FR3011250A1 - NUCLEIC ACIDS BINDING SPECIFICALLY TO HUMAN IX / IXA FACTOR, AND USES - Google Patents

Abstract

The present invention relates to a nucleic acid specifically binding to human factor IX / IXa.

Description

FIELD OF THE INVENTION Nucleic Acids Specifically Binding to Human Factor IX / IXa, and Their Uses FIELD OF THE INVENTION The present invention relates to the field of identifying ligands specific for human factor IX / IXa, these ligands being intended in particular the purification and detection of this protein, as well as their use in the medical field.

PRIOR ART Factor IX (FIX), also called anti-hemophilic factor B, is a vitamin K-dependent glycoprotein which, in its activated form (FIXa), participates in the coagulation process by interacting with thromboplastinogen (coagulation factor VIII ) to form thromboplastin, an enzyme essential for blood clotting. FIX is secreted as a unique peptide chain of 415 amino acid residues, which has a molecular weight of about 57 to 68 kDa. FIXa therefore plays an important role in the mechanisms of coagulation, resulting in the formation of blood clots. FIX is used in the treatment of hemophilia patients with factor IX deficiency (type B hemophilia or Christmas disease). Indeed, hemophilia B is a hereditary deficiency in factor IX coagulation, linked to sex, and is characterized by hemorrhages in the joints, muscles, internal organs, these hemorrhages can be spontaneous, accidental or related to a surgical intervention. The substitution treatment makes it possible to increase the plasma levels of factor IX, temporarily palliating the deficit and correcting the hemorrhagic tendencies. It is therefore necessary to have injectable FIXa concentrates. The oldest method of obtaining FIXa concentrates consisted in the purification of FIXa from plasma proteins resulting from the fractionation. For this purpose, document EP 0 346 241 describes the preparation of a fraction enriched in FIXa, obtained after adsorption then eluting a by-product of the fractionation of plasma proteins containing FIX and FIXa and other proteins such as VII (FVII), X (FX) and II (FII), in particular the pre-eluate of PPSB (P = prothrombin or FII, P = proconvertin or FVII, S = Stuart factor or FX and B = antihemophilic factor B or FIX). The disadvantage of this method is that the FIX obtained still contains some traces of other coagulation factors.

Patent application WO 2002/077161 describes the production of FIXa in a transgenic animal. Such a production method makes it possible to obtain proteins that are secure in terms of contamination by viruses or other pathogens. In addition, such a method makes it possible to obtain proteins whose primary sequence, that is to say an identical sequence between the different amino acids, is identical to the human primary sequence. However, irrespective of the initial source of factor IX / IXa, the methods used generate compositions enriched in human factor IX / IXa comprising contaminating substances. For methods of purification of Factor IX / IXa from human plasma, it would be very advantageous to have a final product that is free, or virtually free of residual thrombogenic activity. For methods for obtaining purified recombinant human factor IX / IXa, it is essential to have a final product that is free, or virtually free, of unwanted cellular proteins. In particular, for compositions of human factor IX / IXa purified from biological fluids from transgenic mammals, it is important to have an end product free or substantially free of the naturally occurring IX / IXa factor produced by these transgenic mammals, likely to be immunogenic in humans. To achieve this objective, it is necessary to have efficient and specific tools for the purification of human factor IX / IXa from a sample comprising said factor IX / IXa, and which can be produced easily and in a reproducible manner. SUMMARY OF THE INVENTION According to the invention, single-stranded nucleic acids specifically binding to human factor IX / IXa are provided. The present invention also relates to various compounds specifically binding to human factor IX / IXa, which comprise in their structure at least one nucleic acid defined above. It also relates to complexes between (i) a nucleic acid or a compound as defined above and (ii) a human factor IX / IXa. This invention also relates to a support for the immobilization of human factor IX / IXa, characterized in that it comprises a solid support material on which are grafted a plurality of nucleic acids or compounds as defined above.

The invention also relates to a method for the immobilization of human factor IX / IXa on a support, comprising a step during which a sample comprising human factor IX / IXa is brought into contact with a support as defined herein. -above. The invention also relates to a method for the purification of human factor IX / IXa comprising the following steps: a) contacting a sample comprising human factor IX / IXa with a nucleic acid or with a support as defined hereinabove. above, to form a complex between (i) said nucleic acid or said support and (ii) human factor IX / IXa, and b) releasing human factor IX / IXa from the complex formed in step a) and recover the purified human IX / IXa Factor. The invention also relates to a method for detecting the presence of human factor IX / IXa in a sample comprising the following steps: a) contacting a nucleic acid or a support as defined above with said sample; and b) detecting the formation of complexes between (i) said nucleic acid or said support and (ii) Factor IX / IXa. The present invention also relates to preventive or curative medical uses of the nucleic acids as defined above.

DESCRIPTION OF THE FIGURES Figure 1 illustrates the consensus sequences of the various nucleic aptamers specifically binding to human Factor IX / IXa identified by the inventors. The sequences shown in FIG. 1 are, from the top to the bottom of the figure, respectively the sequences SEQ ID No. 1 to SEQ ID No. 8, relating, respectively, to the families of aptamers designated by "Nonapta 1" to " Nonapta 8 ". Figure 2 illustrates an alignment of various nucleic aptamers specifically binding to human Factor IX / IXa and resulting from the implementation of two separate SELEX in vitro selection series, designated SELEX 5 and SELEX 6. The sequences shown in FIG. FIG. 2 are, from the top to the bottom of the figure, respectively the sequences SEQ ID No. 9 to SEQ ID No. 20, relating to the aptamer family designated "Nonapta 1". ". The sequences represented in lowercase letters in SEQ ID No. 20 correspond to the complementary sequences of the "primers" (or "primers") used for the amplification of the aptamer of SEQ ID No. 9 by the PCR (Polymerase Chain Reaction) technique. ).

Figure 3 illustrates an alignment of various nucleic aptamers specifically binding to the selected human IX / IXa Factor at the end of the SELEX-type process, designated SELEX 6. The sequences shown in Figure 3 are from the top to the right. the bottom of the figure, respectively the sequences SEQ ID No. 21 and SEQ ID No. 23, relating to the aptamer family designated "Nonapta 2". ". The sequences represented in lowercase letters in SEQ ID No. 23 correspond to the complementary sequences of the "primers" (or "primers") used for the amplification of the aptamer of SEQ ID No. 21 by the PCR technique. Figure 4 illustrates an alignment of various nucleic aptamers specifically binding to the selected human Factor IX / IXa at the end of the implementation of a SELEX type process (designated SELEX 5). The sequences shown in FIG. 4 are, from the top to the bottom of the figure, respectively the sequences SEQ ID No. 24 to SEQ ID No. 44, relating to the aptamer family designated "Nonapta 3". Figure 5 illustrates an alignment of various nucleic aptamers specifically binding to the selected human IX / IXa Factor at the end of a SELEX (SELEX 6) process run. The sequences shown in FIG. 5 are, from the top to the bottom of the figure, respectively the sequences SEQ ID No. 45 to SEQ ID No. 67, relating to the aptamer family designated "Nonapta 3". The sequences represented in lowercase letters in SEQ ID No. 67 correspond to the complementary sequences of the "primers" (or "primers") used for the amplification of the aptamer of SEQ ID No. 45 by the PCR technique. Figure 6 illustrates an alignment of various nucleic aptamers specifically binding to the selected human IX / IXa Factor at the end of a SELEX (ie, SELEX 5) process run. The sequences shown in FIG. 6 are, from the top to the bottom of the figure, respectively the sequences SEQ ID No. 68 to SEQ ID No. 104, relating to the aptamer family designated "Nonapta 4". Figure 7 illustrates an alignment of various nucleic aptamers specifically binding to selected Human Factor IX / IXa at the end of a cycle of SELEX process (i.e. SELEX 6) implementation. The sequences shown in FIG. 7 are, from the top to the bottom of the figure, respectively the sequences SEQ ID No. 105 to SEQ ID No. 131, relating to the aptamer family designated "Nonapta 4". The sequences represented in lowercase letters in SEQ ID No. 131 correspond to the complementary sequences of the "primers" (or "primers") used for the amplification of the aptamer of SEQ ID No. 105 by the PCR technique.

Figure 8 illustrates an alignment of various nucleic aptamers specifically binding to the selected human IX / IXa Factor at the end of a SELEX (SELEX 6) process run. The sequences shown in FIG. 8 are, from the top to the bottom of the figure, respectively the sequences SEQ ID No. 132 to SEQ ID No. 134, relating to the aptamer family designated "Nonapta 5". The sequences represented in lowercase letters SEQ ID No. 132 correspond to the complementary sequences of the "primers" (or "primers") used for the amplification of the aptamer of SEQ ID No. 133 by the PCR technique. Figure 9 illustrates an alignment of various nucleic aptamers specifically binding to selected Human Factor IX / IXa at the end of a cycle of implementation of a SELEX type process. The sequences represented in FIG. 9 are, from the top to the bottom of the figure, respectively the sequences SEQ ID Nos. 135 to 138 relating to the family of aptamers "Nonapta 6", the sequences SEQ ID Nos. 139 to 141 relating to to the "Nonapta 7" aptamer family and the sequences SEQ ID Nos. 142 and 143 relating to the "Nonapta 8" aptamer family. The sequences represented in lowercase letters in SEQ ID Nos. 135, 136, 137, 139 and 142 correspond to the complementary sequences of the "primers" (or "primers") used for the amplification of aptamers, respectively, of SEQ ID No. 138. in whole or in part, 140 and 143 by the PCR technique. Figure 10 illustrates the binding curves of a nucleic acid, belonging to the Nonapta 1 family (ie SEQ ID No. 20 in Figure 2), to the immobilized human plasma factor IX on a support, in a test according to the resonance technique. surface plasmonics. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, curve 1: Nonapta 1 with purified human plasma FIX at a concentration of 500 nM; Curve 2: Nonapta 1 with purified human plasma FIX at a concentration of 250 nM. FIG. 11 illustrates the binding curves of a nucleic acid, belonging to the Nonapta 2 family (ie SEQ ID No. 23 in FIG. 3), to the immobilized human plasma factor IX on a support, in a test according to the resonance technique. surface plasmonics. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, curve 1: Nonapta 2 with purified human plasma FIX at a concentration of 500 nM; Curve 2: Nonapta 2 with purified human plasma FIX at a concentration of 250 nM. FIG. 12 illustrates the binding curves of a nucleic acid, belonging to the Nonapta 3 family (ie SEQ ID No. 67 in FIG. 5) 0, to the immobilized human plasma factor IX on a support, in a test according to the technique of surface plasmon resonance. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, curve 1: Nonapta 3.1 with purified plasma human FIX at a concentration of 500 nM; Curve 2: Nonapta 3.1 with purified human plasma FIX at a concentration of 250 nM. Figure 13 illustrates the binding curves of a nucleic acid belonging to the Nonapta family (ie, SEQ ID No. 132 in Figure 8) to human plasma Factor IX immobilized on a support in a resonance test. surface plasmonics. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, curve 1: Nonapta 5 with purified human plasma FIX at a concentration of 1000 nM; Curve 2: Nonapta 5 with purified human plasma FIX at the concentration of 500 nM; Curve 3: Nonapta 5 with purified human plasma FIX at a concentration of 250 nM; Curve: 4 Nonapta 5 with purified plasma human FIX at the concentration of 100 nM. Figure 14 illustrates the binding curves of a nucleic acid, derived from Nonapta aptamer 5 (ie Nonapta 5.1, SEQ ID NO: 133 in Figure 8), to human plasma Factor IX immobilized on a support, in a test according to the surface plasmon resonance technique. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, curve 1 Nonapta 5.1 with purified human plasma FIX at a concentration of 1000 nM; Curve 2 Nonapta 5.1 with purified human plasma FIX at the concentration of 500 nM; Curve 3 Nonapta 5.1 with purified human plasma FIX at a concentration of 250 nM; Curve 4 Nonapta 5.1 with purified human plasma FIX at a concentration of 100 nM. Figure 15 illustrates the binding curves of a nucleic acid derived from Nonapta aptamer 5 (ie Nonapta 5.7, SEQ ID NO: 134, Figure 8), to human plasma Factor IX immobilized on a support, in a test according to US Pat. surface plasmon resonance technique. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, curve 1 Nonapta 5.7 with purified human plasma FIX at a concentration of 1000 nM; Curve 2 Nonapta 5.7 with purified plasma human FIX at a concentration of 500 nM; Curve 3 Nonapta 5.7 with purified human plasma FIX at a concentration of 250 nM; Curve 4 Nonapta 5.7 with purified human plasma FIX at the concentration of 100 nM. Figure 16 illustrates the binding curves of a nucleic acid, belonging to the Nonapta 6 family (ie SEQ ID No. 135 in Figure 9), to the human plasma Factor IX immobilized on a support, in a test according to the resonance technique. surface plasmonics. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, curve 1 Nonapta 6 with purified human plasma FIX at a concentration of 1000 nM; Curve 2 Nonapta 6 with purified human plasma FIX at a concentration of 500 nM; Curve 3 Nonapta 6 with purified human plasma FIX at the concentration of 200 nM; Curve 4 Nonapta 6 with purified human plasma FIX at the concentration of 100 nM. Figure 17 illustrates the binding curves of nucleic acid derived from Nonapta 6 aptamer (ie Nonapta 6.3, SEQ ID NO: 136, Figure 9), to human plasma Factor IX immobilized on a support, in a test according to US Pat. surface plasmon resonance technique. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, Curve 1 Nonapta 6.3 with purified human plasma FIX at a concentration of 1000 nM; Curve 2 Nonapta 6.3 with purified plasma human FIX at 500 nM concentration; Curve 3 Nonapta 6.3 with purified human plasma FIX at a concentration of 250 nM; Curve 4 Nonapta 6.3 with purified plasma human FIX at the concentration of 100 nM. Figure 18 illustrates the binding curves of nucleic acid derived from Nonapta 6 aptamer (ie Nonapta 6.4, SEQ ID NO: 137, Figure 9), to human plasma Factor IX immobilized on a support, in a test according to US Pat. surface plasmon resonance technique. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, curve 1 Nonapta 6.4 with purified human plasma FIX at a concentration of 1000 nM; curve 2 Nonapta 6.4 with purified human plasma FIX at a concentration of 500 nM; curve 3 Nonapta 6.4 with purified plasma human FIX at a concentration of 250 nM; Curve 4 Nonapta 6.4 with purified plasma human FIX at the concentration of 100 nM. Figure 19 illustrates the binding curves of a nucleic acid, belonging to the Nonapta 7 family (ie SEQ ID No. 139, FIG. 9), to the immobilized human plasma factor IX on a support, in a test according to the resonance technique. surface plasmonics. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, Curve 1 Nonapta 7 with purified human plasma FIX at a concentration of 1000 nM; Curve 2 Nonapta 7 with purified human plasma FIX at a concentration of 500 nM; Curve 3 Nonapta 7 with purified human plasma FIX at a concentration of 250 nM; Curve 4 Nonapta 7 with purified human plasma FIX at the concentration of 100 nM. FIG. 20 illustrates the binding curves of a nucleic acid, belonging to the Nonapta 8 family (ie SEQ ID No. 142, FIG. 9), to the immobilized human plasma factor IX on a support, in a test according to the resonance technique. surface plasmonics. On the abscissa: the time, expressed in seconds; ordinate, resonance signal, expressed in arbitrary resonance units. The curves represent, from the top to the bottom of the figure, Curve 1 Nonapta 8 with purified human plasma FIX at a concentration of 500 nM; Curve 2 Nonapta 8 with purified human plasma FIX at a concentration of 250 nM. Figure 21 represents a purification profile by affinity chromatography of a composition of human factor IX (Betafact® product marketed by LFB, France). On the abscissa: the time, expressed in minutes. On the ordinate: the measurement values of the absorbance at 280 nm, expressed in Arbitrary Units (mAU). On the curve, the injection of the human factor IX is carried out at time 0. On the curve, "1" corresponds to the non-retained fraction, "2" corresponds to the nonspecifically bound fraction and eliminated in the course of time. washing step, "3" corresponds to the peak of the elution fraction. FIG. 22 represents the image of an SDS PAGE electrophoresis gel in which lanes # 1 and 1 'correspond to the Betafact® Factor IX starting material, lanes # 2 and 2' correspond to the fraction of the product. starting point which was not selected on the column and tracks 3 and 3 'correspond to the elution fraction.

DETAILED DESCRIPTION OF THE INVENTION It is provided according to the invention new tools able to bind specifically to human factor IX / IXa, of reduced size, easy and inexpensive to synthesize, and which can be used in all fields in which such tools are useful, including for the purification of human factor IX / IXa, for the detection of human factor IX / IXa and for the use as an active ingredient of medicaments for the prevention or control of treatment of coagulation disorders.

More specifically, the Applicant has obtained a collection of single-stranded nucleic acids capable of binding specifically to the human factor IX / IXa, which nucleic acids are described later in the present description. As will be detailed below, the family of nucleic acids of the invention, which are able to bind specifically to human factor IX / IXa, consist of single-stranded nucleic acids, preferably of the DNA type, that the applicant thinks to be able to adopt conformations in space which contribute to confer to them their Factor IX / IXa binding properties mentioned above. Generically, the nucleic acids of the invention may also be called "aptamers" nucleotides, with reference to a term commonly used by those skilled in the art to designate this type of molecules. Nucleic aptamers capable of binding to various proteins involved in the blood coagulation pathway, including Willebrand factor binding aptamers (PCT Application No. WO 2008/150495), are already known in the art. aptamers binding alpha-thrombin (European Patent Application No. EP 1,972,693) or thrombin (Zhao et al., 2008, Anal Chem, Vol 80 (19): 7586-7593), aptamers binding the factor IX / IXa (Subash et al., 2006, Thromb Haemost, Vol 95: 767-771, Howard et al., 2007, Atherioscl Thromb Vasc Biol, Vol 27: 722-727, PCT Application No. WO 2002/096926 U.S. Patent No. 7,312,325), Factor X / Xa-binding aptamers (PCT Application No. WO 2002/096926, US Patent No. US 7,312,325), Factor VII binding nucleic aptamers Human VIIa (Rusconi et al., 2000, Thromb Haemost, Vol 84 (5): 841-848, Layzer et al., 2007, Spring, Vol 17: 1-11). The present invention relates to a nucleic acid specifically binding to human factor IX / IXa. In the present disclosure, a single-stranded nucleic acid specifically binding to human Factor IX / IXa may also be designated a "nucleic aptamer," "aptamer," "Human Factor IX / IXa binding aptamer," or "anti-FIX / FIXa aptamer. human ". By human factor IX / IXa, a natural human factor IX / IXa (i.e. of plasma origin) and a recombinant human factor IX / IXa are encompassed. For the purposes of the present description, a human factor IX / IXa is considered with reference to its amino acid sequence, that is to say regardless of whether the protein is glycosylated or non-glycosylated, and, if the protein is glycosylated , regardless of the type of glycosylation. The Applicant has isolated families of nucleic aptamers specifically binding to the human factor IX / IXa, the existence of which has been shown to exist between (i) the common structural characteristics and (ii) the common functional characteristic (s). From a structural point of view, a nucleic acid, or nucleic aptamer according to the invention, specifically binding to human factor IX / IXa has the following formula (I): [5'-TER] m - [SEQ ID No. X] - [3'-TER] n (I), in which: - "[5'-TER] II," represents a nucleic acid comprising between 1 and 50 nucleotides, preferably between 1 and 32 nucleotides, - "[5'-TER] 11, 3'-TER] ii "represents a nucleic acid comprising between 1 and 50 nucleotides, preferably between 1 and 40 nucleic acid nucleotides, - m is an integer equal to 0 or 1, - n is an integer equal to 0 or 1, and [SEQ ID NO: X] is a nucleic acid having at least 80% nucleotide identity with a nucleic acid selected from the group consisting of nucleic acids of SEQ ID NO: 5 (= Nonapta). ), SEQ ID No. 6 (= Nonapta 6), SEQ ID No. 1 (= Nonapta 1), SEQ ID No. 2 (= Nonapta 2), SEQ ID No. 3 (= Nonapta 3), SEQ ID No. 4 (= Nonapta 4), SEQ ID No. 7 (= Nonapta 7) and SEQ ID NO: 8 (= Nonapta 8), in which: - "a and A" represent an adenine nucleotide, 20 - "t and T" represent a thymine nucleotide, - "c and C" represent a cytosine nucleotide, - " g and G "represent a guanine nucleotide, and -" N "represents a nucleotide selected from adenine, thymine, cytosine or guanine. In some embodiments, the nucleic acid of SEQ ID NO: X is 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 nucleotides. According to a preferred embodiment, the nucleic acid of sequence SEQ ID NO: X is 39 or 40 nucleotides in length. In some embodiments, the "m" and "n" integers are both 0, and the nucleic acid of formula (I) is a nucleic acid of the following formula (IA): [SEQ ID NO: X] ] (IA), wherein [SEQ ID No. X] has the same meaning as for the nucleic acid of formula (I). In some embodiments, the integer m is 1 and the integer n is 0, and the nucleic acid of formula (I) is a nucleic acid of the following formula (I13): [5'-TER ] ff, - [SEQ ID NO: X] (IB), wherein [5'-TER] and [SEQ ID No. X] have the same meaning as for the nucleic acid of formula (I). In some embodiments, the integer m is 0 and the integer n is 1, and the nucleic acid of formula (I) is a nucleic acid of the following formula (IC): [SEQ ID NO: X] - [3'-TER] '(IB), wherein [3'-TER] and [SEQ ID No. X] have the same meaning as for the nucleic acid of formula (I). In some embodiments, the "5'-TER" nucleic acid, when present, has a length of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, which includes nucleic acids having exactly each of the specified lengths. In some embodiments, the "3'-TER" nucleic acid, when present, has a length of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides, which includes nucleic acids having exactly each of the specified lengths. In some embodiments, the nucleic acid of formula (I) has at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or 140 nucleotides in length, which includes nucleic acids having exactly each of the specified lengths. The collection of anti-FIX / FIXa nucleic aptamers according to the invention comprises a plurality of families of nucleic aptamers, each family of nucleic aptamers being characterized by common structural characteristics, in particular by a common nucleic acid sequence. In other words, two nucleic aptamers belonging to a given family of anti-FIX / FIXa nucleic aptamers according to the invention have between them a common consensus sequence, and in particular a common sequence which is designated "SEQ ID No. X" In the present description. The collection of antiFIX / FIXa nucleic aptamers according to the invention includes, in particular, eight families of nucleic aptamers, designated respectively "Nonapta 1", "Nonapta 2", "Nonapta 3", "Nonapta 4", "Nonapta 5", " Nonapta 6 "," Nonapta 7 "and" Nonapta 8 ", each family of nucleic aptamers comprising a consensus nucleic sequence, and in particular a consensus nucleic sequence designated" SEQ ID No. X ", which is common to all aptamers of said family.

In the collection of nucleic aptamers of the invention, the set of SEQ ID NO X sequences consists of a nucleic acid having at least 80% nucleotide identity with a nucleic acid selected from the group consisting of sequences SEQ ID No. 1 (= Nonapta 1), SEQ ID No. 2 (= Nonapta 2), SEQ ID No. 3 (= Nonapta 3), SEQ ID No. 4 (= Nonapta 4), SEQ ID No. 5 (= Nonapta 5), SEQ ID No. 6 (= Nonapta 6), SEQ ID No. 7 (= Nonapta 7) and SEQ ID No. 8 (= Nonapta 8). In general, a nucleic acid SEQ ID No. X having at least 80% nucleotide identity with a reference nucleic sequence has at least 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity with said nucleic acid sequence. reference. According to a first variant embodiment, the nucleic acid of sequence SEQ ID No. X may be represented by a nucleic acid having at least 80% identity with a nucleic acid of sequence SEQ ID No. 5 (= Nonapta 5). According to this first variant embodiment, the nucleic acid of formula (I) can then be represented by a nucleic acid of sequence SEQ ID No. 133 (= Nonapta 5.1) or SEQ ID No. 134 (= Nonapta 5.7), preferably a nucleic acid of sequence SEQ ID No. 134. In certain embodiments of a nucleic acid of formula (I) of the invention, the sequence of said nucleic acid has at least 80%, 81%, 82%, 83% , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% % nucleotide identity with a nucleic acid of SEQ ID NO: 5, SEQ ID NO: 133 or SEQ ID NO: 134.

According to a second variant embodiment, the nucleic acid of sequence SEQ ID No. X may be represented by a nucleic acid having at least 80% identity with a nucleic acid of SEQ ID No. 6 (= Nonapta 6). In certain embodiments of a nucleic acid of formula (I) of the invention, the sequence of said nucleic acid has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity with a nucleic acid of SEQ ID No. 6. According to a third variant embodiment, the nucleic acid of sequence SEQ ID No. X may be represented by a nucleic acid having at least 80% identity with a nucleic acid of SEQ ID No. 7 (= Nonapta 7). According to this third variant embodiment, the nucleic acid of formula (I) can then be represented by a nucleic acid of SEQ ID No. 7, SEQ ID No. 139, SEQ ID No. 140 or SEQ ID No. 141. In In some embodiments of a nucleic acid of formula (I) of the invention, the sequence of said nucleic acid has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity with a nucleic acid of SEQ ID No. 7, SEQ ID No. 139, SEQ ID No. 140 or SEQ ID No. 141. According to a fourth embodiment variant, the nucleic acid of sequence SEQ ID No. X may be represented by a nucleic acid. having at least 80% identity with a nucleic acid of SEQ ID No. 2 (= Nonapta 2). According to this fourth variant embodiment, the nucleic acid of formula (I) may then be represented by a nucleic acid of SEQ ID No. 2, SEQ ID No. 21, SEQ ID No. 22 or SEQ ID No. 23. 25 In certain embodiments of a nucleic acid of formula (I) of the invention, the sequence of said nucleic acid has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity with a nucleic acid of SEQ ID No. 2, SEQ ID No. 21, SEQ ID No. 22 or SEQ ID No. 23. According to a fifth embodiment variant, the nucleic acid of SEQ ID No. X can be represented by a nucleic acid. having at least 80% identity with a nucleic acid of SEQ ID No. 3 (= Nonapta 3). According to this fifth embodiment variant, the nucleic acid of formula (I) can then be represented by a nucleic acid of SEQ ID No. 3 or SEQ ID No. 24 to SEQ ID No. 67.

In certain embodiments of a nucleic acid of formula (I) of the invention, the sequence of said nucleic acid has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity with a nucleic acid of SEQ ID No. 3 or SEQ ID No. 24 to SEQ ID No. 67. According to a sixth variant embodiment, the nucleic acid of sequence SEQ ID No. X may be represented by a nucleic acid having at least 80% of identity with a nucleic acid of SEQ ID No. 1 (= Nonapta 1). According to this sixth variant embodiment, the nucleic acid of formula (I) can then be represented by a nucleic acid of SEQ ID No. 1 or SEQ ID No. 9 to SEQ ID No. 20. In some embodiments of the invention, a nucleic acid of formula (I) of the invention, the sequence of said nucleic acid has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity with a nucleic acid of SEQ ID No. 1 or SEQ ID No. 9 to SEQ ID No. 20. According to a seventh embodiment variant, the nucleic acid of sequence SEQ ID No. X may be represented by a nucleic acid having at least 80% identity with a nucleic acid. of SEQ ID No. 4 (= Nonapta 4). According to this seventh embodiment, the nucleic acid of formula (I) can then be represented by a nucleic acid of SEQ ID No. 4 or SEQ ID No. 68 to SEQ ID No. 131. In certain embodiments of the invention, a nucleic acid of formula (I) of the invention, the sequence of said nucleic acid has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity with a nucleic acid of SEQ ID No. 4 or SEQ ID No. 68 to SEQ ID No. 131. According to an eighth embodiment, the nucleic acid of sequence SEQ ID No. X may be represented by a nucleic acid having at least 80% identity with a nucleic acid. of SEQ ID No. 8 (= Nonapta 8). According to this eighth embodiment, the nucleic acid of formula (I) can then be represented by a nucleic acid of SEQ ID No. 8, SEQ ID No. 142 or SEQ ID No. 143. In some embodiments, a nucleic acid of formula (I) of the invention, the sequence of said nucleic acid has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity with a nucleic acid of SEQ ID NO: 8, SEQ ID No. 142 or SEQ ID No. 143. In certain embodiments, the sequence SEQ ID No. X of a nucleic acid according to the invention may be chosen from the group consisting of nucleic acids having at least 80% of nucleic acids. nucleotide identity, more preferably at least 90% nucleotide identity, with at least one of SEQ ID NO: 5 or SEQ ID NO: 6, preferably SEQ ID NO: 1. In some embodiments , a nucleic acid according to the inv The ention may have at least 80% nucleotide identity, more preferably at least 90% nucleotide identity, with a nucleic acid selected from the group consisting of the nucleic acids of sequences SEQ ID Nos. 1 to 143. In certain embodiments, embodiment, a nucleic acid according to the invention may have at least 80% nucleotide identity, better still at least 90% nucleotide identity, with a nucleic acid of sequences SEQ ID No. 133 (ie Nonapta 5.1) or SEQ ID No. 134 (ie Nonapta 5.7), preferably the sequence SEQ ID No. 134. The "percentage identity" between two nucleic acid sequences, within the meaning of the present invention, is determined by comparing the two optimally aligned sequences, through a comparison window. The part of the nucleotide sequence in the comparison window may comprise additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or deletions) so as to obtain an alignment optimal between the two sequences. The percent identity is calculated by determining the number of positions at which an identical nucleotide base is observed for the two compared sequences, and then dividing the number of positions at which there is identity between the two nucleobases by the total number of positions in the comparison window, then multiplying the result by one hundred in order to obtain the percentage of nucleotide identity of the two sequences between them. The optimal alignment of the sequences for the comparison can be performed in a computer manner using known algorithms.

Most preferably, the percentage of sequence identity is determined using the CLUSTAL W software (version 1.82) with the parameters set as follows: (1) CPU MODE = ClustalW mp; (2) ALIGNMENT = "full"; (3) OUTPUT FORMAT = "aln w / numbers"; (4) OUTPUT ORDER = "aligned"; (5) COLOR ALIGNMENT = "no"; (6) KTUP (word size) = "default"; (7) WINDOW LENGTH = "default"; (8) SCORE TYPE = "percent"; (9) TOPDIAG = "default"; (10) PAIRGAP = "default"; (11) PHYLOGENETIC TREE / TREE TYPE = "none"; (12) MATRIX = "default"; (13) GAP OPEN = "default"; (14) END GAPS = "default"; (15) GAP EXTENSION = "default"; (16) GAP DISTANCES = "default"; (17) TREE TYPE = "cladogram" and (18) TREE GRAP DISTANCES = "hide". On the basis of a nucleic acid according to the invention, as represented in FIGS. 1 to 9, one skilled in the art can easily determine the possible specific sequences for the sequence SEQ ID No. X, within the whole of the finite number of possible nucleotide sequences.

Those skilled in the art can verify its binding properties to human factor IX / IXa, for example according to one of the methods specified in the present description, in particular in the examples. According to the invention, a human factor IX / IXa binding nucleic acid is a single-stranded nucleic acid capable of forming a complex with human factor IX / IXa when contacted with the human factor IX / IXa. Nucleic acids binding to human factor IX / IXa therefore include those for which complexes can be detected with human factor IX / IXa after a prior step of contacting said respective nucleic and protein partners. The detection of complexes formed between a human factor IX / IXa binding nucleic acid can be easily performed by those skilled in the art, for example by using a surface plasmon resonance detection technique, including the Biacore® technique. as illustrated in the examples. Those skilled in the art can also easily detect the formation of complexes between a nucleic acid of interest and the human factor IX / IXa by conventional techniques of the ELISA type, or by any other laboratory technique capable of highlighting an interaction between a nucleic acid and a protein. As illustrated in the examples, a nucleic acid of formula (I) has a strong binding capacity to any type of human factor IX / IXa. In addition, the binding of a nucleic acid of formula (I) to natural human factor IX / IXa is specific.

Indeed, a nucleic acid of formula (I) has a very weak capacity for binding to Factor VII / VIIa, or even a zero capacity. Without wishing to be bound by any theory, the applicant believes that the results presented in the examples show that a nucleic acid of formula (I) according to the invention has a strong capacity to bind to human factors IX / IXa having distinct types of glycosylation. In other words, the applicant believes that a nucleic acid of formula (I) is capable of efficiently binding not only to Factor IX / IXa from natural sources, including human plasma, but also to a factor IX Recombinant human IXa produced in cell culture in vitro, or in a transgenic animal, preferably a transgenic mammal, of different species, including the rabbit, whose types of glycosylation may differ, even slightly from the type of glycosylation of Factor IX / IXa human produced naturally in the blood plasma. According to the invention, a nucleic acid "specifically" binding to human Factor IX / IXa consists of a nucleic acid having an ability to bind human Factor IX / IXa greater than its ability to bind to any other protein, and particular to Factors VII / VIIa. As used herein, a first nucleic acid has the ability to bind human Factor IX / IXa, which is greater than that of a second nucleic acid, when, using any of the binding detection techniques described above, above, and under the same test conditions, a statistically significant upper binding signal value is obtained with the first nucleic acid, relative to that obtained with the second nucleic acids. Illustratively, when the binding detection technique used is the Biacore technique, as in the examples, a first nucleic acid has an ability to bind human Factor IX / IXa, greater than that of a second nucleic acid, when the resonance signal value for the first nucleic acid, regardless of the expressed resonance unit of measurement, is statistically greater than the resonance signal value measured for the second nucleic acid. Two distinct "statistically" measured values encompass two values which have a greater difference than the measurement error of the link detection technique used.

As illustrated in the examples, the ability of the nucleic acids of the invention to bind specifically to human factor IX / IXa was tested. Moreover, the nucleic acids of formula (I) according to the invention are further advantageous in that they have an ability to bind to human factor IX / IXa which is significantly greater than their binding capacity to any what other factor. In particular, while a nucleic acid of formula (I) has a strong ability to bind to any type of human factor IX / IXa, including natural or recombinant, it has a low ability, and even zero, to bind to a factor VII / VIIa encoded by the genome of a non-human mammal. Without wishing to be bound by any theory, these specificity characteristics of the binding of a nucleic acid of formula (I) according to the invention to human factor IX / IXa illustrate that the aptamer nucleic acids of the invention can be advantageously used. in order to discriminate between a human factor IX / IXa and a human factor VII / VIIa, and, in addition, discriminate between a human factor IX / IXa and a non-human factor IX / IXa. In particular, a nucleic acid of formula (I) according to the invention can be advantageously used in a means for purifying a human factor IX / IXa, including from a complex starting medium capable of containing a factor IX / IXa in combination with other types of coagulation factors, such as, for example, Factor VII / VIIa. In particular, a nucleic acid of formula (I) may be used in a recombinant factor IX / IXa purification means in a biological fluid of a transgenic animal for human factor IX / IXa, said biological fluid being capable of containing other factors of coagulation, and particularly being likely to contain a factor IX / IXa produced naturally by the transgenic animal. As illustrated in the examples, another feature of a nucleic acid of formula (I) is its ability, in a complex with human Factor IX / IXa, to release human Factor IX / IXa by incubating said complex with a metal cation chelating agent such as EDTA (ethylene diamine tetraacetic acid) or EGTA (ethylene glycol-bis (2-aminoethylether) -N, N, N ', N'-tetraacetic acid). In particular, it has been shown that Factor IX / IXa molecules complexed with a nucleic acid of formula (I) are released from said complexes by contact with a metal cation chelating agent such as EDTA. Thus, according to yet another aspect, the binding of a nucleic acid of formula (I) according to the invention to human factor IX / IXa can be dissociated by contacting with a metal cation chelating agent, including by setting contact with EDTA or EGTA.

This additional characteristic of a nucleic acid of formula (I) according to the invention is particularly advantageous for the use of said nucleic acid in a means for purifying human factor IX / IXa. Indeed, in such an application for purification, the human factor IX / IXa, immobilized in a complex with a nucleic acid of formula (I), can then be recovered in purified form by simple contact or incubation with a chelating agent. metal cations, so without requiring the use of substances known to denature, at least partially, the human factor IX / IXa, such as acidic conditions or urea. For use in a human factor IX / IXa purification means, a nucleic acid of formula (I) according to the invention is preferentially immobilized on a solid support. Said solid support includes solid particles, chromatography media, etc. Techniques for immobilizing nucleic acids of interest on a wide variety of types of solid supports are well known to those skilled in the art. The solid support may be an affinity chromatography column composed of a gel derived from agarose, cellulose or a synthetic gel such as an acrylamide, methacrylate or polystyrene derivative, a chip such as a chip adapted to the surface plasmon resonance, a membrane such as a polyamide, polyacrylonitrile or polyester membrane or a magnetic or paramagnetic ball. For its use in a means for purifying human Factor IX / IXa, a nucleic acid of formula (I) according to the invention is preferably included in a chemical structure also comprising a spacer means and, if appropriate, an immobilization means on a solid support. Thus, the present invention also relates to a compound that binds specifically to human factor IX / IXa, characterized in that it has the following formula (II): [SPAC] - [NUCL] (II), in which: [SPAC] means a spacer chain, and - [NUCL] means a nucleic acid specifically binding to human Factor IX / IXa of formula (I). The above compound is a particular embodiment of a human factor IX / IXa purification means. The "spacer chain" designated [SPAC] in the compound of formula (II) may be of any known type. Said spacer chain has the function of physically moving the nucleic acid [NUCL] away from the surface of the solid support on which said compound can be immobilized and to allow a relative mobility of the nucleic acid [NUCL], relative to the surface solid support on which it can be immobilized. The spacer chain limits or avoids that steric hindrances, due to too close proximity of the solid support with the nucleic acid of formula (I), interfere with the binding events between said nucleic acid and human Factor IX / IXa molecules likely to to be put in contact with it. In the compound of formula (II), the spacer chain is preferably bonded to the 5 'end or the 3' end of [NUCL] nucleic acid. Advantageously, the spacer chain is bonded to both an end of the aptamer and the solid support. This spacer construction has the advantage of not directly immobilizing the aptamer on the solid support. Preferably, the spacer chain is a nonspecific oligonucleotide or polyethylene glycol (PEG). When the spacer chain consists of a nonspecific oligonucleotide, said oligonucleotide advantageously comprises at least 5 nucleotides in length, preferably between 5 and 15 nucleotides in length. In embodiments of a compound of formula (II) in which the spacer chain is a polyethylene glycol, said spacer chain includes PEG (C18) type polyethylene glycol. To immobilize the aptamer directly on the solid support, or on the spacer chain, the [NUCL] nucleic acid can be chemically modified with different chemical groups such as groups that make it possible to immobilize said nucleic acid in a covalent manner, such as thiols, amines or any other group capable of reacting with chemical groups present on the solid support. In some embodiments, the spacer chain is itself capable of being immobilized on the solid support, where appropriate after modification of said spacer chain with one or more appropriate chemical groups. In yet other embodiments, the spacer chain is linked to a compound for immobilizing the compound of interest comprising the nucleic aptamer on the mobile support. Thus, the present invention also relates to a compound that binds specifically to human factor IX / IXa, characterized in that it has the following formula (III): [FIX] - [SPAC] - [NUCL] (III), wherein: [FIX] means an immobilization compound on a support, - [SPAC] means a spacer chain, and - [NUCL] means a nucleic acid specifically binding to human Factor IX / IXa of formula (I). The compound of formula (III) above is one embodiment of a human factor IX / IXa purification means. Said means for purifying human Factor IX / IXa of formula (II) or of formula (III) is preferably in a form bound to a solid support. In the compound of formula (III) the compound [FIX] consists of a compound selected from (i) a compound capable of forming one or more covalent bonds with the surface material of a solid support and (ii) a compound capable of to bind specifically to the solid support via weak non-covalent linkages, including hydrogen bonds, electrostatic forces or Van der Waals forces. The first type of compound [FIX] includes the bifunctional coupling agents, such as glutaraldehyde, SIAB or SMCC.

The SIAB compound, described by Hermanson GT (1996, Bioconjugate Technical, San Diego: Academic Press, pp 239-242), is the compound of formula (I) below: (I) The compound SIAB comprises two reactive groups, respectively one iodoacetate group and a sulfo-NHS ester group, these groups reacting respectively on amino and sulfhydryl groups. The SMCC compound, which is described by Samoszuk M.K. et al. (1989, Antibody, Immunoconjugates Radiopharm., 2 (1): 37-46), is the compound of formula (II) below: (11) The SMCC compound comprises two reactive groups, respectively a sulfo-NHS ester group and a maleimide, which react with an amino group and a sulfhydryl group, respectively. The second type of compound [FIX] includes biotin, which is capable of specifically non-covalently binding to avidin or streptavidin molecules present on the solid support. In some embodiments, when immobilized on the solid support via the spacer chain, the aptamer is advantageously modified at its free end (end not bound to the spacer) by virtue of, and not limited to, a chemically nucleotide modified (such as 2'-O-methyl or 2'-fluoropyrimidine, 2'-ribopurine, phosphoramidite), an inverted nucleotide or a chemical group (PEG, polycations, cholesterol). These modifications make it possible to protect certain aptamers from enzymatic degradations. In still some other embodiments, the immobilization of an anti-FIX / IXa nucleic acid according to the invention on a support can be carried out according to the method described in the PCT application published in the name of LFB Biotechnologies under the No. WO 2012/090183 dated July 5, 2012. This immobilization process is carried out by grafting nucleic acids comprising a primary amine on a solid support having activated carboxylic acid groups, said process comprising a step of covalent coupling of said nucleic acids. on said solid support at a pH below 6.

An affinity support on which are immobilized molecules of an anti-FIX / FIXa nucleic acid according to the invention, and prepared according to the teaching of PCT application No. WO 2012/090183 mentioned above, can be represented by the formula following (IV): n-NUCL N (IV) H (SPAC) in which: [SUP] represents the solid support of the affinity support - [SPAC] n and [NUCL] are as previously defined. In other words, -NH- (SPAC) .- NUCL represents a nucleic aptamer in which: n is an index of 0 or 1, ^ SPAC represents a spacer chain, and NUCL represents a nucleic acid binding specifically to the factor Human IX / IXa of formula (I). In some embodiments, a "nucleic aptamer" used for grafting to the pre-activated solid support, according to the method or method described in PCT Application No. WO 2012/090183, may comprise, by definition, a non-nucleotide portion. or nucleotide, for example a non-nucleotide spacer chain, which connects one of the 5 'or 3' ends of the nucleic portion of said aptamer and the reactive amine function used for the chemical grafting of said pre-activated support. In these embodiments, a nucleic aptamer may be of the following formula (V): NH2- [SPAC] .- [NUCL] (V), wherein: n being an index of 1 or 0, 0 meaning that the aptamer does not include a free spacer chain and 1 signifying that the aptamer comprises a spacer chain. - NH2 means the reactive amine function used for grafting on the NHS-pre-activated solid support, - [SPAC] means a spacer chain, and - [NUCL] means a nucleic acid specifically binding to human IX / IXa Factor of formula (I). The solid support may be an affinity chromatography column composed of a gel derived from agarose, cellulose or a synthetic gel such as an acrylamide, methacrylate or polystyrene derivative, a chip such as a chip adapted to the surface plasmon resonance, a membrane such as a polyamide, polyacrylonitrile or polyester membrane, a magnetic or paramagnetic ball. The present invention also relates to a complex between: (i) a substance selected from a nucleic acid of formula (I), a compound of formula (II) and a compound of formula (III), and (ii) a Factor IX / IXa human. The present invention also relates to a support for the immobilization of human factor IX / IXa, characterized in that it comprises a solid support material on which are grafted a plurality of molecules each consisting of or each comprising a nucleic aptamer , said molecules being chosen from (a) a nucleic acid of formula (I), (b) a compound of formula (II) and (c) a compound of formula (III). The above support can be used in virtually any application for which human Factor IX / IXa is to be immobilized, including applications for the Purification of Factor IX / IXa and applications for the detection of Factor IX / IXa. Human IX / IXa factor. The production of supports on which are immobilized nucleic aptamers of the invention specifically binding to human factor IX / IXa is illustrated in the examples, in which the aptamers of the invention are used in particular as Factor IX / IXa capture agents. human, usable for purification or for the detection of human factor IX / IXa in samples. The present invention therefore also relates to a method for immobilizing human factor IX / IXa on a carrier, comprising a step in which a sample comprising human factor IX / IXa is contacted with a solid support on which previously immobilized a substance selected from a nucleic acid of formula (I), a compound of formula (II) and a compound of formula (III). Said method may comprise, according to the technical objectives pursued, an additional step of recovering immobilized human IX / IXa molecules complexed with the nucleic acid molecules of formula (I). The additional step of recovering Factor IX / IXa consists preferentially in a step of contacting Factor IX / IXa complexes with the nucleic acids of formula (I) with a metal cation chelating agent, such as EDTA or EGTA. The present invention further relates to a method for the purification of human factor IX / IXa comprising the following steps: a) contacting a sample comprising human factor IX / IXa with a nucleic acid of formula (I), a compound of formula (II), a compound of formula (III) or with a solid support as defined in the present description, to form a complex between (i) said nucleic acid, or said compound, or said support and (ii) the Human IX / IXa Factor, and b) release the human IX / IXa Factor from the complex formed in step a) and recover the purified human IX / IXa Factor. For carrying out protein purification methods by affinity chromatography using solid supports on which the aptamers of interest are immobilized, one skilled in the art can refer to the works described by Romig et al. (1999, Chomatogr B Biomed Sci Apl, Vol 731 (2): 275-284). In a particular embodiment, the method comprises a step a '), step a' following step a) and preceding step b), which consists of a step of washing the affinity support with a buffer washing.

Advantageously, the process comprises a step a ') of washing the affinity support during which the ionic strength is increased, that is to say that a washing buffer is used whose ionic strength is increased relative to the ionic strength of the buffer used in step a). Advantageously, the ionic strength of the washing buffer used in step a ') is greater by 2 to 500 times with respect to the ionic strength of the buffer used in step a).

Advantageously, the ionic strength of the washing buffer is greater by 100 to 500 times, preferably 200 to 500 times, relative to the ionic strength of the buffer used in step a). A wash buffer having a high ionic strength, particularly a buffer having a high concentration of NaCl, effectively removes nonspecifically bound substances from the affinity support without simultaneously affecting Factor IX binding in a detectable manner. to the affinity support. In step a '), a wash buffer having a final NaCl concentration of at least 0.5M is preferably used.

According to the invention, a wash buffer having a final NaCl concentration of at least 0.5 M includes wash buffers having a final NaCl concentration of at least 1 M, 1.5 M, 2 M, 2 At least 3 M Mg. Preferably a wash buffer used in step a ') of the process has a final NaCl concentration of at most 3.5 M. Advantageously, a wash buffer used in step a ') of the process has a final NaCl concentration of between 0.5 and 3.5, preferably between 1.5 and 3.5, preferably between 2 and 3.5, for example preferably between 2 and 3.5, 5 and 3.5. According to a particular embodiment, the washing buffer used in step a ') contains both NaCl and propylene glycol. In addition, in some embodiments of the above purification method, step b) is performed by contacting the affinity support with an elution buffer containing a divalent ion chelating agent, preferably EDTA or EGTA. By way of illustration, the elution buffer may contain a final EDTA concentration of at least 1 mM and at most 300 mM. The term "at least 1 mM" includes at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 mM. The term "not more than 300 mM" includes not more than 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120 or 110 mM . In addition, the manufacture of an affinity support comprising a nucleic aptamer of the invention and the realization of a method of purifying human Factor IX with said affinity support is illustrated in the examples. In general, the solid supports on which the aptamers of the invention may be immobilized include any type of support having the structure and composition commonly found for filter supports, silicon support for chips, membranes, etc. . Solid supports include resins, resins for affinity chromatography columns, polymer beads, magnetic beads, and the like. Solid supports also include glass or metal-based materials such as steel, gold, silver, aluminum, copper, silicon, glass and ceramics. Solid supports also include, in particular, polymeric materials, such as polyethylene, polypropylene, polyamide, polyvinylidene fluoride, and combinations thereof. The solid support may be coated with a material facilitating attachment, fixation, complex formation, immobilization or interaction with aptamers.

In some embodiments, the solid support is a glass slide whose surface is coated with a gold layer, a carboxymethylation-treated layer, a layer of dextran, collagen, avidin, streptavidin, etc. In this way, the aptamers according to the invention can be immobilized on the solid support by means of an attachment coating, as for example described above, either by chemical reaction with creation of covalent bonds, or by association by non-covalent bonds such as hydrogen bonds, electrostatic forces, Van der Waals forces, etc. According to the invention, the term "affinity support" generally means a support formed of a solid material on which nucleic aptamers as defined in the present description have been immobilized. The following examples describe an embodiment of a solid support on which the aptamers of the invention are immobilized by non-covalent bonds. In the examples, there is described in particular a solid support consisting of a glass slide coated with a layer of streptavidin molecules, and aptamers of the invention conjugated to a biotin molecule which are immobilized on the support by non-covalent association biotin / streptavidin. In certain embodiments, the aptamers of the invention may be immobilized on a solid support suitable for affinity chromatography, electrochromatography and capillary electrophoresis, as described for example by Ravelet et al. (2006, J Chromatogr A, Vol 117 (1): 1-10), Connor et al. (2006, J Chromatogr A, Vol 111 (2): 115-119), Cho et al. (2004, Electrophoresis, Vol 25 (21-22): 3730-3739) or Zhao et al. (2008, Anal Chem, Vol 80 (10): 3915-3920). An aptamer of formula (I) according to the invention and specifically binding to human factor IX / IXa, a compound of formula (II), or a compound of formula (III), can also be advantageously used as a FIX / IXa human, in methods and devices for detection or diagnosis. In yet another aspect, the present invention also relates to a method for detecting the presence of human factor IX / IXa in a sample comprising the following steps: a) contacting (i) a nucleic acid of formula (I), a compound of formula (II) or a compound of formula (III) or a solid support on which a plurality of molecules of said nucleic acid or said compound are immobilized, with (ii) said sample; and b) detecting the formation of complexes between (i) said nucleic acid of formula (I), said compound of formula (II) or said compound of formula (III) or said support and (ii) Factor IX / IXa. The examples of the present patent application provide various embodiments of methods for detecting human FIX / IXa with aptamers of the invention previously immobilized on a solid support. For carrying out a detection method according to the invention, the solid support used may be a solid support chosen from the solid supports described above in connection with the method for purifying human factor IX / IXa.

For the realization of a method or a device for detecting human factor IX / IXa, a person skilled in the art can refer in particular to the techniques described in European Patent Application No. EP 1 972 693, PCT Application No. WO 2008/038696, PCT Application No. WO 2008/025830, or PCT Application No. WO 2007/0322359. In some embodiments, step b) of detecting complex formation between (i) said nucleic acid or said solid support and (ii) human factor IX / IXa can be performed by measuring the plasmonic resonance signal of surface, as described in the examples. In certain other embodiments, the step b) of detecting the formation of complexes between (i) said nucleic acid or said solid support and (ii) the human factor IX / IXa can be carried out by bringing said complexes into contact with each other. optionally formed with a human Factor IX / IXa ligand, said ligand being detectable. As a detectable ligand for human Factor IX / IXa, there may be mentioned monoclonal or polyclonal antibodies antiIX / IXa human, labeled with an enzyme, in this case horseradish peroxidase, as conventionally used in ELISA type tests.

Advantageously, the sample comprising or capable of comprising human factor IX / IXa consists of a liquid sample which contains the human factor IX / IXa, including a liquid sample comprising the human factor IX / IXa and which is capable of containing also factor IX / IXa molecules of a non-human mammal. In some embodiments of the above purification method or detection method, said sample consists of a biological solution, such as a body fluid, a cell, a crushed cell, a tissue, a tissue mill, an organ or an entire organism. In some embodiments of the purification method or detection method above, said sample consists of a liquid biological solution from an animal such as blood, a blood derivative (blood derivative), mammalian milk or a mammalian milk derivative. Said sample may consist of plasma, plasma cryoprecipitate, clarified milk or their derivatives. In particularly preferred embodiments of the above purification method or detection method, said sample is derived from a transgenic animal for human factor IX / IXa. Advantageously, the solution is mammalian milk or a transgenic mammalian milk derivative for human factor IX / IXa. For the purposes of the invention, the transgenic animals include (i) non-human mammals such as cows, goats, rabbits, pigs, monkeys, rats or mice, (ii) birds or (iii) ) insects such as mosquitoes, flies or silkworms. In some preferred embodiments, the human factor IX / IXa transgenic animal is a non-human transgenic mammal, most preferably a transgenic rabbit for human factor IX / IXa. Advantageously, the transgenic mammal produces the recombinant factor IX / IXa in its mammary glands, because of the insertion into its genome of an expression cassette comprising a nucleic acid encoding human factor IX / IXa which is under control. a specific promoter allowing the expression of the transgene protein in the milk of said transgenic mammal. One method of producing human factor IX / IXa in the milk of a transgenic animal may comprise the following steps: a DNA molecule comprising a gene encoding human factor IX / IXa, said gene being under the control of a promoter of a naturally secreted protein in the milk (such as the casein, beta-casein, lactalbumin, beta-lactoglobulin promoter or the WAP promoter) is integrated into an embryo of a non-human mammal. The embryo is then placed in a female mammal of the same species. Once the mammal from the embryo has grown sufficiently, the lactation of the mammal is induced, then the milk collected. The milk then contains human factor IX / IXa. An example of a process for the preparation of protein in the milk of a female mammal other than the human is given in EP 0 527 063, the teaching of which may be repeated for the production of the protein of the invention. . A plasmid containing the WAP (Whey Acidic Protein) promoter is manufactured by introducing a sequence comprising the promoter of the WAP gene, this plasmid being made in such a way as to be able to receive a foreign gene placed under the control of the WAP promoter. The plasmid containing the promoter and the gene coding for the protein of the invention are used to obtain transgenic rabbits by microinjection into the male pronucleus of rabbit embryos. The embryos are then transferred into the oviduct of hormonally prepared females. The presence of the transgenes is revealed by the Southern Blot technique from the DNA extracted from the transgenic rabbits obtained. The concentrations of the transgene protein in animal milk are evaluated using specific radioimmunoassays.

Other documents describe methods for preparing proteins in milk of a female mammal other than man. These include, but are not limited to, US 7,045,676 (transgenic mouse) and EP 1,739,170 (production of von Willebrand factor in a transgenic mammal). According to other aspects of the present invention, a nucleic acid according to the invention and specifically binding to human Factor IX / IXa can be used in vivo, in physiological situations requiring the inhibition of Factor X activation by Factor IXa, for example by inhibiting the formation of a functional complex Factor IXa / Factor VIIIa). In other words, an aptamer nucleic acid as defined in the present description can also be used, as a preventive or curative, as anticoagulant active ingredient of a drug. In particular, an aptamer nucleic acid as defined in the present description may be used as a preventive or curative anticoagulant active ingredient, in particular for the treatment of coagulation disorders including venous thromboses, arterial thromboses, post-surgical thromboses, coronary artery bypass (CABG) disorders, stroke, percutaneous transdermal coronary angioplasty (PTCA), tumor metastasis, non-severe or severe inflammatory reactions, septic shock, hypotension, acute respiratory distress syndrome (ARDS), pulmonary embolisms, disseminated intravascular coagulation (DIC), vascular restenosis, platelet deposition, myocardial infarction, angiogenesis, and any prohylactic treatment of men or women at risk of developing thrombosis. The present invention also relates to a pharmaceutical composition comprising a nucleic acid specifically binding to human factor IX / IXa as defined herein, in combination with one or more pharmaceutically acceptable excipients. The amount of human anti-FIX / FIXa aptamer nucleic acid according to the invention, in a pharmaceutical composition, is adapted to allow the administration of an effective amount of this active ingredient to patients.

The dose ranges of the human anti-FIX / FIXA aptamer of the invention can be readily determined by the physician or pharmacist. In general, the amount of active ingredient to be administered varies with the patient's age, medical condition, and sex, as well as with the degree of spread of the disease or degree of risk of developing the disease, and may be easily determined by those skilled in the art. In general, a pharmaceutical composition comprises an amount of a human anti-FIX / FIXa aptamer ranging from 1 nanogram to 100 milligrams per unit dose, more preferably from 100 nanograms to 10 milligrams per unit dose. In general, a pharmaceutical composition according to the invention comprises from 0.01% to 99.9% by weight of an anti-FIX / FIXa aptamer, or a combination of several anti-FIX / FIXa aptamers, and from 99% to 99.9% by weight of an anti-FIX / FIXa aptamer. , 9% to 0.01% by weight of an excipient, or a combination of excipients, relative to the total weight of said composition. In some embodiments, said pharmaceutical composition comprises 1, 2, 3, 4, 5 or 6 distinct anti-FIX / FIXa aptamers.

In order to produce a pharmaceutical composition according to the invention, those skilled in the art can advantageously refer to Remington's work. The pharmaceutical composition according to the invention can be used prophylactically and / or therapeutically. The administration of a pharmaceutical composition according to the invention can be carried out in several ways, including but not limited to, locally and mucocutaneously, enterally or parenterally. By "local route and mucocutaneous" means in particular the topical route, intra-auricular, intravaginal, intrauterine, inhalation, transdermal, ocular, or intravesical. By "enteral route" is meant in particular the route, intrarectal, sublingual, oral, nasal, intrastagmal or intrajejunal. By "parenteral route" is meant in particular the intradermal, subcutaneous, intramuscular, intracardiac, intravascular, intravenous, intraocular, intraarterial, epidural, intraspinal, extracorporeal, intrathecal, intraperitoneal, intrapleural, intraluminal, intravitreal, intracavernous, intraventricular, intraosseous, palatal, intra- articular, intracellular, pulmonary or intrafoetalous. Preferably, the administration of a pharmaceutical composition according to the invention can be made intravenously. Thus, a human anti-FIX / FIXA aptamer according to the present invention is prepared in a form adapted to the chosen type of administration, for example in liquid form or in freeze-dried form. The pharmaceutical compositions of human anti-FIX / FIXA aptamers according to the present invention may contain an excipient and / or a pharmaceutically acceptable vehicle, preferably an aqueous vehicle. Many pharmaceutically acceptable excipients and / or carriers may be used, for example, water, buffered water, saline solution, glycine solution and their derivatives as well as agents necessary to reproduce physiological conditions such as buffering agents and pH adjusters, surfactants such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, this list not being limiting. In addition, the pharmaceutical composition can be sterilized by sterilization techniques well known to those skilled in the art. In general, to manufacture a pharmaceutical composition according to the invention, one skilled in the art can advantageously also refer to the latest edition of the European Pharmacopoeia, for example to the 5th edition of the European Pharmacopoeia published in January 2005, or still in the 6th edition of the European Pharmacopoeia, available to the public in June 2007. For the manufacture of a pharmaceutical composition comprising an aptamer as an active ingredient, the person skilled in the art can also refer to the contents of PCT application no. WO 2007/058801, PCT Application No. WO 2005/084412 and PCT Application No. WO 2004/047742 or in PCT Application No. WO 2008/150495.

In some embodiments of a pharmaceutical composition according to the invention, the human anti-FIX / FIXa aptamer active ingredient (s) may be used alone or in combination with one or more other pharmaceutically acceptable molecules. active agents, including one or more other anticoagulant active ingredients.

The invention also relates to a nucleic acid specifically binding to human factor IX / IXa, as defined in this specification, for use as a medicament. The invention also relates to the use of a nucleic acid specifically binding to human factor IX / IXa, as defined in the present description, for the manufacture of a medicament for the treatment of coagulation disorders, in particular including those mentioned above, preferably type B hemophilia. The invention also relates to the use of a nucleic acid specifically binding to human factor IX / IXa, as defined in the present description, for treating coagulation disorders, including those mentioned above, preferably hemophilia B. The invention also relates to a method for preventing or treating a coagulation disorder, comprising a step during to which a patient in need of preventive or curative treatment of a coagulation disorder is administered a human anti-FIX / FIXa aptamer pharmaceutical composition as defined herein. escription. In general, a daily dose of a human anti-FIX / FIXa aptamer as defined in the present description ranging from 1 nanogram to 100 milligrams, more preferably from 100 nanograms to 10 milligrams, is administered for a patient of 80 kgs. The present invention is further illustrated hereinafter, but not limited thereto, to the following examples.

EXAMPLES Example 1 Specific Embodiments of an Interaction Protocol of Human Factor IX with an aptamer on Biacore (Resistance to NaCl) Materials and Methods A solid support was made on which nucleic aptamer molecules were immobilized according to the invention. Prior to their attachment to the solid support, the 5 'end of the aptamers considered was chemically coupled to a biotin molecule. There is a solid support containing immobilized streptavidin molecules (Serie S sensor chip SA, GE). Then, the solid support above was brought into contact with the aptamer compounds below in order to immobilize the nucleic acids. by non-covalent association between the streptavidin molecules of the support and the biotin molecules of the aptamer compounds. The aptamers considered are those represented by the sequences SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 67, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135. , SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 139 and SEQ ID NO: 142.

The aptaptor Nonapta 1 is thus immobilized with a immobilization rate of approximately 1830 RU (1 RU corresponds approximately to 1 μg of immobilized product per mm 2). The aptamers Nonapta 2 and Nonapta 3 are thus immobilized with an immobilization rate of approximately 1,950 RU.

Nonapta aptamer 3.1 (ie an aptamer of sequence SEQ ID No. 67 for which the 3 'end was chemically coupled to a biotin molecule) is thus immobilized with an immobilization rate of about 2050 RU. Nonapta aptamers 5, 5.1 and 5.7 are thus immobilized with an immobilization rate of, respectively, about 2 190 RU, 3 080 RU and 1 760 RU. Nonapta aptamers 6, 6.3 and 6.4 are thus immobilized with an immobilization rate of approximately 2 270 RU, 1 720 RU and 2 150 RU, respectively. The aptaptor Nonapta 7 is thus immobilized with an immobilization rate of approximately 2 220 RU.

The Nonapta aptamer 8 is thus immobilized with a immobilization rate of approximately 2,260 RU. Purified human FIX was diluted in running buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl 2, 1 mM MgCl 2, pH 7.4) to obtain 100 and 250 nM FIX samples. , or for some of the above aptamers of concentration 500 and 1000 nM in FIX. Note however that, for Nonapta 6, the same purified human FIX was diluted in running buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl 2, 1 mM MgCl 2, pH 7.4) to obtain 100, 200, 500 and 1000 nM samples in FIX.

Several supports have been manufactured, each comprising a single specific aptamer among those indicated above. All injections were performed with a flow rate of 301.11 / min for 60 sec after the injection.

These analyzes are carried out with the RPS Biacore® T-200 (GE) device under the control of the Biacore® T-200 Control Software (Version 1.0). The binding curves of the immobilized aptamers described above in the human FIX were calculated with the dedicated module of the Biacore® T-200 Evaluation Software (Version 1.0).

These results made it possible to determine that: the aptamers used, once immobilized, actually bind to the FIX. These results are illustrated in FIGS. 10 to 20. Factor IX binding is particularly significant for Nonapta aptamers 5, 5.1, 5.7, 6, 6.3 and 6.4, and more particularly the Nonapta aptamer 5.7. These results are illustrated in FIGS. 13 to 18.

Example 2: Preparation of an Affinity Support The affinity support was made from a solid support material consisting of a matrix onto which streptavidin (streptavidin-agarose-Novagen) was grafted. A volume of 1 ml of gel was introduced into a container consisting of a column (i.d., 11 mm). The gel was washed with purified water to remove the storage solvent. The characteristics of the gel are: - Biotin adsorption capacity> 85 nanomole / ml of gel - Functional test: Capture> 99% of biotinylated thrombin in 30 minutes at RT - Other tests: Protease-free, endo / exonuclease-free, RNase-free. - Preservative: 100 mM sodium phosphate pH7.5 + NaN3 0.02 The conditioned column outlet ("packaged") (gel bed height = 1 cm) is connected to an absorbance detector equipped with a UV filter at 254 nm and a recorder. The biotinylated anti-human FIX nucleic aptamers according to the invention, and in particular those considered in Examples 1 and 2 above, are solubilized in purified water at the final concentration of 0.5 mg / 0.187 ml, ie a concentration final molar of 0.1 mM. The nucleic aptamer solution was activated at 95 ° C according to the standard cycle, for aptamer immobilization on the solid support material. The nucleic aptamer solution was previously diluted with 4.8 ml of purified water and then 1.5 ml of Me ++ buffer (5 × concentrated). The absorbance detector is adjusted to 1 AUFS (Absorbance Unit Full Scale) and the OD at 254 nm of this solution is recorded at 0.575 UA254. The solution of biotinylated nucleic aptamers is injected onto the prepackaged streptavidinagarose gel ("prepackaged") and recirculated with a peristaltic pump at a flow rate of 2.5 ml / minute, ie a contact time on the gel of 24 seconds (input / output I / O). Under these conditions, the OD at 254 nm stabilizes rapidly at 0.05 AU254, a value of 91% theoretical coupling, that is to say 0.455 mg of nucleic aptamers per milliliter of gel. Washing with 10 mM CaCl2 + 4 mM MgCl2 and then 2M NaCl is performed to remove nucleic aptamers that are not specifically attached to streptavidin molecules grafted onto the solid support material. Example 3: Method for Purifying Human Factor IX A. Materials and Methods A.1. The affinity gel affinity chromatography medium on which the Nonapat 5.1 aptamer of sequence SEQ ID No.1, immobilized directly with biotin, was immobilized without a spacer chain between the aptamer and biotin. The aptamer is immobilized on a streptavidin gel (Novagen supplier) using a 5 'terminal biotin with a theoretical ligand density of 0.4 mg / ml: volume 1 ml packed in a column) (K16 (GE).

The starting material of Factor IX is Betafact 94 IU / mL - 1 mL. A.2. Equilibration gel purification protocol: 50 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl 2, 1 mM MgCl 2, pH 7.4, Wash: TSP buffer containing 0.5 M NaCl-2.5 mL, Elution: EDTA 200 mM, pH 8 - 2.5 mL, 1 mL of Betafact is injected with a flow rate of 0.4 mL / min in equilibration buffer.

After detecting the peak of unretained 2 column volumes of elution buffer is injected. Protein peaks are detected by measuring the absorbance value at the wavelength of 280 nanometers. B. Results The results are illustrated in FIGS. 21 and 22. FIG. 21 represents the curve of the measured values of the absorbance at 280 nm as a function of time. In Figure 21, peak # 1 is the fraction of the starting material that was not retained on the column. Peak No. 2 corresponds to the elution fraction.

The starting product as well as the eluted product was analyzed in SDS PAGE with silver nitrate staining. Figure 22 shows this gel: - lanes n ° 1 and l correspond to the starting product of Factor IX Betafact. - Tracks 2 and 2 'correspond to the fraction of the starting product which has not been retained on the column. - Tracks 3 and 3 'correspond to the elution fraction. Tracks 1 to 3 are obtained under non-reducing conditions. lanes # 1 'to 3' are obtained under reducing conditions The results of FIGS. 21 and 22 show that the aptamer of sequence SEQ ID No. 133 (ie Nonapta 5.1) is capable of binding human FIX and elute specifically in the presence of EDTA.

Indeed, the band corresponding to tracks 2 and 2 'is particularly clear or non-existent, which reflects the capacity of said aptamer to bind effectively to human FIX.

Claims (15)

REVENDICATIONS1. Nucleic acid binding specifically to the human factor IX / IXa of the following formula (I): [SEQ ID NO: X] - [3 '-TER] n (I), in which: - "[5'-TER] m Represents a nucleic acid comprising between 1 and 32 nucleotides, - "[3'-TER] '" represents a nucleic acid comprising between 1 and 40 nucleotides, - m is an integer equal to 0 or 1, - n is an integer 0 or 1, and - "[SEQ ID NO: X]" is a nucleic acid having at least 80% nucleotide identity with a nucleic acid selected from the group consisting of nucleic acids of SEQ ID N ° 5, SEQ ID NO: 6, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 8. 2. Nucleic acid according to claim 1, characterized in that the sequence SEQ ID No. X is a nucleic acid having at least 80% identity nucleotides with nucleic acid selected from the sequences SEQ ID No. 5 or SEQ ID No. 6. 3. Nucleic acid according to one of the claims. 1 and 2, characterized in that it has at least 80% nucleotide identity with a nucleic acid selected from the group consisting of the nucleic acids of SEQ ID NO: 1 to 143, preferably a nucleic acid of sequence SEQ ID NO: 134. 4. Nucleic acid according to any one of claims I to 3, characterized in that its binding to human factor IX / IXa can be dissociated by contact with a metal cation chelating agent. `30 5. Compound binding specifically to human factor IX / IXa, characterized in that it has the following formula (II): [SPAC] - [NUCL] (H), in which: [SPAC] signifies a spacer chain, and [NUCL] means a nucleic acid specifically binding to human Factor IX / IXa of formula (I) as defined in one of claims 1 to 4. 6. Compound binding specifically to human factor IX / IXa, characterized in that it has the following formula (III): [FIX] - [SPAC1- [NU CL] (III), wherein: - [FIX] means an immobilization compound on a support, [SPAC] means a spacer chain, and - [NUCL] means a nucleic acid specifically binding to human Factor IX / IXa of formula (I) as defined in one of the claims 1 to 4. 7. Compound binding specifically to human factor IX / IXa, characterized in that it has the following formula (V): NH 2 - [SPAC] - [NUCL] (V), in which: n being a subscript Wherein 0 or 0 means that the aptamer does not comprise a free spacer chain and 1 signifies that the aptamer comprises a spacer chain. - NH2 means the reactive amine function used for grafting on the NHS-pre-activated solid support, - [SPAC] means a spacer chain, and - [NUCL] means a nucleic acid specifically binding to human IX / IXa Factor of formula (I) as defined in one of claims 1 to 4. 8. Complex between (i) a nucleic acid according to any one of claims 1 to 4 or a compound according to any one of claims 5 to 7 and (ii) a human factor IX / IXa, 9. Support for the immobilization of human factor IX / IXa, characterized in that it comprises a solid support material on which are grafted a plurality of nucleic acids according to any one of claims 1 to 4 or compounds according to any one of claims 5 to 7. A method for immobilizing human factor IX / IXa on a carrier, comprising a step of contacting a sample comprising human factor IX / IXa with a carrier according to claim 9. 11. A method for purifying Human Factor IX / lXa comprising the steps of: a) contacting a sample comprising human Factor IX / IXa with a nucleic acid according to any one of claims 1 to 4, or with a compound according to any one of claims 5 to 7, or with a carrier according to claim 9, to form a complex between (i) said nucleic acid, or said compound, or said carrier and (ii) the LX / IXa Factor human, and b) release human Factor IX / lXa from the complex formed in step a) and recover the purified human Factor IX / IXa. 15 A method for detecting the presence of human factor IX / IXa in a sample comprising the following steps: a) contacting a nucleic acid according to any one of claims 1 to 4 or a compound according to any one of the claims 5 to 7 or with a support according to claim 9 with said sample; and b) detecting the formation of complexes between (i) said nucleic acid, or said compound, or said support, and (ii) Factor IX / IXa. 13. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1 to 4, in combination with one or more pharmaceutically acceptable excipients. 14. Nucleic acid according to any one of claims 1 to 4 for its use COrntne.Médicament. 30 15. Use of a nucleic acid according to any one of claims 1 to 4, for the manufacture of a medicament for the treatment of coagulation disorders, preferably type B hemophilia.