Methods and means for prevention and / or treatment of hemophilic arthropathy in hemophilia

Abstract

To provide a composition useful for the prevention, inhibition and / or treatment of hemophilic arthropathy of joints in patients with hemophilia B.SOLUTION: A gene therapy vehicle comprising a nucleic acid encoding a coagulation factor having Factor IX activity is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of blood clotting disorders commonly known as hemophilia, and more particularly to the prevention, inhibition and / or treatment of hemophilic arthropathy in patients with mild, moderate and / or severe hemophilia. [Background technology]

[0002] Hemophilia generally results from a deficiency of a (functional) factor in the coagulation cascade, a complex series of multifactorial enzymatic transformations that ultimately leads to the formation of a blood clot. The two most common forms of hemophilia are known as hemophilia A and B. Hemophilia A is caused by a deficiency of (functional) factor VIII, and hemophilia B is caused by a deficiency of (functional) factor IX. Because both congenital disorders involve single-gene defects (although various mutations are known in the genes encoding factor VIII and factor IX), they have long been considered "ideal" candidates for gene therapy approaches. Early attempts at gene therapy failed due to the inability to establish long-term expression of factors that correct the genetic defect. Now, with improvements in delivery vehicles and vectors (in many respects, including codon usage, promoters, and capsids), gene therapy for hemophilia appears to be fulfilling its promise of correcting the genetic defect and eliminating or at least substantially reducing the risk of bleeding episodes.

[0003] Mild, moderate, and severe disease are defined based on activated clotting factor levels (Srivastava A et al., 2013).

[0004] For patients with severe hemophilia B, 1 IU dL -1 Less than (0.01IU mL -1 These patients have FIX activity levels below normal (less than 1% of normal) or less than 1% of normal. These patients are characterized by spontaneous bleeding into joints or muscles, primarily in the absence of an identifiable hemostatic challenge.

[0005] For patients with moderate hemophilia B, 1-5 IU dL-1 (0.01-0.05IU mL -1 ) or have FIX activity levels of 1-5% of normal. These patients are characterized by occasional spontaneous bleeding and / or prolonged bleeding following minor trauma or surgery.

[0006] For people with mild hemophilia B, 5-40 IU dL -1 (0.05-0.40IU mL -1 ) or have FIX activity levels 5-40% of normal. These patients are characterized by severe bleeding associated with major trauma or surgery. Spontaneous bleeding is rare. Summary of the Invention [Problem to be solved by the invention]

[0007] The risk of bleeding episodes is a major burden for hemophilia patients, but even when this risk is controlled, there are still deleterious effects on hemophilia patients. One of those effects is irreversible hemophilic arthropathy (joint damage). The present invention provides means and methods for ameliorating hemophilic arthropathy in hemophilia patients.

[0008] Hemophilic arthropathy, or joint damage, is a common and disabling complication of severe hemophilia and, to a lesser extent, moderate and mild hemophilia A or B, where repeated bleeding into the joints often results in characteristic arthropathy. In hemophilia patients, these joint changes can lead to arthropathy and ultimately to chronic arthropathy (Knobe K et al., 2011). [Means for solving the problem]

[0009] Hemophilic arthropathy can be caused by internal bleeding in the joints, such as intra-articular and intramuscular bleeding, even when the patient is receiving prophylactic protein therapy. Without wishing to be bound by any theory, the inventors believe that hemophilic arthropathy in hemophilia can be prevented or at least slowed by applying gene therapy that delivers missing and / or defective factors and provides an essentially constant level of corrected clotting factors in the patient's circulation. The inventors (without wishing to be bound by theory) believe that the peaks and troughs seen with conventional protein replacement therapy are the root cause of joint damage, and that avoiding these troughs in particular will prevent intra-articular bleeding that leads to hemophilic arthropathy. To achieve this effect, it appears important to flatten the peaks and troughs in clotting factor activity levels as much as possible. For all categories of hemophilia patients (severe, moderate, mild), the present invention teaches that increasing activity levels in less severe groups (severe to moderate, moderate to mild, mild to asymptomatic) and maintaining the increased activity levels relatively constant and above the lower limit for less severe variants of hemophilia B can also alleviate at least some of the joint damage that would otherwise occur in hemophilia patients. Instead of activity levels, circulating protein concentration levels may be measured. For wild-type coagulation factors, particularly wild-type FIX, and most coagulation factor variants, particularly FIX variants, a concentration-activity correlation is provided based on the intrinsic activity of the variant used. [Effects of the Invention]

[0010] The present invention provides means and methods for at least slowing the rate of progression of hemophilic arthropathy in hemophilia patients. Preferably, the progression of hemophilic arthropathy is prevented. In some cases, it may be possible to reverse at least some of the damage to the joints of hemophilia patients and improve their health scores. According to the present invention, relevant clotting factors are provided such that the activity level of the clotting factors remains relatively constant over time. This may be achieved by gene therapy and / or protein replacement therapy, as described in more detail below. While the present invention focuses specifically on factor IX and hemophilia B, it is also fully applicable to hemophilia A. [Brief explanation of the drawings]

[0011] [Figure 1-1] Sustained increase in FIX activity after 5 years of AAV5-wild-type FIX administration. FIX activity was measured using a one-step activated partial thromboplastin time-based assay. Only values ​​at least 10 days after the last FIX concentrate administration are included. FIX prophylaxis was continued after AAV5-wild-type FIX infusion and tapered from weeks 6 to 12. *Patients 3, 4, and 5 tested positive for AAV5 neutralizing antibodies using a luciferase-based assay. Patient 5 was unable to attend the 4.5-year follow-up due to COVID-19, and the 5-year follow-up blood draw was obtained within 10 days of the use of exogenous FIX for bleeding, so was excluded per protocol. [Figure 1-2] Sustained increase in FIX activity after 5 years of AAV5-wild-type FIX administration. FIX activity was measured using a one-step activated partial thromboplastin time-based assay. Only values ​​at least 10 days after the last FIX concentrate administration are included. FIX prophylaxis was continued after AAV5-wild-type FIX infusion and tapered from weeks 6 to 12. *Patients 3, 4, and 5 tested positive for AAV5 neutralizing antibodies using a luciferase-based assay. Patient 5 was unable to attend the 4.5-year follow-up due to COVID-19, and the 5-year follow-up blood draw was obtained within 10 days of the use of exogenous FIX for bleeding, so was excluded per protocol. [Figure 2] Sustained increase in FIX activity after AAV5-PaduaFIX administration. FIX activity was measured using a one-stage activated partial thromboplastin time-based assay. The week 0 time point reflects FIX activity before AAV5-PaduaFIX treatment. Samples from administration up to week 2 may contain activity due to exogenous FIX replacement. Data labels represent the percentage of normal FIX activity at week 104 for each participant. [Figure 3] Joint health score (HJHS2.1) after AAV5-PaduaFIX administration. [Figure 4-1] Change in joint health score (ΔHJHS2.1) one year after AAV5-PaduaFIX administration. In Figure 4a, joint health status was considered to have improved if ΔHJHS2.1 was greater than or equal to -4, worsened if ΔHJHS2.1 was greater than or equal to 4, remained stable if ΔHJHS2.1 was between -3 and 3, and was considered NA if an HJHS2.1 score was not available. [Figure 4-2] Change in joint health score (ΔHJHS2.1) one year after AAV5-PaduaFIX administration. In Figure 4b, if ΔHJHS2.1<0, joint health status is considered to have improved, if ΔHJHS2.1>0, it is considered to have deteriorated, and if ΔHJHS2.1=0, it is considered to have remained stable. If an HJHS2.1 score was not available, it was considered NA. [Figure 5-1] Changes in joint health score (ΔHJHS2.1) 1 and 5 years after AAV5-wild-type FIX administration. In Figure 5a, joint health status is considered to have improved if ΔHJHS2.1 ≥ -4, worsened if ΔHJHS2.1 ≥ 4, remained stable if ΔHJHS2.1 was -3 to 3, and was considered NA if an HJHS2.1 score was not available. [Figure 5-2] Changes in joint health score (ΔHJHS2.1) 1 and 5 years after AAV5-wild-type FIX administration. In Figure 5b, if ΔHJHS2.1<0, joint health status is considered to have improved; if ΔHJHS2.1>0, joint health status is considered to have deteriorated; if ΔHJHS2.1=0, joint health status is considered to have remained stable. If an HJHS2.1 score was not available, the score was considered NA. [Figure 6-1] Changes in joint health score (ΔHJHS2.1) 1 and 2 years after AAV5-PaduaFIX administration. In Figure 6a, joint health status was considered to have improved if ΔHJHS2.1 was greater than or equal to -4, worsened if ΔHJHS2.1 was greater than or equal to 4, remained stable if ΔHJHS2.1 was between -3 and 3, and was considered NA if an HJHS2.1 score was not available. [Figure 6-2] Changes in joint health score (ΔHJHS2.1) 1 and 2 years after AAV5-PaduaFIX administration. In Figure 6b, if ΔHJHS2.1<0, joint health improved; if ΔHJHS2.1>0, joint health worsened; if ΔHJHS2.1=0, joint health remained stable; and if no HJHS2.1 score was available, it was recorded as NA. DETAILED DESCRIPTION OF THE INVENTION

[0012] In one aspect, the present invention provides a gene therapy vehicle for use in preventing, inhibiting, and / or treating hemophilic arthropathy of the joints in patients with hemophilia B, comprising a nucleic acid encoding a coagulation factor having factor IX activity. It has been shown that long-term stable expression of factor IX can be achieved in this manner. Preferably, the peak and trough activity is no more than 25% above or below the mean activity level, more preferably no more than 10% above or below the mean activity level.

[0013] Hemophilic arthropathy can cause pain, reduced range of motion, and / or muscle atrophy, resulting in inactivity and limited social participation. For example, hemophilic arthropathy can include joint hemorrhages and / or synovitis.

[0014] The hemophilia B patient can be a mild, moderate, or severe hemophilia B patient. Preferably, the patient is a mild or moderate hemophilia B patient. For example, the patient is a moderate hemophilia B patient. For example, the patient can have a pre-treatment (baseline) level of factor IX activity of less than 1 IU / dL, or 1 to a maximum of 5 IU / dL, or 5 to a maximum of 40 IU / dL. Preferably, the patient has a pre-treatment (baseline) level of factor IX activity of 1 to a maximum of 40 IU / dL, for example, 5 to a maximum of 40 IU / dL.

[0015] According to the present invention, a gene delivery vehicle is a package containing a nucleic acid encoding a protein of interest. The claimed protein of interest is factor IX or a functional equivalent thereof. Factor VIII is also contemplated. Gene therapy using factor VIII may rely on different viral delivery systems due to the size of the gene of interest. Typically, viral vectors for full-length factor VIII are retroviral, particularly lentiviral, based. However, it is well known that truncated factor VIII variants can also be used and may be compatible with other viral delivery vectors, such as AAV. Below, the present invention is described with particular reference to factor IX and its variants, and AAV delivery. The same methods and means can also be used for factor VIII, for example, in the context of lentiviruses.

[0016] According to the claimed invention, the coagulation factor has factor IX activity. Typically, the coagulation factor for use according to the invention is factor IX or a functional equivalent thereof for use in hemophilia B. A functional equivalent has essentially the same function (in type, but not necessarily in amount) as a wild-type coagulation factor in the coagulation cascade. In practice, hyperactive variants are preferred, as the essentially steady-state activity levels preferred according to the invention are more easily achieved with such hyperactive variants. Essentially steady-state is defined as an activity level that remains essentially above the border between two severity levels (severe, moderate, mild) and fluctuates over time by less than 25% of the mean level. Preferably, the activity is more than 25% above the borderline. Preferably, the deviation from the mean is less than 10%.

[0017] The package for gene therapy may be a non-viral vehicle such as a DNA / cationic lipid (lipoplex), or a DNA / cationic polymer (polyplex), or a DNA / cationic polymer / cationic lipid (lipopolyplex), containing liposomes and / or exosomes, or an inorganic particle such as an artificial nanoparticle whose size, shape, and porosity can be altered to protect the encapsulated molecule from degradation. In certain embodiments, the gene therapy vehicle is a lentivirus- or parvovirus-based particle or vector. Lentiviral vectors for expressing the FVIII gene are known in the art (see Kafri T et al., 1997).

[0018] In some embodiments, the gene therapy package may be derived from a virus. In a preferred embodiment, the parvovirus-based particle is an adeno-associated virus (AAV)-based particle or vector, preferably a recombinant AAV (rAAV)-based particle or vector. Most preferably, it is an AAV5 particle. AAV is a proven vehicle for gene therapy (Wang D et al., 2019), with no known pathologies associated with it, moderate infection of human cells, and no serious immunogenicity issues.

[0019] For nucleic acids encoding coagulation factors with factor IX activity, AAV vectors are preferred. For nucleic acids encoding full-length coagulation factors with factor VIII activity, lentiviral vectors may be preferred due to their greater ability to accommodate gene sequences with larger kb (kilobase pairs, a unit of length measurement). However, AAV vectors carrying nucleic acids encoding truncated forms of factor VIII are also preferred.

[0020] Adeno-associated virus (AAV) is a small (approximately 25 nm in diameter), non-enveloped, icosahedral, non-pathogenic parvovirus (Wang D et al., 2019). AAV infects cells through a receptor-mediated process, followed by transport of viral DNA to the nucleus. AAV replication requires a helper virus, such as adenovirus or herpesvirus. Wild-type AAV has a linear, single-stranded DNA genome approximately 4.7 kilobases (kb) in length. The genome consists of two coding elements: the replicase (rep) gene (encoding rep78, rep68, rep52, and rep40), which encodes the replicase (Rep proteins) required for AAV replication and packaging, and the capsid (cap) gene, which encodes the capsid proteins (VP1, VP2, and VP3).

[0021] In further specific embodiments, the AAV vector comprises a capsid derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV11, or variants thereof (e.g., capsid variants with amino acid insertions, additions, and substitutions, or hybrid capsids). AAV capsids typically contain a VP1 protein and two short proteins called VP2 and VP3, which are essentially amino-terminal truncations of VP1. The three capsid proteins VP1, VP2, and VP3 are typically present in the capsid in a ratio approaching 1:1:10, respectively, although this ratio, particularly that of VP3, can vary significantly and should not be considered limiting.

[0022] The genome of wild-type AAV is typically flanked by two inverted terminal repeats (ITRs), which serve as substrates for Rep proteins during replication and packaging of the vector genome. Vector genomes consist of either a plus (+) or minus (-) strand. They are packaged into capsids composed of three viral proteins: VP1, VP2, and VP3. Each capsid consists of 60 individual proteins, approximately 80% of which is VP3. For gene therapy purposes, AAV is typically produced recombinantly, typically replacing the wild-type vector genome with a gene of interest. Additionally, elements required for expression, such as a promoter sequence, poly(A) tail, stuffer, introns, and / or additional elements, are typically present.

[0023] Methods for producing recombinant AAV (rAAV) may involve mammalian or insect cell systems (Wang D et al., 2019; Gao G & Sena-Esteves M, 2012; Urabe M et al., 2006).

[0024] Preferably, the gene therapy vehicle of the present invention comprises a viral vector, such as an AAV-based particle.

[0025] A gene of interest can be packaged into an AAV capsid by placing it between two ITRs from a parvovirus. These ITRs can be from the same serotype as the capsid or from different sources. The examples herein disclose a combination of an AAV5 capsid with AAV2 ITRs. Other hybrids are also embodiments of the present invention. Such hybrids include not only combinations of capsids and ITRs from different serotypes, but also combinations of capsid elements from different serotypes, possibly with additional ITRs.

[0026] The dose of rAAV administered to a patient depends on the vehicle, gene of interest, and / or route of administration. Typically, for intravenous administration, the dose is 8×10 10 vg / kg~5×10 13vg / kg range.

[0027] The dosage depends on several factors, including the serotype used (preferred serotypes are AAV5, AAV8, AAV9, AAVrhlO, AAV10, AAV6, and hybrids containing capsid elements of these serotypes, e.g., AAV2 / 8). The importance of the capsid selection is primarily immunogenicity and / or efficacy of infection of target cells. As explained earlier herein, the interior of the AAV gene delivery vehicle may be of a different serotype than the exterior. In particular, the ITRs may be derived from a different serotype, such as AAV2.

[0028] According to the present invention, the AAV-based particles may be AAV5 particles.

[0029] The nucleic acid sequence packaged in the AAV vector, i.e., the vector genome, contains a nucleic acid sequence encoding a coagulation factor. This sequence is preferably codon-optimized, for example, by reducing the number of CpG (cytosine-guanine) dinucleotides or by replacing the original codons with tissue-specific ones, compared to the human wild-type sequence encoding human FIX (Wright JF, 2020). Other types of codon optimization are also possible. It is generally believed that (tissue-specific) codon optimization can result in improved expression levels of the transgene. Many algorithms for achieving codon optimization are known in the art.

[0030] Nucleic acid sequences encoding coagulation factors may include the factor IX (FIX) protein. FIX is a vitamin K-dependent protein synthesized by hepatocytes as a precursor to the serine protease FIXa. The FIX gene, consisting of eight exons and seven introns, is approximately 34 kb in length and is located on the long arm of the X chromosome, Xq27.1 (reviewed in Thompson 2001). FIX is synthesized as a 461-amino acid precursor protein containing a 28-residue signal prepeptide and an 18-residue leader propeptide. The resulting mature protein is a single chain of 415 residues. Structurally, FIX contains an N-terminal Gla domain (residues 1–40), a short hydrophobic stack (residues 41–46), two epidermal growth factor (EGF)-like domains (EGF1: residues 47–83 and EGF2: residues 88–127, which are connected by a linker residues 84–87), an activation peptide (residues 146–180), and a C-terminal protease domain (residues 181–415) ( Schmidt AE et al., 2003 ).

[0031] The gene therapy vehicle comprising a nucleic acid encoding coagulation factor IX is used to treat hemophilia B disease.

[0032] In some embodiments, the FIX protein is wild-type, while in other embodiments, the FIX protein is a mutant containing at least one amino acid substitution that alters the protein's clotting activity. In embodiments according to the present invention, such one or more substitutions generate a hyperactive mutant with increased FIX clotting ability. Hyperactive variants of clotting factors are preferred (Samelson-Jones BJ et al., 2021). In the case of factor IX, these variants include R338L (Padua), R338Q, and the FIX variant CB2679d-GT (R318Y, R338E, T343R). Less active variants, such as R338A, R338E, or FIX-Triple A (V86A / E277A / R338A), are also useful in gene therapy vehicles according to the present invention. R338L is disclosed in Simioni P et al., 2009; R338Q is disclosed in Simioni P et al., 2009 and Wu W et al., 2021; CB2679d-GT (R318Y / R338E / T343R) is disclosed in Nair N et al., 2021; R338E is disclosed in Nichols TC et al., 2020; FIX-Triple A (V86A / E277A / R338A) is disclosed in Lin CN et al., 2010; R338A is disclosed in Chang J et al., 1998.

[0033] Preferably, the nucleic acid comprises a hyperactive variant such as FIX R338L, also referred to as R338L, 338L, FIX Padua, FIX-Padua, Padua-FIX, PaduaFIX, or simply Padua.

[0034] The sequences of wild-type factor IX and preferred hyperactive variants are shown below: SEQ ID NO: 1 Mature FIX 1 YNSGKLEEFV QGNLERECMEEKCSFEEARE VFENTERTTE FWKQYVDGDQ 51 CESNPCLNGG SCKDDINSYE CWCPFGFEGKNCELDXTCNI KNGRCEQFCK 101 NSADNKVVCS CTEGYRLAEN QKSCEPAVPFPCGRVSVVSQT SKLTRAEXVF 151 PDVDYVNSTE AETILDNITQ STQSFNDFTRVVGGEDAKPG QFPWQVVLNG 201 KVDAFCGGSI VNEKWIVTAA HCVETGVKITVVAGEHNIEE TEHTEQKRNV 251 IRIIPHHNYN AAINKYNHDI ALLELDXPLVLNSYVTPICI ADKEYTNIFL 301 KFGSGYVSGW GRVFHKGXSA LVLQYLRVPLVDRATCLXST KFXIYNNMFC 351 AGFHEGGRDS CQGDSGGPHV TEVEGTSFLTGIISWGEECA MKGKYGIYTK 401 VSRYVNWIKE KTKLT At position 86, X can be V(wt) or A At position 148, X can be A (wt) or T (wt). At position 277, X can be E (wt) or A At position 318, X can be R (wt) or Y At position 338, X can be R (wt) or L or Q or E or A At position 343, X can be T (wt) or R In the above, (wt) means wild type.

[0035] The FIX protein used in the context of the present invention may comprise or consist of this sequence.

[0036] In another embodiment, the gene therapy vehicle comprises a nucleic acid encoding a clotting factor having factor VIII activity, which can be either wild-type human factor VIII or modified human factor VIII, and is used to treat hemophilia A.

[0037] Such nucleic acids may further comprise elements such as a promoter / enhancer, an intron, and a polyA tail, which may be present between two inverted terminal repeats (ITRs), particularly in the case of AAV gene therapy vehicles.

[0038] The promoter / enhancer element may be selected from the group of liver-specific promoters, including the human alpha-1-antitrypsin (hAAT) promoter, the HCR-hAAT hybrid promoter, and the apolipoprotein E promoter LP1, Q1 promoter, Q1-prime, C14 promoter, or promoters such as those described in WO 2020 / 104424. Preferably, the promoter / enhancer element is LP1, as described in WO 2006 / 036502.

[0039] A preferred LP1 promoter / enhancer element according to the present invention comprises the core liver-specific element from a contiguous segment of the human apolipoprotein hepatic control region (HCR, base pairs 134-442 of GenBank record HSU32510) and the human alpha-1-antitrypsin (hAAT) gene promoter (base pairs 1747-2001 of GenBank record K02212), including the 5' untranslated region (Nathwani AC et al., 2006). Because the liver is a target organ for gene therapy for hemophilia, a liver-specific promoter is preferred.

[0040] The ITRs are typically located at the left and right ends (i.e., the 5' and 3' ends, respectively) of the vector genome. Each ITR may be separated from the remaining sequences by a nucleic acid sequence of variable length. Preferably, the ITRs are selected from the group consisting of adeno-associated virus (AAV) ITR sequences. More preferably, the ITR sequences comprise ITR sequences of AAV1, AAV2, AAV5, AAV6, or AAV8. Optionally, the two ITR sequences comprise both AAV1, both AAV2, both AAV5, both AAV6, or both AAV8 ITR sequences. Also optionally, the ITR sequence at the 5' end of the nucleic acid sequence is different from the ITR sequence at the 3' end of the nucleic acid sequence, and the ITR sequence is selected from the ITR sequences of AAV1, AAV2, AAV5, AAV6, or AAV8.

[0041] In some embodiments, the AAV vector is defined as a "hybrid," meaning that the viral ITRs and the viral capsid are derived from different AAV parvoviruses: the viral ITRs are preferably derived from AAV2, and the capsid is preferably derived from another, typically AAV5, virus.

[0042] Hemophilia patients who are at low risk of bleeding events but who experience persistent joint damage will also benefit from treatment according to the present invention, as providing the required high (steady-state) levels of clotting factors will prevent or reduce joint damage in these patients. This benefit also exists in patients with moderate and severe hemophilia.

[0043] According to the present invention, the gene therapy vehicle is administered in a dose of 8×10 vector genomes per kg body weight (vg / kg). 10 vg / kg~5×10 13The viral vector is administered at a dose ranging from 1×10 to 1×10 vg / kg. The range depends on several factors, including, but not limited to, the expression level required to achieve a steady-state level of activity with therapeutic efficacy, any host immune response to the viral vector, the host immune response to the expressed protein, and the stability of the expressed protein. Preferably, only a single dose is given. In certain circumstances, follow-up administration may be necessary. In this case, it is particularly important to pay attention to the possibility of an immune response. This can be done using a different serotype or by other methods known to those skilled in the art. The preferred dose is 1×10 11 vg / kg, more preferably 5×10 11 vg / kg, max 5×10 12 vg / kg, or up to 2 x 10 13 vg / kg range.

[0044] Typically, gene therapy is administered as a single dose, i.e., a single administration. This means that the patient is treated only once over a long period of time. Preferably, long-term means at least 1 year, more preferably at least 5 years, at least 10 years, or at least 15 years. Most preferably, long-term means the patient's lifespan.

[0045] The effect achieved by the present invention is, as explained above, a more steady state at levels higher than the borderline between different severity stages, and therefore this effect does not necessarily have to be achieved by gene therapy. For example, a steady state may be achieved by selecting factor IX variants with different activity and / or half-life. Such techniques are well known in the diabetes field using insulin derivatives (Madsbad S, 2002). These effects can also be achieved using infusion devices (as is also known from the diabetes field). Gene therapy that does not reach the desired steady state level can also be supplemented in this way.

[0046] Thus, in a further aspect, the present invention provides a clotting factor for use in the prevention, inhibition, and / or treatment of hemophilic arthropathy in patients with mild, moderate, and / or severe hemophilia. In particular, the present invention provides said uses of clotting factors having factor IX activity or factor VIII activity. More specifically, the present invention provides a clotting factor with human factor IX activity for use in the prevention, inhibition, and / or treatment of hemophilic arthropathy in patients with mild, moderate, and / or severe hemophilia B. In another aspect, the clotting factor has factor VIII activity, more particularly human factor VIII activity, and is used in the prevention, inhibition, and / or treatment of hemophilic arthropathy in patients with mild, moderate, and / or severe hemophilia A. In a further embodiment, the clotting factor having either factor IX or factor VIII activity has an increased half-life.

[0047] As mentioned above, a longer half-life makes it easier to achieve higher, more steady-state levels. Therefore, it is one aspect of the present invention that the coagulation factor has a longer half-life. Longer half-life coagulation factor variants have been described (Young G et al., 2016; Santagostino E et al., 2016; Graf L, 2018). Coagulation factors with longer half-lives may also be hyperactive variants. The best way to achieve steady-state levels is to have a combination of different half-lives in the composition. Such compositions are also part of the present invention. All protein compositions according to the present invention can also be used to supplement gene therapy treatments that lead to activity levels in the patient's circulation that are insufficient to achieve less severe forms of hemophilia.

[0048] During the course of this invention, and as shown in the Examples, the inventors have surprisingly established that a gene therapy approach for the treatment of hemophilia B prevents, inhibits, and / or treats hemophilic arthropathy in such patients.

[0049] In some embodiments, gene therapy vehicles for use in preventing, inhibiting and / or treating joint damage in patients with mild, moderate and / or severe hemophilia may be non-viral in origin.

[0050] The patient treated in accordance with the present invention is preferably a human.

[0051] According to some embodiments, a therapeutically effective dose of an AAV vector is one that, when administered to a human subject with hemophilia B, is sufficient to provide a steady-state level of clotting factor FIX activity that reduces the hemophilia from severe to moderate or mild, and that should be maintained for at least 2-3 years.

[0052] According to certain embodiments, a therapeutically effective dose of an AAV vector reduces or eliminates the need for recombinant human factor IX replacement therapy in a human subject with hemophilia.

[0053] In certain embodiments, a therapeutically effective dose of an AAV vector prevents or reduces hemophilic arthropathy by reducing the severity and frequency of joint bleeds in a human patient with hemophilia.

[0054] The joints may be one or both elbows, one or both knees, one or both ankles, one or both shoulders, one or both hips, one or both wrists, one or more joints of the hand, one or more joints of the foot, or any combination thereof. Preferably, the joints are selected from the group consisting of one or both elbows, one or both knees, one or both ankles, and any combination thereof.

[0055] Joint health status can be measured using the Hemophilia Joint Health Score (HJHS). In this study, the HJHS version 2.1 (HJHS2.1) was used. The HJHS2.1 consists of eight joint-level scores and an overall ambulation score. Scores range from 0 to 20 for each joint, with the overall ambulation score ranging from 0 to 4. Focusing on the elbow, knee, and ankle, the total HJHS2.1 score ranges from 0 to 124. Higher scores indicate worse joint health. Changes in joint health status can be quantified using the ΔHJHS2.1. Typically, a baseline HJHS2.1 score is obtained before treatment (T0), and another HJHS2.1 score is obtained at each follow-up visit (T1, T2, etc.) after treatment. ΔHJHS2.1 is defined as the HJHS2.1 at each follow-up visit minus the baseline HJHS2.1.

[0056] According to the present invention, patients have a ΔHJHS2.1 of 0 or less than 0. Preferably, patients have a ΔHJHS2.1 of less than 0, less than -2, and more preferably less than -4. Follow-up time can be 1 year, 2 years, or 5 years after the last administration of a gene therapy vehicle.

[0057] In yet another aspect, the present invention further provides a pharmaceutical composition for use in preventing, inhibiting, and / or treating hemophilic arthropathy of the joints in patients with hemophilia B, comprising a gene therapy vehicle according to the present invention. The pharmaceutical composition allows for the administration of the gene therapy vehicle, preferably an AAV vector of the AAV5 serotype, to a human patient. Such administration preferably involves administration via the bloodstream, e.g., via intravenous infusion. Thus, preferably, the pharmaceutical composition is in a form suitable for intravenous infusion. For example, the pharmaceutical composition may be a liquid, or may be, for example, a lyophilized formulation for injection. The liquid or solid may then be combined with, for example, a solution for injection or infusion. Preferably, the pharmaceutical composition is administered in a single dose.

[0058] In yet another aspect, the present invention further provides a method for preventing, inhibiting and / or treating hemophilic arthropathy of the joints in a patient with hemophilia B, comprising the step of administering to the patient an effective amount of a gene therapy vehicle according to the present invention or an effective amount of a pharmaceutical composition according to the present invention.

[0059] In one embodiment of the invention, the method reduces the patient's HJHS2.1 score by at least 0, preferably at least 2, and more preferably at least 4 points. In one embodiment of the invention, the reduction in HJHS2.1 score occurs at least 1 year, or at least 2 years, or at least 5 years after administration of the gene therapy vehicle.

[0060] All embodiments and features described above with respect to one aspect of the invention also apply to other aspects of the invention. [Example]

[0061] Example 1 This example describes 5-year efficacy results for FIX protein levels and assessment of joint health in a Phase I / II clinical trial.

[0062] In this study, 10 adult hemophilia B subjects with FIX activity ≤2 IU / dL received a single intravenous dose of an adeno-associated virus serotype 5 (AAV5) vector encoding a codon-optimized wild-type human blood clotting factor IX (FIX) gene driven by a liver-specific promoter. Five subjects received 5 × 10 12 vg / kg (Cohort 1), and the remaining 5 patients received 2 × 10 13 vg / kg (Cohort 2).

[0063] FIX protein levels were estimated using ELISA (Spronck EA et al., 2019). Joint health status was assessed using the Hemophilia Joint Health Score (HJHS) version 2.1 (Kuijlaars I et al., 2017).

[0064] Mean FIX protein concentrations 4 years after infusion also confirmed persistent transgene expression; these data were generally consistent with FIX activity, which varied between 1.37% and 10.71% in seven of the ten study participants. The mean FIX antigen-to-activity ratio in the seven participants was 0.85 (SD 0.28). The remaining three patients (presumed cross-reactant positive) had FIX antigen-to-activity ratios of 9.28, 2.42, and 25.56 (see Table 1).

[0065] At 5 years post-infusion, the mean endogenous FIX activity in the low-dose cohort (cohort 1) was 5.2% and in the high-dose cohort (cohort 2) was 7.4%; see Figure 1 .

[0066] Overall joint health improved in Cohort 1, decreasing from a mean baseline score of 24.4 (SD 17.5) to 19.2 (SD 15.0) after 5 years (see Table 2). In the high-dose cohort (Cohort 2), overall joint health also improved somewhat, decreasing from a mean score of 6.8 (SD 6.5) to 4.4 (SD 5). An increase of 4 in the total HJHS score is defined as joint deterioration (Kuijlaars et al., 2017).

[0067] In conclusion, transgene expression was maintained for 5 years, and joint health assessment scores improved by approximately 21% and 35% in cohorts 1 and 2, respectively.

[0068] Example 2 This example describes 2-year efficacy results for assessment of FIX activity levels and joint health in a Phase I / II clinical trial. In this study, three adult hemophilia B subjects received 2 x 10 FIX containing a codon-optimized Padua variant human factor IX (Padua-FIX) gene with a liver-specific promoter. 13 A single intravenous injection of adeno-associated virus serotype 5 (AAV5) vector was administered at a dose of 1000 mg / kg. All participants had severe or moderate FIX deficiency with FIX activity <2%.

[0069] FIX activity was assessed using a one-stage activated partial thromboplastin time (aPTT)-based assay and a chromogenic assay.FIX activity was measured using a one-stage activated partial thromboplastin time (aPTT)-based assay and a chromogenic assay (Spronck EA et al., 2019).

[0070] Joint health status was assessed using the Hemophilia Joint Health Score (HJHS) version 2.1 (Kuijlaars I et al., 2017) at baseline and annually as part of long-term follow-up.

[0071] The mean endogenous FIX activity at 2 years was 44.2% (minimum to maximum, 36.3% to 51.6%) (Figure 2). Participants 1 and 3 maintained FIX activity in the nonhemophilic range (≥40%). Participant 2 maintained FIX activity in the high-to-mild range. FIX activity measured at 2 years (estimated using a one-stage aPTT-based assay) was 10.2-fold higher than FIX antigen levels.

[0072] Two of the three participants had a decrease in total joint health score from baseline (35, 36, and 1 for participants 1-3, respectively) compared to scores two years after vector injection (24, 30, and 6, respectively). Participant 3, who had a low baseline score of 1, increased to a score of 6 two years after treatment (see Figure 3). An increase of 4 or more in the total HJHS is indicative of joint degeneration (Kuijlaars et al., 2017).

[0073] In conclusion, patients treated with a single infusion of AAV5-Padua-FIX were shown to have a stable and sustained increase in FIX activity. Participants 1 and 2 experienced improvement in joint health, with total joint scores decreasing by 11 and 6, respectively (from baseline to the second year of follow-up). Participant 3 experienced an increase of 5 (total HJHS score) over the course of the study. However, this patient's avascular hip necrosis worsened, requiring two surgeries during the two-year follow-up period.

[0074] Example 3 This example demonstrates improved joint health in hemophilia B patients who received a gene therapy vehicle according to the present invention in a Phase II / III clinical trial. Each patient received 5 x 10 mAbs containing the relevant FIX gene with a liver-specific promoter. 12 or 2 x 10 13 A single dose of adeno-associated virus serotype 5 (AAV5) vector was administered intravenously at 1000 mg / kg. HJHS2.1 scores were obtained at T0 (baseline) (before treatment) and T1 (1, 2, or 5 years after treatment). ΔHJHS2.1 was defined as the HJHS2.1 score at T1 minus the HJHS2.1 score at T0. Changes in joint health status were assessed using ΔHJHS2.1. For Figures 4a, 5a, and 6a, a ΔHJHS2.1 of -4 or greater indicated improvement in joint health status, a ΔHJHS2.1 of 4 or greater indicated deterioration in joint health status, and a ΔHJHS2.1 of -3 to 3 indicated stable joint health status. NA was used when no appropriate data were available. For Figures 4b, 5b, and 6b, joint health status was considered to improve if ΔHJHS2.1<0, worsen if ΔHJHS2.1>0, and remain stable if ΔHJHS2.1=0, or NA if no suitable data were available. The proportion of patients meeting the criteria associated with "improvement," "worsening," "stable," and "NA" was calculated and shown in the figures.

[0075] As shown in the figure, using more stringent criteria, 18.5% (n=54) of participants reported improved joint health one year after administration of a gene therapy vehicle containing AAV5-PaduaFIX (Figure 4a). Using more lenient criteria, 44.4% of participants reported improved joint health one year after treatment (Figure 4b). In a smaller group (n=3) (Figure 6a), one of three participants reported improved joint health one year after treatment with the present invention, and one additional participant reported improvement two years later. Participant 3's total HJHS score increased by 5 points over the course of the study. However, this patient's avascular hip necrosis worsened, requiring two surgeries during the two-year follow-up period.

[0076] Similarly, using more stringent criteria, 10% (n = 10) of participants reported improved joint health 1 year after administration of a gene therapy vehicle containing AAV5-wild-type FIX (Figure 5a), a percentage that increased to 50% 5 years after treatment. Using more lenient criteria, 40% of participants reported improved joint health already 1 year after treatment. By 5 years after treatment, 80% of participants reported improved joint health (Figure 5b). [Table 1] [Table 2]

[0077] Legend for tables and figures Table 1: Mean % steady-state FIX protein concentration after AAV5-wild-type FIX administration. Values ​​less than 1.23 were set to 1.23 for calculation of summary statistics. Contaminating values ​​were excluded from calculations. Additional / unscheduled visits were included in calculating the mean. Values ​​are obtained after FIX exogenous tapering. CI, confidence interval. *Participants 1, 2, and 9 are presumed cross-reacting material positive (CRM+).

[0078] Table 2: Joint Health Score (HJHS) after AAV5-wild-type FIX administration. HJHS status was assessed using the Hemophilia Joint Health Score version 2.1. SD, standard deviation; N, number of participants.

[0079] Reference list 1. Chang J, Jin J, Lollar P, Bode W, Brandstetter H, Hamaguchi N,Straight DL, Stafford DW. Changing residue 338 in human factor IX from arginineto alanine causes an increase in catalytic activity. J Biol Chem. 1998 May15;273(20):12089-94. doi: 10.1074 / jbc.273.20.12089. PMID: 9575152. 2. GaO G & Sena-Esteves M, in MolecularCloning (eds Green, M. R. & Sambrook, J. R.) 1209-1330 (Cold Spring HarborLaboratory Press, 2012). 3. Graf L. Extended Half-Life Factor VIIIand Factor IX Preparations. Transfus Med Hemother. 2018;45(2):86-91.doi:10.1159 / 000488060. 4. Kafri T, Blomer U,Peterson DA, Gage FH, Verma IM. Sustained expression ofgenes delivered directly into liver and muscle by lentiviral vectors. NatGenet. 1997 Nov;17(3):314-7. doi: 10.1038 / ng1197-314. PMID: 9354796. 5. Knobe K, Berntorp E. Haemophilia andjoint disease: pathophysiology, evaluation, and management. J Comorb.2011;1:51-59. Published 2011 Dec 27. doi:10.15256 / joc.2011.1.2. 6. Kuijlaars I a. R, Timmer MA, de KleijnP, Pisters MF, Fischer K. Monitoring joint health in haemophilia: Factorsassociated with deterioration. Haemophilia. 2017;23(6):934-940. 7. Lin CN, Kao CY, Miao CH, Hamaguchi N, WuHL, Shi GY, Liu YL, High KA, Lin SW. Generation of a novel factor IX withaugmented clotting activities in vitro and in vivo. J Thromb Haemost. 2010Aug;8(8):1773-83. doi: 10.1111 / j.1538-7836.2010.03913.x. Epub 2010 May 21.PMID: 20492477. 8. Madsbad S. Insulin analogues: have theychanged insulin treatment and improved glycaemic control? Diabetes Metab ResRev. 2002 Jan-Feb;18 Suppl 1:S21-8. doi: 10.1002 / dmrr.206. PMID: 11921426. 9. Nair N, De Wolf D, Nguyen PA, Pham QH,Samara E, Landau J, Blouse GE, Chuah MK, VandenDriessche T. Gene Therapy ForHemophilia B Using CB 2679d-GT: A Novel Factor IX Variant With Higher PotencyThan Factor IX Padua. Blood. 2021 Mar 18:blood.2020006005. doi:10.1182 / blood.2020006005. Epub ahead of print. PMID: 33735915. 10. Nathwani AC, Gray JT, Ng CY, et al.Self-complementary adeno-associated virus vectors containing a novelliver-specific human factor IX expression cassette enable highly efficienttransduction of murine and nonhuman primate liver. Blood. 2006;107(7):2653-2661.doi:10.1182 / blood-2005-10-4035. 11. Nichols TC, Levy H, Merricks EP, RaymerRA, Lee ML. Preclinical evaluation of a next-generation, subcutaneouslyadministered, coagulation factor IX variant, dalcinonacog alfa. PLoS One. 2020Oct 28;15(10):e0240896. doi: 10.1371 / journal.pone.0240896. PMID: 33112889;PMCID: PMC7592742. 12. Samelson-Jones BJ, Finn JD, RaffiniLJ, Merricks EP, Camire RM, Nichols TC, Arruda VR. Evolutionary insights intocoagulation factor IX Padua and other high-specific-activity variants. BloodAdv. 2021 Mar 9;5(5):1324-1332. doi: 10.1182 / bloodadvances.2019000405. PMID:33656538; PMCID: PMC7948292. 13. Santagostino E, Martinowitz U,Lissitchkov T, et al. Long-acting recombinant coagulation factor IX albuminfusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial. Blood.2016;127(14):1761-1769. 14. Schmidt AE, Bajaj SP.Structure-function relationships in factor IX and factor IXa. Trends CardiovascMed. 2003 Jan;13(1):39-45. doi: 10.1016 / s1050-1738(02)00210-4. PMID: 12554099. 15. Simioni P, TormeneD, Tognin G, et al. X-linked thrombophilia with amutant factor IX (factor IX Padua). N Engl J Med. 2009;361(17):1671-1675. [ PubMed ] 16. Spronck EA, Liu YP, Lubelski J,Ehlert E, Gielen S, Montenegro-Miranda P, de Haan M, Nijmeijer B, Ferreira V,Petry H, van Deventer SJ. Enhanced Factor IX Activity following Administrationof AAV5-R338L "Padua" Factor IX versus AAV5 WT Human Factor IX inNHPs. Mol Ther Methods Clin Dev. 2019 Sep 26;15:221-231. doi:10.1016 / j.omtm.2019.09.005. PMID: 31709273; PMCID: PMC6834974. [ PubMed ] 17. Srivastava A, Brewer AK,Mauser-Bunschoten EP, Key NS, Kitchen S, Llinas A, Ludlam CA, Mahlangu JN, Mulder K, Poon MC, Street A; Treatment Guidelines Working Group on Behalf ofThe World Federation Of Hemophilia. Guidelines for the management ofhemophilia. Haemophilia. 2013 Jan;19(1):e1-47. doi:10.1111 / j.1365–2516.2012.02909.x. Epub 2012 Jul 6. PMID:22776238. 178. Urabe M, Nakakura T, Xin KQ, ObaraY, Mizukami H, Kume A, Kotin RM, Ozawa K. Scalable generation of high-titerrecombinant adeno-associated virus type 5 in insect cells. J Virol. 2006Feb;80(4):1874-85. doi: 10.1128 / JVI.80.4.1874-1885.2006. PMID: 16439543; PMCID:PMC1367135. 19. Wang D, Tai PWL, Gao G.Adeno-associated virus vector as a platform for gene therapy delivery. Nat RevDrug Discov. 2019 May;18(5):358-378. doi: 10.1038 / s41573-019-0012-9. PMID:30710128; PMCID: PMC6927556. 20. Wright JF. Codon Modification and PAMPsin Clinical AAV Vectors: The Tortoise or the Hare?. 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[0080] A preferred embodiment of the present invention is as follows. [1] A gene therapy vehicle for use in preventing, inhibiting and / or treating hemophilic arthropathy of the joints in patients with hemophilia B, comprising a nucleic acid encoding a clotting factor having factor IX activity. [2] A gene therapy vehicle for use according to [1], wherein the patient with hemophilia B has a factor IX activity level of less than 1 IU / dL, or 1 to a maximum of 5 IU / dL, or 5 to a maximum of 40 IU / dL; preferably, the patient has a pre-treatment (baseline) level of factor IX activity of 1 to a maximum of 40 IU / dL. [3] A gene therapy vehicle for use according to [1] or [2], wherein the vehicle is a viral vector, preferably an AAV-based particle. [4] The gene therapy vehicle for use according to [3], wherein the AAV-based particles are AAV5 particles. [5] The gene therapy vehicle for use according to any one of [1] to [4], wherein the nucleic acid comprises wild-type factor IX or a hyperactive variant of factor IX; preferably, the nucleic acid comprises a hyperactive variant of factor IX, such as FIX-R338L. [6] The gene therapy vehicle for use according to any one of [1] to [5], wherein the nucleic acid further comprises a promoter, for example, a liver-specific promoter. [7] The gene therapy vehicle is 8×10 10 vg / kg~2×10 13 The gene therapy vehicle for use according to any one of [1] to [6], which is administered at a dose of 100 mg / kg. [8] The gene therapy vehicle for use according to any one of [1] to [7], wherein the gene therapy vehicle is administered in a single dose. [9] A gene therapy vehicle for use according to any one of [1] to [8], wherein the joints are selected from the group consisting of one or both elbows, one or both knees, one or both ankles, and combinations thereof.

[10] The gene therapy vehicle for use according to any one of [1] to [9], wherein the hemophilic arthropathy includes joint bleeding and / or synovitis.

[11] The gene therapy vehicle for use according to any one of [1] to

[10] , wherein the patient is a human.

[12] A pharmaceutical composition for use in the prevention, inhibition and / or treatment of hemophilic arthropathy of the joints in patients with hemophilia B, comprising a gene therapy vehicle for use according to any one of [1] to

[11] .

[13] The pharmaceutical composition for use according to

[12] , wherein the pharmaceutical composition is in a form suitable for intravenous injection.

[14] The pharmaceutical composition for use according to

[13] , wherein the pharmaceutical composition is a liquid or a freeze-dried solid.

[15] A method for preventing, inhibiting and / or treating hemophilic arthropathy of the joints in a patient with hemophilia B, comprising the step of administering to the patient an effective amount of a gene therapy vehicle for use according to any one of [1] to

[11] or a pharmaceutical composition for use according to any one of

[12] to

[14] .

[16] The method according to

[15] , wherein the gene therapy vehicle or pharmaceutical composition for said use is administered by intravenous infusion.

[17] The method described in

[15] or

[16] , wherein the patient has a pre-treatment baseline HJHS2.1 score and a post-treatment HJHS2.1 score, and the post-treatment HJHS2.1 score is not higher than the pre-treatment baseline HJHS2.1 score.

[18] The method described in

[17] , wherein the post-treatment HJHS2.1 score is lower than the pre-treatment baseline HJHS2.1 score; preferably, the post-treatment HJHS2.1 score is at least 2 points lower than the pre-treatment baseline HJHS2.1 score; more preferably, the post-treatment HJHS2.1 score is at least 4 points lower than the pre-treatment baseline HJHS2.1 score.

[19] The method of

[17] or

[18] , wherein the post-treatment HJHS2.1 score is measured at least one year after administration of the gene therapy vehicle for use or the pharmaceutical composition for use; preferably, the post-treatment HJHS2.1 score is measured at least two years after administration of the gene therapy vehicle for use or the pharmaceutical composition for use.

Claims

1. A gene therapy vehicle for the prevention, inhibition and / or treatment of hemophilic arthropathy of the joints in patients with hemophilia B, comprising a nucleic acid encoding factor IX, A gene therapy vehicle wherein, after administration to the patient, the patient's factor IX activity is maintained at at least 75% of the patient's average factor IX activity level after administration.

2. The gene therapy vehicle according to claim 1, wherein the patient's factor IX activity is maintained at a level of at least 90% of the patient's average factor IX activity level after administration.

3. The gene therapy vehicle according to claim 1 or 2, wherein the IX factor is a hyperfunctional variant of the IX factor.

4. The gene therapy vehicle according to claim 3, wherein the hyperfunctional variant of factor IX is FIX-R338L.

5. The gene therapy vehicle according to claim 3, wherein the hyperfunctional variant of factor IX has A at position 148.

6. The gene therapy vehicle according to claim 1 or 2, wherein the patient having hemophilia B has a factor IX activity level of less than 1 IU / dL, 1 to a maximum of 5 IU / dL, or 5 to a maximum of 40 IU / dL.

7. The gene therapy vehicle according to claim 1 or 2, wherein the vehicle is an AAV-based particle.

8. The gene therapy vehicle according to claim 7, wherein the AAV-based particles are AAV1 particles, AAV2 particles, AAV3 particles, AAV4 particles, AAV5 particles, AAV6 particles, AAV7 particles, AAV8 particles, AAV9 particles, AAVrh10 particles, or AAV11 particles.

9. The gene therapy vehicle according to claim 1 or 2, wherein the nucleic acid further comprises a promoter.

10. The gene therapy vehicle according to claim 1 or 2, wherein the gene therapy vehicle is administered at a dose of 8 × 10¹⁰ vg / kg to 2 × 10¹³ vg / kg.

11. The gene therapy vehicle according to claim 1 or 2, wherein the gene therapy vehicle is administered in a single dose.

12. The gene therapy vehicle according to claim 1 or 2, wherein the joint is selected from the group consisting of one or both elbows, one or both knees, one or both ankles, and any combination thereof; and / or the hemophilic arthropathy is associated with joint hemorrhage, synovitis, or both.

13. The gene therapy vehicle according to claim 1 or 2, wherein the gene therapy vehicle is administered by intravenous infusion.

14. The gene therapy vehicle according to claim 1 or 2, wherein the patient has a pre-treatment baseline HJHS2.1 score and a post-treatment HJHS2.1 score, and the post-treatment HJHS2.1 score is not higher than the pre-treatment baseline HJHS2.1 score.

15. The gene therapy vehicle according to claim 14, wherein the post-treatment HJHS2.1 score is at least 2 points lower than the pre-treatment baseline HJHS2.1 score.

16. The gene therapy vehicle according to claim 14, wherein the post-treatment HJHS2.1 score is measured at least one year after administration of the gene therapy vehicle.

17. A pharmaceutical composition for the prevention, inhibition and / or treatment of hemophilic arthropathy of the joints in patients having hemophilia B, comprising the gene therapy vehicle described in Claim 1.

18. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is in a form suitable for intravenous injection.

19. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is to be administered by intravenous injection.

20. The pharmaceutical composition according to claim 17, wherein the patient has a pre-treatment baseline HJHS2.1 score and a post-treatment HJHS2.1 score, and the post-treatment HJHS2.1 score is not higher than the pre-treatment baseline HJHS2.1 score.

21. The pharmaceutical composition according to claim 20, wherein the post-treatment HJHS2.1 score is at least 2 points lower than the pre-treatment baseline HJHS2.1 score.

22. The pharmaceutical composition according to claim 20, wherein the post-treatment HJHS2.1 score is measured at least one year after administration of the pharmaceutical composition.

23. The pharmaceutical composition according to claim 19, wherein the patient has an HJHS2.1 score that is not higher than a previously assessed HJHS2.1 score.

24. The gene therapy vehicle according to claim 1 or 2, or the pharmaceutical composition according to claim 17, wherein, after administering the gene therapy vehicle or the pharmaceutical composition to the patient, the peak and trough levels of the FIX activity level are within 25% above or below the average activity level.

25. The gene therapy vehicle or pharmaceutical composition according to claim 24, wherein, after administering the gene therapy vehicle or pharmaceutical composition to the patient, the peak and trough levels of the FIX activity level are within 10% above or below the average activity level.

26. The gene therapy vehicle or pharmaceutical composition according to claim 24, wherein the hemophilic arthropathy effect is at least one of pain, reduced range of motion, loss of activity, limited social participation, joint bleeding, synovitis, chronic arthropathy, or a combination thereof.

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