 6-7-fold lower median maximal platelet accumulation (median fluorescence intensity: 0.2x109 (95%-CI: 0.1-0.3x109) versus 1.1x109 (95%-CI: 0.8-1.5x109); p<0.0001; FIG. 5B, 5C). In parallel, fibrin deposition was abnormal in f10lowmice compared to f10WTmice FIGS. 5D and 5E). Fibrin deposition was reduced by >4-fold in the f10lowmice median fluorescence intensity: 0.5x109(95%-CI: 0.5-0.8x109) versus 2.3x109(95%-CI: 1.3-3.0x109); p=0.0008; FIGS. 5D and 5E). These shows that the strongly reduced FX activity levels in plasma result in defective thrombus formation in vivo. Importantly, the notion that some fibrin is generated indicates that FX activity levels are not absolutely zero, but that minor levels of FX (which are below our limit of detection) are still present to allow some thrombin-mediated fibrin formation. Tail-Clip Bleeding ModelAttorney Docket: 15979-20206.40

[0077] To get insight into the bleeding phenotype, both f10WTand f10lowmice were subjected to the tail-clip bleeding model in which the distal tip of the tail (3 mm) is excised. In all cases, mice were monitored for 30 min after injury. Blood loss in f10WTmice was minimal, and did not exceed 20μl in the majority of mice (12±16 μl; 95%-CI: 6-18 μl; n=30; FIG. 6A). In contrast, blood loss was more pronounced in the f10lowmice (590±335 μl; 95%- CI: 473-706 μl; n=34; p<0.0001; FIG. 6A). Blood loss was corrected upon infusion of 50 U / kg human FX (108±315 μl; 95%-CI: -103-320 μl; n=11; p=0.65 vs. f10WTand p<0.0001 vs. f10low), demonstrating that the increased bleeding tendency originated from the FX deficiency (FIG. 6A). Interestingly, blood loss in f10lowmice was much more variable compared to FVIII-knock out mice, with 40% of the f10lowmice having less blood loss compared to the FVIII-knock out mice (FIG. 6A). Moreover, the majority of the FVIII-knock out mice were characterized by the presence of a primary arrest, whereas this happened in only 50% of the f10lowmice (p=0.0009; FIGS. 6B and 6C). This suggests that the presence of FX might be relevant in the primary hemostatic response. Saphenous Vein Puncture Model

[0078] From studies with FVIII-deficient mice, it was determined that the saphenous vein puncture model is more severe compared to the tail clip model, as they require 50% and 20% FVIII for full correction, respectively. The bleeding phenotype of the f10low-mice in this particular model was also examined. A severe bleeding phenotype of the f10low-mice was observed, 2±2 clots / 30 min being formed (n=28; FIG. 7). f10WT-mice generated 19±5 clots / 30 min (n=14; p<0.0001). Interestingly, the f10low-mice displayed a similar impeded hemostasis as the f8KO-mice, which also generated 2±2 clots / 30 min (FIG. 7). Treatment of f10low-mice with a low-dose FX (1 U / kg) partially corrected bleeding to an intermediate phenotype (11±6 clots / 30 min; n=13; p<0.0001 compared to f10WT-mice and f10low-mice; FIG. 7). A five-fold higher dose (5 U / kg) resulted in complete normalization of the bleeding phenotype (21 ± 8 clots / min; n=8; p=0.933 compared to f10WT-mice). These data show that the saphenous vein puncture model is an attractive method to evaluate whether procoagulant agents are able to rescue the bleeding phenotype in FX-deficiency. Fitusiran Increases Thrombin Generation in FX Deficiency

[0079] Guided by the knowledge that antithrombin is the main inhibitor of FXa, it was hypothesized that by reducing antithrombin expression, the residual amounts of FX would be active over a longer period of time, and therefore would improve thrombin generation. This was first tested in in TG on assays using plasma from f10low-mice treated with fitusiran. Two to three weeks after induction of FX-deficiency, f10low-mice (having <1% FX activity)Attorney Docket: 15979-20206.40 received two weekly doses of fitusiran (10 mg / kg). Seven days after the last fitusiran injection, plasma samples were obtained and analyzed for residual antithrombin activity levels. Antithrombin levels were 96±20% (n=13) in f10low-mice that did receive vehicle- injections, whereas antithrombin levels were reduced to 17±6% (n=21) in fitusiran-treated f10low-mice (p<0.0001; FIG. 8A). Next, these samples were analyzed for TG. Compared to thrombin generation in plasma of f10low-mice, plasma of f10low-mice treated with fitusiran displayed marked increased thrombin generation (Table 2; FIG. 8B). Lag-time was shortened (p=0.0229), while ETP was increased 6-fold (p=0.0002) and Peak thrombin was increased 2.6-fold (p=0.0136). Since FX-levels below 1% are very rare, we also compared the effect of fitusiran in plasmas spiked with 2% FX. Despite TG being more efficient in the presence of 2% FX compared to <1%, the reduced levels of antithrombin due to fitusiran treatment still had a beneficial effect on thrombin generation under these conditions (Table 4; FIG. 8C). Both ETP (783 nM·min vs 1724±260 nM·min; p=0.0179) and Peak thrombin (31±11 nM vs 59±10 nM; p=0.0193) were increased in plasma of fitusiran-treated mice. Thus, at low or virtually absent FX levels, reduction of antithrombin concentrations is associated with increased thrombin generation. Table 4. Thrombin GenerationETP: endogenous thrombin potential; Data represent mean ±S D. Statistical analysis was performed using unpaired Student’s t-test.

[0080] Next, the effect of fitusiran-treatment in f10low-mice was evaluated. Compared to untreated f10low-mice, fitusiran-treated f10low-mice had a significantly increased number of clots in the saphenous vein puncture model: 8±6 clots / 30 min (n= 14) versus 2±2 clots / 30 min (n=28; p=0.0029; FIG. 8D). An analysis was performed, in which fitusiran-treated and untreated f10low-mice received low dose FX (1 U / kg) 5 min before injury. As expected, infusion of 1 U / kg FX resulted in increased clot formation (11±6 clots / 30 min; n=13; FIG. 8E). However, combined with fitusiran treatment, a significant increase in the number ofAttorney Docket: 15979-20206.40 clots was obtained (17±4 clots / 30 min; n=6; p=0.0262; FIG. 8E). Interestingly, this number of clots was no longer different from that observed in f10WT-mice (p=0.35). These data show that fitusiran has a substantial procoagulant effect in a mouse model for FX-deficiency. Conclusion

[0081] Factor X (FX)-deficiency is a rare bleeding disorder manifesting a bleeding tendency that is inversely associated with residual FX levels. To explore the use of fitusiran in increasing the in vivo hemostatic potential under conditions of FX-deficiency, we developed a novel model of inducible FX-deficiency, generating mice expressing <1% FX activity and antigen (f10low-mice). Compared to control f10WT-mice, f10low-mice had 6- and 4- fold prolonged clotting times in prothrombin time- and activated Partial Prothrombin Time- assays, respectively (p<0.001). Thrombin generation was also severely reduced, irrespective whether tissue factor or factor XIa was used as initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury-model. Furthermore, in two distinct bleeding models (tail clip- and saphenous vein puncture (SVP)-model), f10low-mice displayed an increased bleeding tendency compared to f10WT-mice. In the tail-clip assay blood loss was increased from 12±16 microliter to 590±335 microliter (p<0.0001), whereas the number of clots was reduced from 19±5 clots / 30 min for f10WT-mice to 2±2 clots / 30 min (p<0.0001) for f10low-mice, respectively.

[0082] In both models, bleeding was corrected upon infusion of purified FX. F10low-mice then received fitusiran (2x10 mg / kg at one-week interval) resulting in residual antithrombin activity being 14±2%, and was associated with increased thrombin generation (endogenous thrombin potential 4-fold, thrombin peak 2-3-fold). In the SVP-model, the number of clots was increased to 8±6 clots / 30 min (p=0.0029).

[0083] Together, we have demonstrated that under conditions of severe FX-deficiency, reduction of antithrombin levels is associated with improved hemostatic activity.

Claims

Attorney Docket: 15979-20206.40 CLAIMS 1. A method of treating factor X deficiency in a mammal, comprising administering fitusiran to the mammal in need thereof.

2. A method of prophylaxis to prevent or reduce bleeding in a mammal having factor X deficiency, comprising administering fitusiran to the mammal in need thereof.

3. A method of reducing use of factor X replacement factor to treat a bleeding event in a mammal with factor X deficiency, comprising administering fitusiran to the mammal in need thereof.

4. A method of increasing thrombin generation in a mammal having factor X deficiency, comprising administering fitusiran to the mammal in need thereof.

5. A method of improving hemostatic activity in a mammal having factor X deficiency, comprising administering fitusiran to the mammal in need thereof.

6. The method of any one of claims 1-5, wherein the factor X deficiency is type I factor X deficiency.

7. The method of claim 6, wherein the factor X deficiency is severe.

8. The method of claim 6 or claim 7, wherein the factor X activity in the mammal is less than 1% of a normal level.

9. The method of any one of claims 1-5, wherein the factor X deficiency is type II factor X deficiency.

10. The method of any one of claims 1-9, wherein the mammal is a mouse.

11. The method of any one of claims 1-9, wherein the mammal is a human.Attorney Docket: 15979-20206.40 12. The method of any one of claims 1-11, wherein fitusiran is administered, optionally in a pharmaceutical composition comprising phosphate-buffered saline, to the mammal by subcutaneous injection.

13. Fitusiran for use in treating factor X deficiency, in prophylaxis to prevent or reduce bleeding in a mammal having factor X deficiency, in reducing use of factor X replacement factor to treat a bleeding event in a mammal with factor X deficiency, in increasing thrombin generation in a mammal having factor X deficiency, or in improving hemostatic activity in a mammal having factor X deficiency, in the method of any one of claims 1-12.

14. Use of fitusiran in the manufacture of a medicament for treating factor X deficiency, in prophylaxis to prevent or reduce bleeding in a mammal having factor X deficiency, in reducing use of factor X replacement factor to treat a bleeding event in a mammal with factor X deficiency, in increasing thrombin generation in a mammal having factor X deficiency, or in improving hemostatic activity in a mammal having factor X deficiency, in the method of any one of claims 1-12.

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