Pharmaceutical composition of recombinant adeno-associated virus vector and application thereof

Abstract

The invention provides a pharmaceutical composition of a recombinant adeno-associated virus vector and application of the pharmaceutical composition. The pharmaceutical composition comprises a recombinant adeno-associated virus (rAAV), ionic salt, a buffer agent, a stabilizer and a surfactant. The rAAV vector carries hFIX and can be used for treating hemophilia B.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of Chinese Application No. CN202211467932.8, filed on November 22, 2022, the content of which is incorporated herein by reference in its entirety. Technical field

[0003] This article belongs to the field of recombinant adeno - associated virus vector drug preparations. This article relates to a pharmaceutical composition of a recombinant adeno - associated virus vector and its applications, in particular to a liquid preparation of a gene therapy drug with a recombinant adeno - associated virus as a vector and its applications. Background art

[0004] Cell and Gene Therapy (CGT) is a therapy that overcomes the limitations of traditional small - molecule and antibody drugs in regulating at the protein level through gene expression, silencing, or in vitro modification means, achieving therapy upgrade or rare - disease treatment. For CGT therapies, a very important part is the in - vivo delivery of gene vectors. Currently, common gene vectors are divided into non - viral vectors and viral vectors. Non - viral vectors include plasmids or naked DNA (non - viral), LNP delivery systems, etc., but poor in - vivo transfection efficiency and excessive toxicity severely restrict the clinical transformation of non - viral vectors. Viral vectors include commonly used adenovirus AdV, adeno - associated virus AAV, lentivirus LV, and retrovirus RV vectors.

[0005] Adeno - associated virus (AAV) was first discovered in the mid - 1960s from laboratory adenovirus (AdV) preparations and was soon found in human tissues. Recombinant adeno - associated virus (rAAV) vectors usually replace the viral coding sequence with the gene of interest. These vectors have been shown to have efficient expression and gene targeting in some different parts in vitro and in vivo. Studies have shown that AAV is safe in studies of the respiratory tract, central nervous system, skeletal muscle, liver, and eyes and can express stably and continuously. With the improvement of the titer and purity of rAAV preparations, the efficiency of rAAV - mediated transduction is also increasing.

[0006] Currently, the total number of rAAV - related clinical trials conducted globally generally shows an increasing trend. So far, four gene therapy products with rAAV as a vector have obtained marketing approvals:

[0007] In 2012, Glybera was approved by the EMA for the treatment of lipoprotein lipase deficiency (LPLD), becoming the first officially approved human AAV gene therapy product.

[0008] At the end of 2017, the FDA approved Luxturna from Spark Therapeutics for the treatment of inherited retinal diseases, making it the first "in vivo administration" gene therapy approved in the United States.

[0009] In May 2019, Novartis' Zolgensma was approved by the US FDA for marketing for the treatment of children under 2 years old with spinal muscular atrophy (SMA) who have bi-allelic mutations in the survival motor neuron 1 (SMN1) gene.

[0010] On August 24, 2022, BioMarin Pharmaceutical Company (BioMarin) announced that the European Commission (EC) approved the conditional marketing of the hemophilia A gene therapy ROCTAVIAN TM (valoctocogene roxaparvovec) for the treatment of adult patients with severe hemophilia A who have no history of FVIII inhibitor and are negative for AAV5 antibodies.

[0011] All of the above gene therapy products need to be stored in a frozen state, and their transportation and storage temperatures are generally -20°C or -60°C. Maintaining the freezer temperature at -20°C or -60°C is a challenge, and from a logistics perspective, it requires a relatively large cost. Such low temperature requirements will have a negative impact on the ability to distribute the product to a wide range of clinical sites. Therefore, it is desirable to provide a pharmaceutical composition of a recombinant adeno-associated virus vector-based gene therapy drug that is stable for more than 1 year under general refrigeration temperature conditions, suitable for thawing during transportation or at the clinical site and storing the product in a freezer until it is used for patient administration.

[0012] To solve the above problems, the present invention provides a pharmaceutical composition of a recombinant adeno-associated virus vector, especially a liquid preparation of a gene therapy drug based on a recombinant adeno-associated virus. This pharmaceutical composition can be stored for more than 1 year under general refrigeration temperature conditions, such as 2 - 8°C, can keep indicators such as genomic titer and biological activity stable, and also has good stability when stored at room temperature for two weeks. Summary of the Invention

[0013] The present invention provides a pharmaceutical composition of a recombinant adeno-associated virus vector and its application. The pharmaceutical composition of the recombinant adeno-associated virus vector includes recombinant adeno-associated virus (rAAV), ionic salt, buffer, stabilizer, and surfactant.

[0014] In some embodiments, the recombinant adeno-associated virus (rAAV) comprises components from one or more adeno-associated virus serotypes selected from the group consisting of: AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAVrh10. In some embodiments, the rAAV comprises a capsid protein of the AAV5 serotype.

[0015] In some embodiments, the ionic salt can be one or more components selected from the group consisting of: sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium sulfate, magnesium sulfate, calcium sulfate, and their hydrates. In some embodiments, the buffer can be one or more components selected from the group consisting of: potassium dihydrogen phosphate, potassium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate hexahydrate, sodium dihydrogen phosphate monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acids, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium citrate, potassium citrate, calcium citrate. The stabilizer is one or more of sucrose, sorbitol, methionine, trehalose, mannose, mannitol, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, lactulose, maltose, lactose, isomaltose, maltitol, stachyose, melezitose, dextran. The surfactant is poloxamer and / or polysorbate (Tween), and the preferred surfactant is one or more of poloxamer 188, polysorbate 20, polysorbate 80.

[0016] In some embodiments, the concentration of the ionic salt can be at least 0.5 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 50 mM, at least 100 mM, or at least 150 mM present in the solution. In some embodiments, the concentration of the ionic salt can be 0.5 - 600 mM, or 1 - 300 mM, or 50 - 200 mM present in the solution. In some embodiments, the ionic salt can be about 1 mM present in the solution. In some embodiments, the ionic salt can be about 2 mM present in the solution. In some embodiments, the ionic salt can be about 10 mM present in the solution. In some embodiments, the ionic salt can be about 50 mM present in the solution. In some embodiments, the ionic salt can be about 150 mM present in the solution. In some embodiments, the ionic salt can be about 200 mM present in the solution. In some embodiments, the ionic salt can be about 250 mM present in the solution.

[0017] In some embodiments, the concentration of the buffer can be at least 1 mM, at least 5 mM, at least 10 mM, or at least 20 mM present in the solution. In some embodiments, the buffer can be 1 - 50 mM, or 5 - 30 mM, or 10 - 25 mM present in the solution. In some embodiments, the buffer can be 10 mM present in the solution. In some embodiments, the buffer can be about 12.5 mM present in the solution. In some embodiments, the buffer can be about 15 mM present in the solution. In some embodiments, the buffer can be about 17.5 mM present in the solution. In some embodiments, the buffer can be about 20 mM present in the solution. In some embodiments, the buffer can be about 22.5 mM present in the solution. In some embodiments, the buffer can be about 25 mM present in the solution.

[0018] In some embodiments, the concentration of the stabilizer can be at least 1 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 50 mM, at least 100 mM, or at least 200 mM present in the solution. In some embodiments, the concentration of the stabilizer can be 10 - 500 mM, or 50 - 300 mM, or 100 - 250 mM present in the solution. In some embodiments, the concentration of the stabilizer can be about 50 mM present in the solution. In some embodiments, the concentration of the stabilizer can be about 100 mM present in the solution. In some embodiments, the concentration of the stabilizer can be about 150 mM present in the solution. In some embodiments, the concentration of the stabilizer can be about 200 mM present in the solution. In some embodiments, the concentration of the stabilizer can be about 250 mM present in the solution. In some embodiments, the concentration of the stabilizer can be about 300 mM present in the solution.

[0019] In some embodiments, the mass - volume concentration (w / v) of the surfactant can be at least 0.001%, at least 0.002%, at least 0.005% or at least 0.01% present in the solution. In some embodiments, the mass - volume concentration of the surfactant can be 0.001% - 0.1%, or 0.002% - 0.05%, or 0.005% - 0.02%. In some embodiments, the mass - volume concentration of the surfactant can be 0.001%. In some embodiments, the mass - volume concentration of the surfactant can be about 00.002%. In some embodiments, the mass - volume concentration of the surfactant can be about 0.001%. In some embodiments, the mass - volume concentration of the surfactant can be about 0.002%. In some embodiments, the mass - volume concentration of the surfactant can be about 0.003%. In some embodiments, the mass - volume concentration of the surfactant can be about 0.005%. In some embodiments, the mass - volume concentration of the surfactant can be about 0.007%. In some embodiments, the mass - volume concentration of the surfactant can be about 0.008%. In some embodiments, the mass - volume concentration of the surfactant can be about 0.01%.

[0020] In some embodiments, the pH of the pharmaceutical composition is about 7.0 - 9.0. In some embodiments, the pH of the pharmaceutical composition is about 7.2 - 8.8. In some embodiments, the pH of the pharmaceutical composition is about 7.4 - 8.6. In some embodiments, the pH of the pharmaceutical composition is about 7.5 - 8.5. In some embodiments, the pH of the pharmaceutical composition is about 7.7 - 8.3. In some embodiments, the pH of the pharmaceutical composition is about 7.7. In some embodiments, the pH of the pharmaceutical composition is about 7.8. In some embodiments, the pH of the pharmaceutical composition is about 7.9. In some embodiments, the pH of the pharmaceutical composition is about 8.0. In some embodiments, the pH of the pharmaceutical composition is about 8.1. In some embodiments, the pH of the pharmaceutical composition is about 8.2. In some embodiments, the pH of the pharmaceutical composition is about 8.3. In some embodiments, the pH of the pharmaceutical composition is about 8.4. In some embodiments, the pH of the pharmaceutical composition is about 8.5. In some embodiments, the pH of the pharmaceutical composition is about 8.6.

[0021] Differential scanning fluorimetry (DSF) as an application of good manufacturing practice (GMP) that can determine the melting temperature (T m )), AAV5 has a narrow T mRanges located in different buffers. Vector stability is determined only by VP3 of AAV, especially the ratio of basic / acidic amino acids, and is independent of the VP1 and VP2 content or the packaged genome. In addition, the stability of rAAV varies depending on individual basic or acidic amino acid residues and can be distinguishable. Comparative stability analysis of rAAV1–rAAV9 and rAAVrh.10 in common formulations and storage buffers revealed serotype-specific stability. Comparative analysis of rAAV in different buffers showed that each buffer had a different effect on each serotype, i.e., no buffer resulted in consistent stabilization or destabilization of all ten viruses tested (see Bennett, Antonette, et al. "Thermal stability as a determinant of AAV serotype identity." Molecular Therapy-Methods & Clinical Development 6 (2017): 171-182.).

[0022] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which comprises rAAV, an ionic salt, a buffer, a stabilizer, and a surfactant. The ionic salt is sodium chloride, potassium chloride. The buffer is tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl). The stabilizer is sucrose. The surfactant is poloxamer 188. The pH of the pharmaceutical composition is about 7.0-9.0.

[0023] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which comprises rAAV, 0.5-500 mM of an ionic salt, 1-50 mM of a buffer, 10-500 mM of a stabilizer, and 0.001%-0.1% (w / v) of a surfactant. The pH of the pharmaceutical composition is about 7.0-9.0.

[0024] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which comprises rAAV, 1-500 mM of sodium chloride, 0.1-100 mM of magnesium chloride, 1-50 mM of Tris-HCl, 10-500 mM of sucrose or sorbitol, 0.001%-0.1% (w / v) of poloxamer 188. The pH of the pharmaceutical composition is about 7.0-9.0.

[0025] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which comprises rAAV, 10 - 300 mM of sodium chloride, 0.5 - 5 mM of magnesium chloride, 10 - 25 mM of Tris-HCl, 50 - 300 mM of sucrose or sorbitol, and 0.001% - 0.01% (w / v) of poloxamer 188. The pH of the pharmaceutical composition is about 7.5 - 8.5.

[0026] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, characterized by comprising rAAV, 1 - 500 mM of sodium chloride, 0.1 - 100 mM of magnesium chloride, 1 - 50 mM of Tris-HCl, 10 - 500 mM of sucrose or sorbitol, 0.001% - 0.1% (w / v) of poloxamer 188, and the pH of the pharmaceutical composition is 7.0 - 9.0. Preferably, the concentration of the sodium chloride is 10 - 300 mM. Preferably, the concentration of the magnesium chloride is 0.5 - 5 mM. Preferably, the concentration of the Tris-HCl is 10 - 25 mM. Preferably, the concentration of the sucrose or sorbitol is 50 - 300 mM. Preferably, the concentration of the poloxamer 188 is 0.001% - 0.01% (w / v). Preferably, the pH of the pharmaceutical composition is 7.6 - 8.6. More preferably, the concentration of the sodium chloride is 100 - 150 mM; more preferably, the concentration of the magnesium chloride is 1.0 - 1.5 mM; more preferably, the concentration of the Tris-HCl is 20 - 25 mM; more preferably, the concentration of the poloxamer 188 is 0.003% - 0.005% (w / v). More preferably, the pH of the pharmaceutical composition is 8.2 - 8.6.

[0027] In some embodiments, the recombinant adeno-associated virus (rAAV) used herein carries human coagulation factor IX (hFⅨ), namely rAAV-hFⅨ. Coagulation factor IX is one of the main factors in the coagulation cascade, encoded by the FⅨ gene located on the X chromosome. Mutations resulting in its loss of function cause hemophilia B (HB). Hemophilia B is a bleeding disorder caused by a deficiency of highly active coagulation factor IX. In severe patients, the activity of coagulation factor IX is often less than 1% of normal, and spontaneous bleeding often occurs, leading to muscle hematomas or joint deformities. Infusing coagulation factor IX preparations (currently usually coagulation factor IX protein recombinantly expressed in vitro) to supplement the level of coagulation factor IX in patients is currently the only effective treatment method, but frequent administration is required. Gene therapy is a treatment method currently in clinical trials. By introducing the normal coagulation factor IX gene into patients through a viral vector for long-term expression, the level of coagulation factor IX can be increased and bleeding can be prevented. It should be noted that the sequence of human coagulation factor IX (hFⅨ) used herein is the sequence of the highly active coagulation factor IX mutant (SEQ ID NO:1) or the coagulation factor IX expression-encoding optimized sequence (SEQ ID NO:3) in the patent with the application number CN201610898732.6 (filing date: October 14, 2016, invention name: Preparation and Application of a Highly Active Coagulation Factor IX Mutant, Recombinant Protein and Fusion Protein). The sequence of hFⅨ in the embodiments of the present application is the coagulation factor IX expression-encoding optimized sequence (SEQ ID NO:3). hFⅨ is only used as a model target gene introduced by the recombinant adeno-associated virus vector (rAAV). The recombinant adeno-associated virus vector can also carry other suitable target genes for treating other corresponding diseases.

[0028] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein. It is characterized by comprising rAAV-hFⅨ, 1 - 500 mM sodium chloride, 0.1 - 100 mM magnesium chloride, 1 - 50 mM Tris-HCl, 10 - 500 mM sucrose or sorbitol, 0.001% - 0.1% (w / v) poloxamer 188, and the pH of the pharmaceutical composition is 7.0 - 9.0. The pharmaceutical composition of the recombinant adeno-associated virus vector can be used to treat hemophilia B. Preferably, the concentration of the sodium chloride is 10 - 300 mM. Preferably, the concentration of the magnesium chloride is 0.5 - 5 mM. Preferably, the concentration of the Tris-HCl is 10 - 25 mM. Preferably, the concentration of the sucrose or sorbitol is 50 - 300 mM. Preferably, the concentration of the poloxamer 188 is 0.001% - 0.01% (w / v). Preferably, the pH of the pharmaceutical composition is 7.6 - 8.6.

[0029] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which comprises rAAV-hFⅨ, 100 - 200 mM of sodium chloride, 1.0 - 2.0 mM of magnesium chloride, 15 - 25 mM of Tris-HCl, 50 - 100 mM of sucrose or sorbitol, 0.001% - 0.005% (w / v) of poloxamer 188, and the pH of the pharmaceutical composition is 7.6 - 8.6. The pharmaceutical composition of the recombinant adeno-associated virus vector can be used for the treatment of hemophilia B. Preferably, the concentration of the sodium chloride is 100 - 150 mM. Preferably, the concentration of the magnesium chloride is 1.0 - 1.5 mM. Preferably, the concentration of the Tris-HCl is 20 - 25 mM. Preferably, the concentration of the poloxamer 188 is 0.003% - 0.005% (w / v). Preferably, the pH of the pharmaceutical composition is 8.2 - 8.6.

[0030] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which comprises rAAV5, an ionic salt, a buffer, a stabilizer, and a surfactant. The ionic salt is sodium chloride, potassium chloride. The buffer is tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl). The stabilizer is sucrose. The surfactant is poloxamer 188. The pH of the pharmaceutical composition is about 7.0 - 9.0.

[0031] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which comprises rAAV5, 0.5 - 500 mM of an ionic salt, 1 - 50 mM of a buffer, 10 - 500 mM of a stabilizer, and 0.001% - 0.1% (w / v) of a surfactant. The pH of the pharmaceutical composition is about 7.0 - 9.0.

[0032] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which comprises rAAV5, 1 - 500 mM of sodium chloride, 0.1 - 100 mM of magnesium chloride, 1 - 50 mM of Tris-HCl, 10 - 500 mM of sucrose or sorbitol, 0.001% - 0.1% (w / v) of poloxamer 188. The pH of the pharmaceutical composition is about 7.0 - 9.0.

[0033] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which comprises rAAV5, 10 - 300 mM of sodium chloride, 0.5 - 5 mM of magnesium chloride, 10 - 25 mM of Tris-HCl, 50 - 300 mM of sucrose or sorbitol, 0.001% - 0.01% (w / v) of poloxamer 188. The pH of the pharmaceutical composition is about 7.5 - 8.5.

[0034] In some embodiments, the recombinant adeno-associated virus (rAAV) used herein carries human coagulation factor IX (hFⅨ), i.e., rAAV-hFⅨ, preferably rAAV5-hFⅨ. Coagulation factor IX is one of the main factors in the coagulation cascade, encoded by the FⅨ gene located on the X chromosome. Mutations causing loss of its function lead to hemophilia B (HB). Hemophilia B is a hemorrhagic disease caused by a defect in highly active coagulation factor IX. In severe patients, the activity of coagulation factor IX is often less than 1% of normal, and spontaneous bleeding often occurs, leading to muscle hematomas or joint deformities. Infusing coagulation factor IX preparations (currently usually coagulation factor IX protein recombinantly expressed in vitro) to supplement the level of coagulation factor IX in patients is currently the only effective treatment method, but frequent administration is required. Gene therapy is a treatment method currently in clinical trials. By introducing the normal coagulation factor IX gene into patients through a viral vector for long-term expression, the level of coagulation factor IX can be increased to prevent bleeding. It should be noted that the sequence of human coagulation factor IX (hFⅨ) used herein is the sequence of the highly active coagulation factor IX mutant (SEQ ID NO:1) or the coagulation factor IX expression coding optimized sequence (SEQ ID NO:3) in the patent with the application number CN201610898732.6 (application date: October 14, 2016, invention name: Preparation and Application of a Highly Active Coagulation Factor IX Mutant, Recombinant Protein and Fusion Protein). The sequence of hFⅨ in the embodiments of this application is the coagulation factor IX expression coding optimized sequence (SEQ ID NO:3). hFⅨ is only used as a model target gene introduced by the recombinant adeno-associated virus vector (rAAV). The recombinant adeno-associated virus vector can also carry other suitable target genes for treating other corresponding diseases.

[0035] A pharmaceutical composition as described herein may comprise one or more recombinant vectors that, upon delivery to a mammal (suitable human), are capable of inducing an immune response, such as a humoral (e.g., antibody) response and / or a cell-mediated (e.g., cytotoxic T cell) response, against the transgenic product delivered by the vector. A recombinant adeno-associated virus may comprise (appropriately in any of its gene deletions) a gene encoding a desired immunogen and can thus be used in a vaccine. A recombinant adeno-associated virus can be used as a prophylactic or therapeutic vaccine against any pathogen for which an antigen has been identified that is critical for the induction of an immune response and for which the cDNA is available and that can limit the spread of the pathogen.

[0036] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which is characterized by comprising rAAV5, 1-500 mM of sodium chloride, 0.1-100 mM of magnesium chloride, 1-50 mM of Tris-HCl, 10-500 mM of sucrose or sorbitol, 0.001%-0.1% (w / v) of poloxamer 188, and the pH of the pharmaceutical composition is 7.0-9.0. Preferably, the concentration of the sodium chloride is 10-300 mM. Preferably, the concentration of the magnesium chloride is 0.5-5 mM. Preferably, the concentration of the Tris-HCl is 10-25 mM. Preferably, the concentration of the sucrose or sorbitol is 50-300 mM. Preferably, the concentration of the poloxamer 188 is 0.001%-0.01% (w / v). Preferably, the pH of the pharmaceutical composition is 7.6-8.6. More preferably, the concentration of the sodium chloride is 100-150 mM; more preferably, the concentration of the magnesium chloride is 1.0-1.5 mM; more preferably, the concentration of the Tris-HCl is 20-25 mM; more preferably, the concentration of the poloxamer 188 is 0.003%-0.005% (w / v). More preferably, the pH of the pharmaceutical composition is 8.2-8.6.

[0037] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which is characterized by comprising rAAV5-hFⅨ, 1-500 mM of sodium chloride, 0.1-100 mM of magnesium chloride, 1-50 mM of Tris-HCl, 10-500 mM of sucrose or sorbitol, 0.001%-0.1% (w / v) of poloxamer 188, and the pH of the pharmaceutical composition is 7.0-9.0. The pharmaceutical composition of the recombinant adeno-associated virus vector can be used for the treatment of hemophilia B. Preferably, the concentration of the sodium chloride is 10-300 mM. Preferably, the concentration of the magnesium chloride is 0.5-5 mM. Preferably, the concentration of the Tris-HCl is 10-25 mM. Preferably, the concentration of the sucrose or sorbitol is 50-300 mM. Preferably, the concentration of the poloxamer 188 is 0.001%-0.01% (w / v). Preferably, the pH of the pharmaceutical composition is 7.6-8.6.

[0038] In some embodiments, a pharmaceutical composition of a recombinant adeno-associated virus vector is disclosed herein, which comprises rAAV5-hFⅨ, 100-200 mM sodium chloride, 1.0-2.0 mM magnesium chloride, 15-25 mM Tris-HCl, 50-100 mM sucrose or sorbitol, 0.001%-0.005% (w / v) poloxamer 188, and the pH of the pharmaceutical composition is 7.6-8.6. The pharmaceutical composition of the recombinant adeno-associated virus vector can be used for the treatment of hemophilia B. Preferably, the concentration of the sodium chloride is 100-150 mM. Preferably, the concentration of the magnesium chloride is 1.0-1.5 mM. Preferably, the concentration of the Tris-HCl is 20-25 mM. Preferably, the concentration of the poloxamer 188 is 0.003%-0.005% (w / v). Preferably, the pH of the pharmaceutical composition is 8.2-8.6.

[0039] In some embodiments, the compositions described herein are used in the immunization of a subject (e.g., a human). The immune levels of selected genes can be monitored to determine the need for boosting. After assessing the antibody titers in the serum, optional booster immunizations may be desirable.

[0040] Optionally, the compositions of the present invention can be formulated to contain other components, including pharmaceutically acceptable carriers, e.g., adjuvants, preservatives, etc.

[0041] In certain embodiments, the compositions as described herein are administered to a subject by intramuscular injection, intravaginal administration, intravenous injection, intraperitoneal injection, subcutaneous injection, intradermal administration, intradermal injection, nasal administration, or oral administration.

[0042] If the treatment regimen involves co-administration of one or more adeno-associated virus vectors and / or other components, these can be co-formulated (i.e., in the same mixture or composition) or formulated separately in different compositions. When formulated separately, they are advantageously co-administered at or near the same site. For example, the components can be administered to the same side limb (e.g., intramuscularly, transdermally, intradermally, subcutaneously) ("ipsilateral" administration) or to opposite side limbs ("contralateral" administration).

[0043] The dose of the viral vector depends mainly on factors such as the disorder being treated, the age, weight, and health of the patient, and can thus vary between patients. For example, a therapeutically effective adult or veterinary dose of the viral vector typically contains 1×10 5 to 1×10 15 viral particles, e.g., 1×10 8 -1×10 13 (e.g., 1×10 8 、1×10 9, 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 particles). Alternatively, it can generally be 1×10 8 to 1×10 13 genome titer (vg / ml), (e.g., 1×10 8 vg / ml, 1×10 9 vg / ml, 1×10 10 vg / ml, 1×10 11 vg / ml, 1×10 12 vg / ml or 1×10 13 vg / ml) of the viral vector is administered. The dose can vary according to the size of the animal and the route of administration. For example, for a single site, a suitable human or veterinary dose for intramuscular injection (for an animal of about 80 kg) is in the range of about 1×10 9 to about 5×10 12 particles / mL. Optionally, multiple administration sites can be used. In some embodiments, for an oral formulation, a suitable human or veterinary dose can be in the range of about 1×10 11 to about 1×10 15 particles / mL.

[0044] In order to develop a formulation prescription for the long-term stable storage of a liquid formulation of a recombinant adeno-associated virus vector and ensure the quality of the product within the validity period (tentatively 24 months), prescription research experiments such as pH screening, excipient screening, and surfactant screening were carried out, and the final prescription of the liquid formulation of the recombinant adeno-associated virus vector was determined. According to the literature, viruses are prone to aggregation during repeated freezing and thawing, the particle size also becomes larger, and they are prone to inactivation at high temperatures. During the prescription screening process, conditions such as repeated freezing and thawing, high temperatures of 25°C or 40°C were used to accelerate the physical and biochemical property changes of the virus in each prescription, so as to screen out the most stable prescription.

[0045] The stability of the pharmaceutical composition of the recombinant adeno-associated virus vector can be characterized by observing or detecting indicators such as the clarity of appearance, osmotic pressure, fluorescence quantitative PCR, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the number of insoluble particles, and the in vitro activity of hFⅨ expression.

[0046] Fluorescence quantitative polymerase chain reaction analysis (qPCR) is a method in DNA amplification sensing that measures the total amount of products after each polymerase chain reaction (PCR) cycle with a fluorescent chemical substance, and is a method for quantitatively analyzing specific DNA sequences in test samples by internal reference or external reference methods. qPCR can quantify adeno-associated virus vectors.

[0047] Insoluble particles can be determined using a microscope insoluble particle detector.

[0048] Size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is used to detect the purity of the pharmaceutical composition of the recombinant adeno-associated virus vector.

[0049] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most commonly used protein expression analysis techniques in polyacrylamide gel electrophoresis. The principle of this technique is to separate proteins in the electrophoresis gel according to their different molecular weights in the sample. SDS-PAGE is usually used to detect the protein expression (expression level, expression distribution), and analyze the purity of the target protein, etc.

[0050] The in vitro active hFⅨ expression level is used to determine the protein content of hFⅨ in the pharmaceutical composition of the recombinant adeno-associated virus vector by the double antibody sandwich ELISA method. Specific antibodies (i.e., primary antibody, capture antibody) are coated on the solid phase carrier to form a solid phase antibody. After blocking and washing, the test sample is added. After incubation and washing, a biotin-labeled antibody (i.e., secondary antibody, detection antibody) is added. After further incubation and washing, streptavidin-peroxidase (SA-HRP) is added. After incubation and washing, a chromogenic substrate is added for determination, and the protein content of hFⅨ in the test sample is obtained by colorimetry. Description of the Drawings

[0051] Figure 1A , 1B Figures 1C and 1D are graphs of the experimental results of Examples 1-10, where N / A represents not applicable or not measured.

[0052] Figure 2 It is a graph of the experimental results of the long-term stability study of the liquid preparation of the recombinant adeno-associated virus vector of Examples 1-10 placed at 2-8°C. Detailed Description of the Invention

[0053] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content disclosed in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

[0054] Example 1

[0055] A liquid preparation of a recombinant adeno-associated virus vector, which comprises rAAV5-hFⅨ, 100 mM sodium chloride, 1.5 mM magnesium chloride, 15 mM Tris-HCl, 50 mM sucrose, 0.003% (w / v) poloxamer 188, and the pH of the pharmaceutical composition is 7.6.

[0056] Example 2

[0057] A liquid preparation of a recombinant adeno-associated virus vector, which comprises rAAV5-hFⅨ, 150 mM sodium chloride, 1.0 mM magnesium chloride, 20 mM Tris-HCl, 50 mM sucrose, 0.005% (w / v) poloxamer 188, and the pH of the pharmaceutical composition is 8.2.

[0058] Example 3

[0059] A liquid preparation of a recombinant adeno-associated virus vector, which comprises rAAV5-hFⅨ, 200 mM sodium chloride, 2.0 mM magnesium chloride, 25 mM Tris-HCl, 100 mM sucrose, 0.001% (w / v) poloxamer 188, and the pH of the pharmaceutical composition is 8.6.

[0060] Example 4

[0061] A liquid preparation of a recombinant adeno-associated virus vector, which comprises rAAV5-hFⅨ, 150 mM sodium chloride, 1.0 mM magnesium chloride, 20 mM Tris-HCl, 50 mM sorbitol, 0.005% (w / v) poloxamer 188, and the pH of the pharmaceutical composition is 8.2.

[0062] Example 5

[0063] A liquid preparation of a recombinant adeno-associated virus vector, which comprises rAAV5-hFⅨ, 200 mM sodium chloride, 1.0 mM magnesium chloride, 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate, 50 mM sucrose, 0.005% (w / v) poloxamer 188, and the pH of the pharmaceutical composition is 6.5.

[0064] Example 6

[0065] A liquid preparation of a recombinant adeno-associated virus vector, which comprises rAAV5-hFⅨ, 200 mM sodium chloride, 1.0 mM magnesium chloride, 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate, 50 mM sucrose, 0.005% (w / v) poloxamer 188, and the pH of the pharmaceutical composition is 7.5.

[0066] Example 7

[0067] A liquid preparation of a recombinant adeno-associated virus vector, which comprises rAAV5-hFⅨ, 150 mM of sodium chloride, 1.0 mM of magnesium chloride, 20 mM of Tris-HCl, 0.005% (w / v) of poloxamer 188, and the pH of the pharmaceutical composition is 8.2.

[0068] Example 8

[0069] A liquid preparation of a recombinant adeno-associated virus vector, which comprises rAAV5-hFⅨ, 150 mM of sodium chloride, 10 mM of magnesium chloride, 20 mM of Tris-HCl, 50 mM of sucrose, 0.005% (w / v) of poloxamer 188, and the pH of the pharmaceutical composition is 8.2.

[0070] Example 9

[0071] A liquid preparation of a recombinant adeno-associated virus vector, which comprises rAAV5-hFⅨ, 150 mM of sodium chloride, 1 mM of methionine, 20 mM of Tris-HCl, 50 mM of sucrose, 0.005% (w / v) of poloxamer 188, and the pH of the pharmaceutical composition is 8.2.

[0072] Example 10

[0073] A liquid preparation of a recombinant adeno-associated virus vector, which comprises rAAV5-hFⅨ, 150 mM of sodium chloride, 1.0 mM of magnesium chloride, 20 mM of Tris-HCl, 50 mM of sucrose, 0.005% (w / v) of polysorbate 80, and the pH of the pharmaceutical composition is 8.2.

[0074] The experimental results of the stability study of Examples 1-10 are as Figure 1A 、 1BAs shown in Figures 1C and 1D, the liquid preparations of the recombinant adeno-associated virus vectors of Examples 1-4 have good stability, a small number of insoluble particles, and the highest in vitro active hFⅨ expression level. Compared with the liquid preparation of Example 2, the pH of the liquid preparation of Example 5 is 6.5, and its SEC-HPLC data shows a decrease in purity and also a decrease in the in vitro active hFⅨ expression level. Compared with the liquid preparation of Example 1, the pH value of the liquid preparation of Example 5 is close, except that the buffer is different, which is disodium hydrogen phosphate / sodium dihydrogen phosphate, and its SEC-HPLC data shows a decrease in purity and also a decrease in the in vitro active hFⅨ expression level. Compared with the liquid preparation of Example 2, the liquid preparation of Example 7 does not contain the stabilizer sucrose, and its SEC-HPLC data shows a slight decrease in purity and also a decrease in the in vitro active hFⅨ expression level. Compared with the liquid preparation of Example 2, the liquid preparation of Example 8 increases the concentration of magnesium chloride to 10 mM, and its SEC-HPLC data shows a slight decrease in purity and also a decrease in the in vitro active hFⅨ expression level. Compared with the liquid preparation of Example 2, the liquid preparation of Example 9 replaces magnesium chloride with methionine, and its SEC-HPLC data shows a slight decrease in purity and also a decrease in the in vitro active hFⅨ expression level. Compared with the liquid preparation of Example 2, the liquid preparation of Example 10 replaces magnesium chloride with methionine, and its SEC-HPLC data shows a slight decrease in purity and also a decrease in the in vitro active hFⅨ expression level.

[0075] Example 11

[0076] In the long-term stability experiment, the liquid preparation of the recombinant adeno-associated virus vector of Example 2 was placed at 2-8 °C, and key indicators such as the in vitro activity and genomic titer of the product were measured at 0, 1, 2, 3, 6, 9, and 12 months. There was no obvious downward trend, indicating that the liquid preparation of the recombinant adeno-associated virus vector of Example 2 can maintain stability for a long time at 2-8 °C, which has more advantages than the current storage condition that requires freezing, facilitates the transportation and clinical use of the product, and reduces the investment and use costs of all relevant parties.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing this invention, the preferred materials and methods are described herein. When describing and claiming this invention, the following terms will be used.

[0078] It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0079] As used herein, when referring to measurable values such as amounts, lengths of time, etc., the term "about" means including variations of ±20% or ±10% from the specified value, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, whereby such variations are suitable for carrying out the disclosed methods.

[0080] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one active ingredient useful in the present invention with other chemical components, such chemical components being, for example, carriers, stabilizers, diluents, adjuvants, dispersants, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates the administration of the active ingredient to an organism. There are various techniques for administering compounds in the art, including but not limited to: intravenous, oral, aerosol, parenteral, intraocular, pulmonary, and topical administration.

[0081] As used herein, the term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, which participate in transporting or delivering the compound(s) of the present invention within or to a subject so that it can perform its desired function. Generally, such compounds are transported or delivered from one organ or part of the body to another organ or part of the body. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and harmless to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; diols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solution; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifying agents; coloring agents; demolding agents; coating agents; sweetening agents; flavoring agents; perfuming agents; preservatives; antioxidants; plasticizers; gelling agents; thickening agents; hardening agents; sedimenting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, etc., that are compatible with the activity of the compound and physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the composition.

[0082] Although the present invention has been described in detail with reference to its specific embodiments, it should be understood that functionally equivalent variations are within the scope of the present invention. In fact, various modifications of the present invention other than those shown and described herein will become apparent to those skilled in the art in light of the foregoing description and drawings. Those skilled in the art will recognize or be able to ascertain using only routine experimentation, many equivalent forms of the specific embodiments of the present invention described herein.

[0083] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application were specifically and individually incorporated by reference in its entirety herein.

Claims

1. A pharmaceutical composition of a recombinant adeno-associated virus (rAAV) vector, comprising: (a) rAAV, (b) sodium chloride, (c) magnesium chloride, (d) Tris-HCl, (e) sucrose or sorbitol, and (f) poloxamer 188 or polysorbate 80.

2. The pharmaceutical composition of the rAAV vector according to claim 1, wherein the rAAV comprises one or more components of adeno-associated virus serotypes selected from the group consisting of AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAVrh10.

3. The pharmaceutical composition of the rAAV vector according to claim 1 or 2, wherein the rAAV is rAAV5, rAAV8, or rAAV9.

4. The pharmaceutical composition of the rAAV vector according to any one of claims 1-3, wherein the concentration of the sodium chloride is 100-300 mM.

5. The pharmaceutical composition of the rAAV vector according to any one of claims 1-4, wherein the concentration of the sodium chloride is 100-150 mM.

6. The pharmaceutical composition of the rAAV vector according to any one of claims 1-5, wherein the concentration of the magnesium chloride is 0.5-5.0 mM.

7. The pharmaceutical composition of the rAAV vector according to any one of claims 1-6, wherein the concentration of the magnesium chloride is 1.0-1.5 mM.

8. The pharmaceutical composition of the rAAV vector according to any one of claims 1-7, wherein the concentration of Tris-HCl is 10-25 mM.

9. The pharmaceutical composition of the rAAV vector according to any one of claims 1-8, wherein the concentration of Tris-HCl is 20-25 mM.

10. The pharmaceutical composition of the rAAV vector according to any one of claims 1-9, wherein the concentration of sucrose or sorbitol is 50-300 mM.

11. The pharmaceutical composition of the rAAV vector according to any one of claims 1-10, wherein the concentration of sucrose or sorbitol is 50-100 mM.

12. The pharmaceutical composition of the rAAV vector according to any one of claims 1-11, wherein the concentration of poloxamer 188 or polysorbate 80 is 0.001%-0.005% (w / v).

13. The pharmaceutical composition of the rAAV vector according to any one of claims 1-12, wherein the concentration of poloxamer 188 or polysorbate 80 is 0.003%-0.005% (w / v).

14. The pharmaceutical composition of the rAAV vector according to any one of claims 1-13, wherein the pH of the pharmaceutical composition is 7.6-8.

6.

15. The pharmaceutical composition of the rAAV vector according to any one of claims 1-14, wherein the pH of the pharmaceutical composition is 8.2-8.

6.

16. A method for treating a disease corresponding to a target gene defect in a subject, comprising administering to the subject a pharmaceutical composition of the rAAV vector according to any one of claims 1-15, wherein the rAAV encodes a target gene for treating or otherwise improving, preventing or slowing down the disease corresponding to the target gene defect.

17. A pharmaceutical composition of a recombinant adeno-associated virus (rAAV) vector, comprising: (a) rAAV-hFIX, (b) sodium chloride, (c) magnesium chloride, (d) Tris-HCl, (e) sucrose or sorbitol, and (f) poloxamer 188 or polysorbate 80.

18. The pharmaceutical composition of the rAAV vector according to claim 17, wherein the rAAV comprises one or more components selected from the group consisting of adeno-associated virus serotypes AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAVrh10.

19. The pharmaceutical composition of the rAAV vector according to claim 17 or 18, wherein the rAAV is rAAV5, rAAV8 or rAAV9.

20. The pharmaceutical composition of the rAAV vector according to any one of claims 11-19, wherein the concentration of the sodium chloride is 100-300 mM.

21. The pharmaceutical composition of the rAAV vector according to any one of claims 17-20, wherein the concentration of the sodium chloride is 100-150 mM.

22. The pharmaceutical composition of the rAAV vector according to any one of claims 17-21, wherein the concentration of the magnesium chloride is 0.5-5.0 mM.

23. The pharmaceutical composition of the rAAV vector according to any one of claims 17-22, wherein the concentration of the magnesium chloride is 1.0-1.5 mM.

24. The pharmaceutical composition of the rAAV vector according to any one of claims 17-23, wherein the concentration of Tris-HCl is 10-25 mM.

25. The pharmaceutical composition of the rAAV vector according to any one of claims 17-24, wherein the concentration of Tris-HCl is 20-25 mM.

26. The pharmaceutical composition of the rAAV vector according to any one of claims 17-25, wherein the concentration of sucrose or sorbitol is 50-300 mM.

27. The pharmaceutical composition of the rAAV vector according to any one of claims 17-26, wherein the concentration of sucrose or sorbitol is 50-100 mM.

28. The pharmaceutical composition of the rAAV vector according to any one of claims 17-27, wherein the concentration of poloxamer 188 or polysorbate 80 is 0.001%-0.005% (w / v).

29. The pharmaceutical composition of the rAAV vector according to any one of claims 17-28, wherein the concentration of poloxamer 188 or polysorbate 80 is 0.003%-0.005% (w / v).

30. The pharmaceutical composition of the rAAV vector according to any one of claims 17-29, wherein the pH of the pharmaceutical composition is 7.6-8.

6.

31. A pharmaceutical composition of an rAAV vector according to any one of claims 17 - 30, wherein the pH of the pharmaceutical composition is 8.2 - 8.

6.

32. A method of treating hemophilia B in a subject, comprising administering to the subject a pharmaceutical composition of an rAAV vector according to any one of claims 18 - 31, wherein the rAAV encodes the target gene hFIX for treating hemophilia B.

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