Pharmaceutical compositions of recombinant adeno-associated virus vectors and their applications

Abstract

A pharmaceutical composition of a recombinant adeno-associated virus vector, including a recombinant adeno-associated virus (rAAV), an ionic salt, a buffer, a stabilizer, and a surfactant, and uses thereof are provided. The rAAV vector carries hFIX and can be used to treat hemophilia B.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Chinese application CN202211467932.8, filed November 22, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical Field The present invention relates to the field of pharmaceutical formulations of recombinant adeno-associated virus vectors. The present invention relates to pharmaceutical compositions of recombinant adeno-associated virus vectors and their applications, and more particularly to liquid formulations of gene therapy drugs using recombinant adeno-associated viruses as vectors and their applications. [Background technology]

[0003] background Cell and gene therapy (CGT) is a therapeutic approach for improving medical treatments or treating rare diseases. It overcomes the limitations of traditional small molecule and antibody drugs in controlling protein levels through gene expression, silencing, or in vitro modification. A crucial aspect of CGT therapy is the in vivo delivery of gene vectors. Currently, common gene vectors are divided into non-viral and viral vectors. Non-viral vectors include plasmids, naked DNA (non-viral), and LNP delivery systems. However, due to their low in vivo transfection efficiency and high toxicity, the clinical translation of non-viral vectors has been severely limited. Commonly used viral vectors include adenovirus (AdV), adeno-associated virus (AAV), lentivirus (LV), and retrovirus (RV).

[0004] Adeno-associated virus (AAV) was first identified in laboratory adenovirus (AdV) preparations in the mid-1960s and was soon found in human tissues. Recombinant adeno-associated virus (rAAV) vectors typically replace viral coding sequences with a gene of interest. These vectors have been shown to be capable of efficient expression and gene targeting at several different sites in vitro and in vivo. Studies have shown that AAV is safe and can be stably and continuously expressed in respiratory, central nervous system, skeletal muscle, liver, and ocular studies. As the titer and purity of rAAV preparations increase, so does the efficiency of rAAV-mediated transduction.

[0005] Currently, the number of rAAV-related clinical trials is generally increasing worldwide, and to date, four gene therapy products using rAAV as a vector have been approved for sale.

[0006] In 2012, Glybera was approved by the EMA for the treatment of lipoprotein lipase deficiency disease (LPLD), the first human AAV gene therapy product to receive formal marketing approval.

[0007] At the end of 2017, the FDA approved Spark Therapeutics' Luxturna for the treatment of inherited retinal diseases, making it the first "in vivo" gene therapy drug approved in the United States.

[0008] In May 2019, Novartis' Zolgensma was approved by the U.S. FDA for the treatment of children under the age of 2 with spinal muscular atrophy (SMA) who have biallelic mutations in the survival motor neuron 1 (SMN1) gene.

[0009] On August 24, 2022, BioMarin Pharmaceuticals, Inc. (BioMarin) announced that the European Commission (EC) approved its hemophilia A gene therapy drug, ROCTAVIAN, for the treatment of adult patients with severe hemophilia A who are FVIII inhibitor-naive and AAV5 antibody-negative.TM The company announced that it has granted conditional marketing approval for valoctocogene roxaparvovec (valoctocogene roxaparvovec).

[0010] The above gene therapy products must be stored frozen, and their transportation and storage temperatures are generally −20°C or −60°C. Maintaining freezer temperatures at −20°C or −60°C is logistically challenging and costly. Such low temperature requirements can adversely affect the ability to distribute the products to a wide range of clinical sites. Therefore, it would be desirable to provide a recombinant adeno-associated virus vector gene therapy pharmaceutical composition that is stable for more than one year under typical refrigeration temperature conditions, making it suitable for transportation or for freezer storage until the product is thawed and used for patient administration at the clinical site.

[0011] To solve the above problems, the present disclosure provides a pharmaceutical composition of a recombinant adeno-associated virus vector, particularly a liquid formulation of a gene therapy drug using a recombinant adeno-associated virus as a vector. The pharmaceutical composition can be stored for more than one year at a common refrigeration temperature, such as 2 to 8°C, and can maintain stable indicators such as genome titer and biological activity. It also has good stability when stored at room temperature for two weeks. Summary of the Invention

[0012] overview The present disclosure provides pharmaceutical compositions of recombinant adeno-associated virus vectors, including recombinant adeno-associated virus (rAAV), ionic salts, buffers, stabilizers, and surfactants, and applications thereof.

[0013] In some embodiments, the recombinant adeno-associated virus (rAAV) comprises one or more components from an adeno-associated virus serotype selected from the group consisting of AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAVrhlO. In some embodiments, the rAAV comprises capsid proteins of the AAV5 serotype.

[0014] In some embodiments, the ionic salt can be one or more members from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium sulfate, magnesium sulfate, calcium sulfate, and hydrates thereof. In some embodiments, the buffer can be one or more members 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, and calcium citrate. In some embodiments, 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, and dextran. In some embodiments, the surfactant is a poloxamer and / or polysorbate (Tween), with preferred surfactants being one or more of poloxamer 188, polysorbate 20, and polysorbate 80.

[0015] In some embodiments, the ionic salt can be present in solution at a concentration of 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. In some embodiments, the ionic salt can be present in solution at a concentration of 0.5-600 mM, or 1-300 mM, or 50-200 mM. In some embodiments, the ionic salt can be present in solution at about 1 mM. In some embodiments, the ionic salt can be present in solution at about 2 mM. In some embodiments, the ionic salt can be present in solution at about 10 mM. In some embodiments, the ionic salt can be present in solution at about 50 mM. In some embodiments, the ionic salt can be present in solution at about 150 mM. In some embodiments, the ionic salt can be present in solution at about 200 mM. In some embodiments, the ionic salt may be present in solution at about 250 mM.

[0016] In some embodiments, the buffering agent can be present in the solution at a concentration of at least 1 mM, at least 5 mM, at least 10 mM, or at least 20 mM. In some embodiments, the buffering agent can be present in the solution at 1-50 mM, or 5-30 mM, or 10-25 mM. In some embodiments, the buffering agent can be present in the solution at 10 mM. In some embodiments, the buffering agent can be present in the solution at about 12.5 mM. In some embodiments, the buffering agent can be present in the solution at about 15 mM. In some embodiments, the buffering agent can be present in the solution at about 17.5 mM. In some embodiments, the buffering agent can be present in the solution at about 20 mM. In some embodiments, the buffering agent can be present in the solution at about 22.5 mM. In some embodiments, the buffering agent can be present in the solution at about 25 mM.

[0017] In some embodiments, the stabilizer may be present in solution at a concentration of 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. In some embodiments, the stabilizer may be present in solution at a concentration of 10-500 mM, or 50-300 mM, or 100-250 mM. In some embodiments, the stabilizer may be present in solution at a concentration of about 50 mM. In some embodiments, the stabilizer may be present in solution at a concentration of about 100 mM. In some embodiments, the stabilizer may be present in solution at a concentration of about 150 mM. In some embodiments, the stabilizer may be present in solution at a concentration of about 200 mM. In some embodiments, the stabilizer may be present in solution at a concentration of about 250 mM. In some embodiments, the stabilizer may be present in solution at a concentration of about 300 mM.

[0018] In some embodiments, the surfactant may be present in the solution at a weight-to-volume concentration (w / v) of at least 0.001%, at least 0.002%, at least 0.005%, or at least 0.01%. In some embodiments, the weight-to-volume concentration of the surfactant may be 0.001% to 0.1%, or 0.002% to 0.05%, or 0.005% to 0.02%. In some embodiments, the weight-to-volume concentration of the surfactant may be 0.001%. In some embodiments, the weight-to-volume concentration of the surfactant may be about 0.002%. In some embodiments, the weight-to-volume concentration of the surfactant may be about 0.001%. In some embodiments, the weight-to-volume concentration of the surfactant may be about 0.002%. In some embodiments, the weight-to-volume concentration of the surfactant may be about 0.003%. In some embodiments, the weight-to-volume concentration of the surfactant may be about 0.005%. In some embodiments, the weight-to-volume concentration of the surfactant may be about 0.007%. In some embodiments, the surfactant weight volume concentration can be about 0.008%. In some embodiments, the surfactant weight volume concentration can be about 0.01%.

[0019] In some embodiments, the pH of the pharmaceutical composition is about 7.0 to 9.0. In some embodiments, the pH of the pharmaceutical composition is about 7.2 to 8.8. In some embodiments, the pH of the pharmaceutical composition is about 7.4 to 8.6. In some embodiments, the pH of the pharmaceutical composition is about 7.5 to 8.5. In some embodiments, the pH of the pharmaceutical composition is about 7.7 to 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.

[0020] Differential scanning fluorescence (DSF) is a Good Manufacturing Practice (GMP) application that can determine the melting temperature (Tm). AAV5 has a narrow range of Tm in different buffers. Vector stability is determined solely by AAV VP3, specifically the ratio of basic to acidic amino acids, and is independent of the VP1 and VP2 content or the packaged genome. Furthermore, rAAV stability can be altered by a single basic or acidic amino acid residue, and can be differentiated. Comparative stability analysis of rAAV1-rAAV9 and rAAVrh.10 in commonly used formulations and storage buffers revealed serotype-specific stability. Comparative analysis of rAAV in different buffers showed that each buffer affected each serotype differently; no buffer consistently stabilized or destabilized all 10 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).

[0021] In some embodiments, disclosed herein is a pharmaceutical composition of a recombinant adeno-associated virus vector, comprising rAAV, an ionic salt, a buffer, a stabilizer, and a surfactant. The ionic salt is sodium chloride or 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 to 9.0.

[0022] In some embodiments, disclosed herein is a recombinant adeno-associated virus vector pharmaceutical composition comprising an rAAV, an ionic salt in an amount of 0.5-500 mM, a buffering agent in an amount of 1-50 mM, a stabilizer in an amount of 10-500 mM, and a surfactant in an amount of 0.001%-0.1% (w / v). The pH of the pharmaceutical composition is about 7.0-9.0.

[0023] In some embodiments, disclosed herein is a pharmaceutical composition of a recombinant adeno-associated virus vector comprising rAAV, 1-500 mM sodium chloride, 0.1-100 mM magnesium chloride, 1-50 mM Tris-HCl, 10-500 mM sucrose or sorbitol, and 0.001%-0.1% (w / v) poloxamer 188. The pH of the pharmaceutical composition is about 7.0-9.0.

[0024] In some embodiments, disclosed herein is a pharmaceutical composition of a recombinant adeno-associated virus vector comprising rAAV, 10-300 mM sodium chloride, 0.5-5 mM magnesium chloride, 10-25 mM Tris-HCl, 50-300 mM sucrose or sorbitol, and 0.001%-0.01% (w / v) poloxamer 188. The pH of the pharmaceutical composition is about 7.5-8.5.

[0025] In some embodiments, disclosed herein is a pharmaceutical composition for a recombinant adeno-associated virus vector, comprising rAAV, 1 to 500 mM sodium chloride, 0.1 to 100 mM magnesium chloride, 1 to 50 mM Tris-HCl, 10 to 500 mM sucrose or sorbitol, and 0.001% to 0.1% (w / v) poloxamer 188, wherein the pH of the pharmaceutical composition is 7.0 to 9.0. Preferably, the sodium chloride concentration is 10 to 300 mM. Preferably, the magnesium chloride concentration is 0.5 to 5 mM. Preferably, the Tris-HCl concentration is 10 to 25 mM. Preferably, the sucrose or sorbitol concentration is 50 to 300 mM. Preferably, the poloxamer 188 concentration is 0.001% to 0.01% (w / v). Preferably, the pH of the pharmaceutical composition is 7.6 to 8.6. More preferably, the sodium chloride concentration is 100 to 150 mM; more preferably, the magnesium chloride concentration is 1.0 to 1.5 mM; more preferably, the Tris-HCl concentration is 20 to 25 mM; and more preferably, the poloxamer 188 concentration is 0.003% to 0.005% (w / v). More preferably, the pH of the pharmaceutical composition is 8.2 to 8.6.

[0026] In some embodiments, the recombinant adeno-associated virus (rAAV) used herein carries human coagulation factor IX (hFIX), i.e., rAAV-hFIX. Coagulation factor IX is one of the key factors in the coagulation cascade and is encoded by the FIX gene located on chromosome X. Loss-of-function mutations in this gene cause hemophilia B (HB). Hemophilia B is a bleeding disorder caused by a deficiency of hyperactive coagulation factor IX. In severely affected patients, coagulation factor IX activity is often less than 1% of normal activity, resulting in frequent spontaneous bleeding and muscle hematomas or joint deformities. Infusion of factor IX concentrates (currently usually recombinantly expressed in vitro) to replenish factor IX levels in patients is currently the only effective treatment, but it requires frequent administration. Gene therapy is a treatment method currently undergoing clinical trials. The normal coagulation factor IX gene is introduced into the patient's body via a viral vector and expressed for a long period of time, thereby increasing coagulation factor IX levels and preventing bleeding. It should be noted that the sequence of human coagulation factor IX (hFIX) used herein is the sequence of a highly active coagulation factor IX variant (SEQ ID NO: 1) or the coding sequence optimized for the expression of coagulation factor IX (SEQ ID NO: 3) in the application entitled "Preparation and Application of Highly Active Blood Coagulation Factor IX Variants, Recombinant Proteins, and Fusion Proteins," filed on October 14, 2016, with application number CN201610898732.6. The hFIX sequence in the examples of the present invention is the coding sequence optimized for the expression of coagulation factor IX (SEQ ID NO: 3). hFIX is used only as a model target gene to be introduced by recombinant adeno-associated virus vectors (rAAV), which can also carry other suitable target genes for treating other corresponding diseases.

[0027] In some embodiments, disclosed herein is a pharmaceutical composition of a recombinant adeno-associated virus vector, comprising rAAV-hFIX, 1 to 500 mM sodium chloride, 0.1 to 100 mM magnesium chloride, 1 to 50 mM Tris-HCl, 10 to 500 mM sucrose or sorbitol, and 0.001% to 0.1% (w / v) poloxamer 188, wherein the pH of the pharmaceutical composition is 7.0 to 9.0. The pharmaceutical composition of a recombinant adeno-associated virus vector can be used for the treatment of hemophilia B. Preferably, the sodium chloride concentration is 10 to 300 mM. Preferably, the magnesium chloride concentration is 0.5 to 5 mM. Preferably, the Tris-HCl concentration is 10 to 25 mM. Preferably, the sucrose or sorbitol concentration is 50 to 300 mM. Preferably, the concentration of poloxamer 188 is 0.001% to 0.01% (w / v). Preferably, the pH of the pharmaceutical composition is 7.6 to 8.6.

[0028] In some embodiments, disclosed herein is a pharmaceutical composition of a recombinant adeno-associated virus vector comprising rAAV-hFIX, 100-200 mM sodium chloride, 1.0-2.0 mM magnesium chloride, 15-25 mM Tris-HCl, 50-100 mM sucrose or sorbitol, and 0.001%-0.005% (w / v) poloxamer 188, wherein the pH of the pharmaceutical composition is 7.6-8.6. The pharmaceutical composition of a recombinant adeno-associated virus vector can be used for the treatment of hemophilia B. Preferably, the sodium chloride concentration is 100-150 mM. Preferably, the magnesium chloride concentration is 1.0-1.5 mM. Preferably, the Tris-HCl concentration is 20-25 mM. Preferably, the poloxamer 188 concentration is 0.003%-0.005% (w / v). Preferably, the pH of the pharmaceutical composition is 8.2 to 8.6.

[0029] In some embodiments, disclosed herein is a pharmaceutical composition of a recombinant adeno-associated virus vector comprising rAAV5, an ionic salt, a buffer, a stabilizer, and a surfactant. The ionic salt is sodium chloride or 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 to 9.0.

[0030] In some embodiments, disclosed herein is a recombinant adeno-associated virus vector pharmaceutical composition comprising rAAV5, an ionic salt in an amount of 0.5 to 500 mM, a buffering agent in an amount of 1 to 50 mM, a stabilizer in an amount of 10 to 500 mM, and a surfactant in an amount of 0.001% to 0.1% (w / v). The pH of the pharmaceutical composition is about 7.0 to 9.0.

[0031] In some embodiments, disclosed herein is a pharmaceutical composition of a recombinant adeno-associated virus vector comprising rAAV5, 1 to 500 mM sodium chloride, 0.1 to 100 mM magnesium chloride, 1 to 50 mM Tris-HCl, 10 to 500 mM sucrose or sorbitol, and 0.001% to 0.1% (w / v) poloxamer 188. The pH of the pharmaceutical composition is about 7.0 to 9.0.

[0032] In some embodiments, disclosed herein is a pharmaceutical composition of a recombinant adeno-associated virus vector comprising rAAV5, 10-300 mM sodium chloride, 0.5-5 mM magnesium chloride, 10-25 mM Tris-HCl, 50-300 mM sucrose or sorbitol, and 0.001%-0.01% (w / v) poloxamer 188. The pH of the pharmaceutical composition is about 7.5-8.5.

[0033] 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 key factors in the coagulation cascade and is encoded by the FIX gene located on chromosome X, and loss-of-function mutations in this gene cause hemophilia B (HB). Hemophilia B is a bleeding disorder caused by a deficiency of highly active coagulation factor IX. In severely affected patients, coagulation factor IX activity often falls below 1% of normal activity, resulting in frequent spontaneous bleeding and muscle hematomas or joint deformities. Infusion of factor IX concentrates (currently usually recombinantly expressed in vitro) to replenish factor IX levels in patients is currently the only effective treatment, but it requires frequent administration. Gene therapy is a treatment approach currently undergoing clinical trials. The normal coagulation factor IX gene is introduced into the patient's body via a viral vector for long-term expression, thereby increasing the level of coagulation factor IX and preventing bleeding. It should be noted that the human coagulation factor IX (hFIX) sequence used herein refers to the sequence of the highly active coagulation factor IX variant (SEQ ID NO: 1) or the coding sequence optimized for coagulation factor IX expression (SEQ ID NO: 3) in the patent application "Preparation and Application of Highly Active Blood Coagulation Factor IX Variants, Recombinant Proteins, and Fusion Proteins," filed on October 14, 2016, with application number CN201610898732.6. The hFIX sequence in the examples of the present invention is the coding sequence optimized for coagulation factor IX expression (SEQ ID NO: 3). hFIX is used only as a model target gene delivered by recombinant adeno-associated viral vectors (rAAVs), which can also carry other suitable target genes for treating other corresponding diseases.

[0034] The pharmaceutical compositions described herein may contain one or more recombinant vectors capable of inducing an immune response, such as a humoral (e.g., antibody) and / or cell-mediated (e.g., cytotoxic T cell) response against the gene delivered by the vector after administration to a mammal (suitably a human). The recombinant adeno-associated virus may contain a gene encoding a desired immunogen (suitably in one of its gene deletions) and thus may be used in a vaccine. The recombinant adeno-associated virus may be used as a prophylactic or therapeutic vaccine against any pathogen for which an antigen important in inducing an immune response and capable of limiting the spread of the pathogen has been identified and its cDNA is available.

[0035] In some embodiments, disclosed herein is a pharmaceutical composition for a recombinant adeno-associated virus vector, comprising rAAV5, 1 to 500 mM sodium chloride, 0.1 to 100 mM magnesium chloride, 1 to 50 mM Tris-HCl, 10 to 500 mM sucrose or sorbitol, and 0.001% to 0.1% (w / v) poloxamer 188, wherein the pH of the pharmaceutical composition is 7.0 to 9.0. Preferably, the concentration of sodium chloride is 10 to 300 mM. Preferably, the concentration of magnesium chloride is 0.5 to 5 mM. Preferably, the concentration of Tris-HCl is 10 to 25 mM. Preferably, the concentration of sucrose or sorbitol is 50 to 300 mM. Preferably, the concentration of poloxamer 188 is 0.001% to 0.01% (w / v). Preferably, the pH of the pharmaceutical composition is 7.6 to 8.6. More preferably, the sodium chloride concentration is 100 to 150 mM; more preferably, the magnesium chloride concentration is 1.0 to 1.5 mM; more preferably, the Tris-HCl concentration is 20 to 25 mM; and more preferably, the poloxamer 188 concentration is 0.003% to 0.005% (w / v). More preferably, the pH of the pharmaceutical composition is 8.2 to 8.6.

[0036] In some embodiments, disclosed herein is a pharmaceutical composition of a recombinant adeno-associated virus vector, comprising rAAV5-hFIX, 1 to 500 mM sodium chloride, 0.1 to 100 mM magnesium chloride, 1 to 50 mM Tris-HCl, 10 to 500 mM sucrose or sorbitol, and 0.001% to 0.1% (w / v) poloxamer 188, wherein the pH of the pharmaceutical composition is 7.0 to 9.0. The pharmaceutical composition of a recombinant adeno-associated virus vector can be used for the treatment of hemophilia B. Preferably, the sodium chloride concentration is 10 to 300 mM. Preferably, the magnesium chloride concentration is 0.5 to 5 mM. Preferably, the Tris-HCl concentration is 10 to 25 mM. Preferably, the sucrose or sorbitol concentration is 50 to 300 mM. Preferably, the concentration of poloxamer 188 is 0.001% to 0.01% (w / v). Preferably, the pH of the pharmaceutical composition is 7.6 to 8.6.

[0037] In some embodiments, disclosed herein is a pharmaceutical composition of a recombinant adeno-associated virus vector comprising 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, and 0.001%-0.005% (w / v) poloxamer 188, wherein the pH of the pharmaceutical composition is 7.6-8.6. The pharmaceutical composition of a recombinant adeno-associated virus vector can be used for the treatment of hemophilia B. Preferably, the sodium chloride concentration is 100-150 mM. Preferably, the magnesium chloride concentration is 1.0-1.5 mM. Preferably, the Tris-HCl concentration is 20-25 mM. Preferably, the poloxamer 188 concentration is 0.003%-0.005% (w / v). Preferably, the pH of the pharmaceutical composition is 8.2 to 8.6.

[0038] In some embodiments, the compositions described herein are used to immunize a subject (e.g., a human). The level of immunity to the selected gene can be monitored to determine whether a booster shot is required. After assessing antibody titers in the serum, an optional booster shot may be desired.

[0039] Optionally, the compositions of the present invention can be formulated to include other ingredients such as pharmaceutically acceptable vectors, for example, adjuvants, preservatives, and the like.

[0040] In certain embodiments, the compositions described herein are administered to a subject by intramuscular injection, intravaginal administration, intravenous injection, intraperitoneal injection, subcutaneous injection, intradermal administration, intranasal administration, or oral administration.

[0041] When a therapeutic regimen involves simultaneous administration of one or more adeno-associated viral vectors and / or other components, they may be co-formulated (i.e., in the same mixture or composition) or separately formulated in different compositions. If separately formulated, they may be advantageously administered simultaneously at or near the same site. For example, the components may be administered (e.g., intramuscularly, transdermally, intradermally, subcutaneously) into the same limb ("ipsilateral" administration) or into opposite limbs ("contralateral" administration).

[0042] The dosage of a viral vector may vary from patient to patient, depending primarily on factors such as the disease being treated, the patient's age, weight, and health status. For example, a therapeutically effective adult or veterinary dose of a viral vector is typically 1×10 5 ~1×10 15 virus particles, e.g., 1 x 10 8 ~1×10 13 virus particles (e.g., 1 x 10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 Alternatively, viral vectors typically contain 1 x 10 8 ~1×1013 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 The dose can be administered at a dose of approximately 1 x 10 vg / ml. The dose may vary depending on the size of the animal and the route of administration. For example, a suitable human or veterinary dose for intramuscular injection (for an animal weighing approximately 80 kg) is approximately 1 x 10 vg / ml for a single site. 9 ~Approx. 5×10 12 10 particles / mL. Optionally, multiple administration sites can be used. In some embodiments, a suitable human or veterinary dose is about 1 x 10 particles / mL for oral formulations. 11 ~Approx. 1×10 15 The concentration can range from 0.01 to 0.01 particles / mL.

[0043] To develop a formulation for long-term storage stability of recombinant adeno-associated virus vector liquid formulations and ensure product quality throughout their shelf life (tentatively 24 months), we conducted formulation studies, including pH screening, additive screening, and surfactant screening. The final formulation for the recombinant adeno-associated virus vector liquid formulation was finalized. Literature has shown that this virus is prone to aggregation, has large particle size, and is easily inactivated by high temperatures after repeated freezing and thawing. The formulation screening process used repeated freezing and thawing, high temperatures of 25°C or 40°C, and other investigational conditions to accelerate changes in the physical and biochemical properties of the virus in each formulation, thereby screening for the most stable formulation.

[0044] The stability of recombinant adeno-associated viral vector pharmaceutical compositions can be characterized by observing or detecting the appearance of clarity, osmolality, fluorescent 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 in vitro activity (as indicated by the expression of hFIX).

[0045] Fluorescent quantitative polymerase chain reaction (qPCR) is a method that uses fluorescent chemicals to induce DNA amplification and measure the total amount of product after each cycle of PCR. It is a method for quantitatively analyzing specific DNA sequences in a test sample using internal or external reference methods. qPCR can be used to quantify adeno-associated virus vectors.

[0046] Insoluble particles can be measured using an insoluble particle detector on a microscope.

[0047] Size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) detects the purity of recombinant adeno-associated viral vector pharmaceutical compositions.

[0048] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the most commonly used protein expression analysis technique in polyacrylamide gel electrophoresis. The principle of this technique is to separate proteins in the electrophoresis gel according to the different molecular weights of the proteins in the sample. SDS-PAGE is usually used to detect protein expression levels (expression level, expression distribution) and analyze the purity of target proteins.

[0049] The in vitro expression level of active hFIX was measured by measuring the hFIX protein content in a recombinant adeno-associated virus vector pharmaceutical composition using a double-antibody sandwich ELISA. A specific antibody (i.e., primary antibody, capture antibody) was coated onto a solid support to form a solid-phase antibody. After blocking and washing, the test sample was added. After incubation and washing, a biotin-labeled antibody (i.e., secondary antibody, detection antibody) was added. After incubation and washing, streptavidin peroxide (SA-HRP) was added. After incubation and washing, a quantitative colorimetric substrate was added, and the hFIX protein content in the test sample was determined by colorimetric analysis. [Brief explanation of the drawings]

[0050] [Figure 1A] The experimental results of Examples 1 to 3 are shown. [Figure 1B] The experimental results of Examples 4 to 6 are shown, where N / A means not applicable or not tested. [Figure 1C] The experimental results of Examples 7 to 9 are shown, where N / A means not applicable or not tested. [Figure 1D] 1 shows the experimental results of Example 10, where N / A means not applicable or not tested. [Figure 2] FIG. 2 shows the experimental results of investigating the long-term stability of the liquid formulations of recombinant adeno-associated virus vectors of Examples 1 to 10 when stored at 2 to 8°C. DETAILED DESCRIPTION OF THE INVENTION

[0051] Detailed Description The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, after reading the disclosure of the present invention, those skilled in the art can make various changes or modifications to the present invention, and it should be understood that these equivalents are also included in the scope defined by the appended claims of this application.

[0052] Example 1 The liquid formulation of the recombinant adeno-associated virus vector consisted of rAAV5-hFIX, 100 mM sodium chloride, 1.5 mM magnesium chloride, 15 mM Tris-HCl, 50 mM sucrose, and 0.003% (w / v) poloxamer 188. The pH of the formulation was 7.6.

[0053] Example 2 The liquid formulation of the recombinant adeno-associated virus vector consisted of rAAV5-hFIX, 150 mM sodium chloride, 1.0 mM magnesium chloride, 20 mM Tris-HCl, 50 mM sucrose, and 0.005% (w / v) poloxamer 188. The pH of the formulation was 8.2.

[0054] Example 3 The liquid formulation of the recombinant adeno-associated virus vector consisted of rAAV5-hFIX, 200 mM sodium chloride, 2.0 mM magnesium chloride, 25 mM Tris-HCl, 100 mM sucrose, and 0.001% (w / v) poloxamer 188. The pH of the formulation was 8.6.

[0055] Example 4 The liquid formulation of the recombinant adeno-associated virus vector consisted of rAAV5-hFIX, 150 mM sodium chloride, 1.0 mM magnesium chloride, 20 mM Tris-HCl, 50 mM sorbitol, and 0.005% (w / v) poloxamer 188, and the pH of the formulation was 8.2.

[0056] Example 5 The liquid formulation of the recombinant adeno-associated virus vector consisted of rAAV5-hFIX, 200 mM sodium chloride, 1.0 mM magnesium chloride, 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate, 50 mM sucrose, and 0.005% (w / v) poloxamer 188. The pH of the formulation was 6.5.

[0057] Example 6 The liquid formulation of the recombinant adeno-associated virus vector consisted of rAAV5-hFIX, 200 mM sodium chloride, 1.0 mM magnesium chloride, 10 mM disodium hydrogen phosphate / sodium dihydrogen phosphate, 50 mM sucrose, and 0.005% (w / v) poloxamer 188. The pH of the formulation was 7.5.

[0058] Example 7 The liquid formulation of the recombinant adeno-associated virus vector consisted of rAAV5-hFIX, 150 mM sodium chloride, 1.0 mM magnesium chloride, 20 mM Tris-HCl, and 0.005% (w / v) poloxamer 188. The pH of the formulation was 8.2.

[0059] Example 8 The liquid formulation of the recombinant adeno-associated virus vector consisted of rAAV5-hFIX, 150 mM sodium chloride, 10 mM magnesium chloride, 20 mM Tris-HCl, 50 mM sucrose, and 0.005% (w / v) poloxamer 188. The pH of the formulation was 8.2.

[0060] Example 9 The liquid formulation of the recombinant adeno-associated virus vector consisted of rAAV5-hFIX, 150 mM sodium chloride, 1.0 mM methionine, 20 mM Tris-HCl, 50 mM sucrose, and 0.005% (w / v) poloxamer 188. The pH of the formulation was 8.2.

[0061] Example 10 The liquid formulation of the recombinant adeno-associated virus vector consisted of rAAV5-hFIX, 150 mM sodium chloride, 1.0 mM magnesium chloride, 20 mM Tris-HCl, 50 mM sucrose, and 0.005% (w / v) polysorbate 80. The pH of the formulation was 8.2.

[0062] The experimental results of the stability studies of Examples 1 to 10 are shown in Figures 1A, 1B, 1C, and 1D. The recombinant adeno-associated virus vector liquid formulations of Examples 1 to 4 exhibited better stability and fewer insoluble particles. Furthermore, they exhibited the highest in vitro expression of active hFIX. Compared to the liquid formulation of Example 2, the liquid formulation of Example 5 had a pH of 6.5, and the SEC-HPLC data of Example 5 indicated lower purity and lower in vitro expression of active hFIX. Compared to the liquid formulation of Example 1, the liquid formulation of Example 5 had a similar pH value, but a different buffer, disodium hydrogen phosphate / sodium dihydrogen phosphate, was used. The SEC-HPLC data of Example 7 indicated lower purity and lower in vitro expression of active hFIX. Compared to the liquid formulation of Example 2, the liquid formulation of Example 7 did not contain sucrose as a stabilizer. The SEC-HPLC data of Example 7 indicated slightly lower purity and lower in vitro expression of active hFIX. Compared with the liquid formulation of Example 2, the liquid formulation of Example 8 had an increased magnesium chloride concentration of 10 mM, and the SEC-HPLC data for Example 8 indicated slightly lower purity and reduced expression of in vitro active hFIX. Compared with the liquid formulation of Example 2, the liquid formulation of Example 9 had magnesium chloride replaced with methionine, and the SEC-HPLC data for Example 9 indicated slightly lower purity and reduced expression of in vitro active hFIX. Compared with the liquid formulation of Example 2, the liquid formulation of Example 10 had magnesium chloride replaced with methionine, and the SEC-HPLC data for Example 10 indicated slightly lower purity and reduced expression of in vitro active hFIX.

[0063] Example 11 For long-term stability testing, the recombinant adeno-associated virus vector liquid formulation of Example 2 was stored at 2-8°C, and important indicators such as the product's in vitro activity and genome titer were measured after 0, 1, 2, 3, 6, 9, and 12 months. No significant decline was observed. This indicates that the recombinant adeno-associated virus vector liquid formulation of Example 2 can be kept stable for long periods at 2-8°C, which is more advantageous than the current storage condition that requires frozen storage, facilitating product transportation and clinical use and reducing investment and usage costs for all parties involved.

[0064] 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 methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0065] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0066] As used herein, when referring to a measurable value such as an amount, duration, etc., the term "about" is meant to include variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value, whereby these variations are suitable for practicing the disclosed methods.

[0067] 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 as carriers, stabilizers, diluents, adjuvants, dispersing agents, suspending agents, thickening agents, and / or additives. Pharmaceutical compositions facilitate administration of the active ingredient to an organism. Various techniques exist in the art for administering compounds, including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

[0068] As used herein, the term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, additives, solvents, or encapsulating materials that are involved in delivering or transporting a compound(s) of the invention into or to a subject so that the compound(s) can perform their intended function. Typically, such compounds are transported or carried from one organ or part of the body to another. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Examples of substances 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; powdered tragacanth; malt; gelatin; talc; additives 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; glycols such as propylene glycol; glycerin, sorbitol, mannitol, polyethylene Examples of suitable carriers include polyols such as glycols; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; diluents; granulating agents; lubricants; binders; disintegrating agents; wetting agents; emulsifiers; coloring agents; release agents; coating agents; sweeteners; flavorings; preservatives; antioxidants; plasticizers; gelling agents; thickening agents; hardening agents; precipitating agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic compatible substances used in pharmaceutical formulations, or combinations thereof. As used herein, "pharmaceutically acceptable carriers" also include any coatings, antibacterial agents, antifungal agents, and absorption delaying agents 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.

[0069] Although the present invention has been described in detail with reference to specific embodiments, it should be understood that functionally equivalent variations are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

[0070] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

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

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

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

5. 5. The rAAV vector pharmaceutical composition of claim 1, wherein the concentration of sodium chloride is 100 to 150 mM.

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

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

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

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

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

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

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

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

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

6.

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

6.

16. 16. A method for treating a disease of interest in a subject, comprising administering to the subject a pharmaceutical composition described in any one of claims 1 to 15, wherein the rAAV encodes a gene of interest that treats, otherwise ameliorates, prevents, or slows the progression of the disease of interest.

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

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

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

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

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

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

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

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

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

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

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

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

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

6.

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

6.

32. 32. A method for treating hemophilia B in a subject, comprising administering to the subject a pharmaceutical composition described in any one of claims 18 to 31, wherein the rAAV encodes the gene hFIX intended to treat hemophilia B.

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