Gene delivery vehicle pharmaceutical formulations

Abstract

The present invention relates to a miRNA-containing formulation having improved stability for the treatment of diseases including neurodegenerative diseases such as spinocerebellar ataxia type 3.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy. Further, the present invention relates to the field of interfering RNA and / or microRNA (miRNA). In particular, the present invention relates to gene therapy comprising such miRNA and, in particular, to pharmaceutical formulations having improved stability for the treatment of diseases, including neurodegenerative diseases such as spinocerebellar ataxia.

Background Art

[0002] [Gene Therapy] With the elucidation of DNA as a carrier of genetic information and thus as a cause of genetic diseases, therapies are envisioned in which mutant and damaged genes can be replaced or at least silenced. Many genes and / or other nucleic acid sequences are currently confirmed to play a role in (gene) diseases. If a mutant gene can be replaced by a healthy gene or a gene expressing an abnormal (sometimes toxic) product can be silenced, the disease can be treated, and in some cases cured, at the molecular level. Gene therapy provides a promising concept, especially for diseases caused by mutations in single genes.

[0003] However, delivering the nucleic acid of interest to the cells that need to be targeted is not an easy task. Many (viral) delivery systems have been studied, and all of them have advantages and disadvantages. One of the viral delivery vehicles used in gene therapy is adeno-associated virus (AAV).

[0004] [AAV] AAV has a single-stranded DNA genome of approximately 4.8 kilobases (kb). AAV belongs to the Parvoviridae family and is dependent on co-infection with other viruses, particularly adenoviruses, for replication. The genome contains Rep (replication) and Cap (capsid) genes. These coding sequences are flanked by inverted repeat sequences (ITRs) necessary for genome replication and packaging. The Rep genes encode four proteins (Rep78, Rep68, Rep52, and Rep40) that replicate and facilitate the packaging of the viral genome, while Cap expression generates viral capsid proteins (VP; VP1, VP2, VP3) and forms the outer capsid shell.

[0005] Regarding gene therapy, the viral DNA of AAV is almost completely removed. Recombinant AAV (rAAV) for gene therapy is formed by a protein capsid containing the target nucleic acid and the transgene to be delivered to the target cell. The target nucleic acid is flanked by the ITR of the AAV. The ITR-flanking transgene encoded by rAAV may form a circular concatemer that remains in the nucleus of the transcellular cell as an episome. Because this episome remains almost episomal, the expression of the AAV delivery nucleic acid sequence may weaken over time once the target cell replicates, and if it has replicated. This dilution may not generally apply to postmittal cells such as nerve cells, which are the target cells of many neurodegenerative diseases. An overview of AAV vectors for gene therapy is provided in Nasoetal, Biodrugs 2017 (pp. 317-334).

[0006] [miRNA] RNA interference (RNAi) is a naturally occurring mechanism involving sequence-specific downregulation of messenger RNA (mRNA). This downregulation of mRNA results in a reduction in the amount of protein expressed. RNA interference is induced by double-stranded RNA. One strand of the double-stranded RNA is substantially or completely complementary to its target mRNA. This strand is called the guide strand. The mechanism of RNA interference involves the incorporation of the guide strand into the RNA-induced silencing complex (RISC). This complex is a high-turnover complex that can bind to its target mRNA via complementary base pairs of the guide strand. Upon binding to its target mRNA, the mRNA may be cleaved or its translation efficiency reduced. Since its discovery, RNA interference has been widely used to knock down specific target genes. The triggers used to induce RNA interference involve the use of small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs). Furthermore, naturally occurring RNAi-inducing molecules, so-called microRNAs (miRNAs), are being used to create artificial (genetically modified) miRNAs that mimic their natural counterparts. These approaches share the commonality of providing substantially double-stranded RNA molecules designed to target the optimal mRNA. RNAi-based therapeutic approaches that utilize the sequence-specific nature of RNAi are under development, and some are currently in clinical trials (see, in particular, Davidson and McCray, Nature Reviews-Genetics, 2011; Vol. 12; 329-340).

[0007] [Spinocerebellar ataxia type 3 (SCA3)] Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is an autosomal dominant monogenic, fatal disorder. The disease is characterized by progressive degeneration of brain regions caused by the expansion of the CAG repeat sequence in the human attaxin 3 gene, also known as the ATXN3 gene (OMIM:607047, reference sequence Homo sapiens attaxin 3 (ATXN3) on chromosome 14, NCBI reference sequence:NG_008198.2). A cytosine-adenine-guanine (CAG) repeat region is present in the 3' region of the gene. This CAG region is within a frame, resulting in the production of the attaxin 3 protein, which contains a poly-Q region and glutamine repeat sequences. Healthy or non-symptomatic individuals may have up to 44 CAG repeat sequences in the ATXN3 gene. It is known that individuals with the disease exhibit elongation, and these can have 52-86 or more CAG repeat sequences. Individuals with 45-51 CAG repeat sequences should exhibit symptoms of incomplete penetrance of the disease. As a result of this elongation, an ataxin 3 protein with an elongated polyQ region and the full length of the CAG repeat sequence is produced, and therefore, the polyQ region within ataxin 3 may correlate with disease progression; that is, the longer the region, the higher the disease progression in general.

[0008] Ataxin 3 proteins with extended poly-Q domains acquire toxic properties (acquisition of toxic function), and the formation of neuronal aggregates in the brain is a prominent neuropathological feature. Neuropathological studies have revealed widespread neuronal loss in various regions, including the cerebellum, thalamus, midbrain, pons, medulla oblongata, and spinal cord, in SCA3 patients (Riess et al., Cerebellum 2008). Although a wide range of pathological conditions have been reported, it is commonly understood that the main pathological conditions are located in the cerebellum and brainstem (Eichler et al., AJNR Am J Neuroradiol, 2011). The disease has complete penetration, meaning that if a person has more than 52 CAG extensions, they will inevitably develop the disease and have a 50% chance of inheriting it to their offspring.

[0009] [Challenges in formulation and administration] The use of AAV vectors to deliver microRNA directly to a patient's brain for ATXN3 gene knockdown could represent a highly groundbreaking and promising approach to SCA3 treatment. However, direct administration to the patient's brain or spinal cord imposes special challenges to the formulation and stability of the appropriate pharmaceutical product, including the need to avoid the undesirable formation of aggregates or lumps in the formulation, which could be harmful to the patient. For example, such a formulation may be stored frozen and then thawed before use. Aggregates or lumps can likely form during or after thawing of the formulation, and before administration of the formulation, but also during the period after thawing. Therefore, it would be highly desirable to provide a groundbreaking formulation of AAV vectors for the treatment of spinocerebellar ataxia type 3 or other diseases that eliminates or substantially eliminates aggregates or lumps in order to extend the shelf life. [Overview of the project]

[0010] [Overview of the prefecture] In the first embodiment, buffer; Adeno-associated virus vectors containing transgenes encoding microRNAs; Isotonic agent; Freeze-protecting substances; and surfactant An isotonic pharmaceutical preparation containing the above is provided.

[0011] In some embodiments, the formulation has an osmolality of 250-330 milliosmoles (mOsm) / kg and a pH of 5-8.

[0012] In some embodiments, the formulation is substantially free of visible particles.

[0013] In some embodiments, the adeno-associated virus vector having a transgene encoding a microRNA targets ATXN mRNA, for example, ATXN3 mRNA. In some embodiments, the adeno-associated virus vector comprises AAV2 serotype, AAV5 serotype, AAV9 serotype, hybrid AAV serotype, or a combination thereof, preferably comprising AAV5 serotype or AAV9 serotype, more preferably AAV5 serotype.

[0014] In some embodiments, the concentration of the adeno-associated virus vector is at least 1E13 gc / ml, for example, 3E13 gc / ml. In some embodiments, the concentration of the adeno-associated virus vector is 0.5E13 to 6E13 gc / ml, for example, 1E13 to 5E13 gc / ml.

[0015] In some embodiments, the cryoprotective substance is a carbohydrate selected from the group consisting of trehalose, sucrose, glucose, dextran, and combinations thereof; present in amounts up to 8% (w / v).

[0016] In some embodiments, the isotonic agent is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, and combinations thereof; present in an amount of 5 mM to 150 mM. In some embodiments, the surfactant is a nonionic surfactant; present in an amount of 0.001 to 0.1% (v / v), preferably 0.003 to 0.08% (v / v), more preferably 0.005 to 0.02% (v / v), for example, about 0.01% (v / v).

[0017] In some embodiments, the buffer is selected from the group consisting of artificial cerebrospinal fluid (aCSF), acetate buffer, citrate buffer, phosphate buffer, HEPES, Tris, and combinations thereof.

[0018] In some embodiments, the formulation remains stable at room temperature (15°C to 25°C) for at least 12 hours after melting without forming aggregates or lumps.

[0019] In a second aspect, there is provided a formulation of the present invention for use in the treatment of spinocerebellar ataxia (SCA).

[0020] In some embodiments, the formulation is administered by intrathecal (CSF) delivery.

[0021] In some embodiments, the formulation is administered to the cisterna magna of the patient.

[0022] [Detailed Description of the Invention] The present invention endeavors to provide a new pharmaceutical formulation in which in-process aggregate or clot formation is reduced and which essentially contains no visually observable particles for a long time at room temperature after thawing. The present invention also endeavors to provide a pharmaceutical formulation containing one or more excipients that are substantially isotonic with human cerebrospinal fluid (CSF) in order to improve administration to the patient's brain. The present invention also endeavors to provide a method for treating spinocerebellar ataxia type 3 (SCA3) by administering the formulation of the present invention to the patient's brain.

[0023] In one aspect of the present invention, there is provided an isotonic pharmaceutical formulation comprising a transgene encoding a microRNA, a tonicity agent, a cryoprotectant, a surfactant, together with a buffer and an adeno-associated virus vector.

[0024] Thus, unexpectedly, it has been found that the addition of a cryoprotectant and a surfactant to the formulation helps to enhance the stability of the pharmaceutical product, particularly at high concentrations. The formulation can maintain a stable state without substantially forming aggregates or clots for a period of at least about 6 months, preferably at least about 12 months, more preferably at least about 24 months, for example at least about 36 months, at a storage temperature of -65°C or lower. The formulation can also maintain a stable state at room temperature (15 - 25°C) without substantially forming aggregates or clots for a long time, preferably at least about 12 hours, more preferably at least about 24 hours, after thawing.

[0025] In some embodiments, an adeno-associated virus vector having a transgene encoding a microRNA can target ATXN mRNA, thereby potentially enabling the treatment of spinocerebellar ataxia with its formulation. For example, an adeno-associated virus vector having a transgene encoding a microRNA can target the target ATXN3, thereby reducing ATXN3 mRNA and protein levels in the treatment of spinocerebellar ataxia type 3. Thus, in some embodiments, an adeno-associated virus vector having a transgene encoding a microRNA targets ATXN mRNA. In a further embodiment, an adeno-associated virus vector having a transgene encoding a microRNA targets ATXN3 mRNA.

[0026] In some embodiments, the adeno-associated virus vector comprises an AAV2 serotype, an AAV5 serotype, an AAV9 serotype, a hybrid AAV serotype, or a combination thereof. Said serotypes have been found to be particularly preferred for the treatment of spinocerebellar ataxia type 3. In some embodiments, the adeno-associated virus vector comprises the AAV9 serotype.

[0027] In some embodiments, the adeno-associated virus vector comprises the AAV5 serotype.

[0028] In some embodiments, the adeno-associated virus vector comprises a hybrid AAV serotype. As an example, the hybrid AAV serotype can be a hybrid AAV2 / AAV5; AAV2 / AAV9; or AAV5 / AAV9 serotype.

[0029] An example of a method and means for delivering miRNA to target cells is provided in WO 2020 / 104469 A1, which is incorporated herein by reference.

[0030] In some embodiments, the concentration of the adeno-associated virus vector is at least 1E13 gc / mL; preferably at least 2E13 gc / mL; more preferably at least 1E14 gc / mL; for example, at least 3E14 gc / mL. In some embodiments, the concentration of the adeno-associated virus vector is at least 6E14 gc / mL. In some embodiments, the concentration of the adeno-associated virus vector is 4E14 to 5E14 gc / mL.

[0031] In some embodiments, the formulation contains AAV5 having a transgene encoding a microRNA, and the concentration of AAV5 is at least 6E14 gc / mL.

[0032] In some embodiments, the formulation contains AAV5 having a transgene encoding a microRNA, and the concentration of AAV5 is at least 3E14 gc / mL.

[0033] In another example, the formulation contains AAV5 having a transgene encoding a microRNA, and the concentration of AAV5 is at least 6E14 gc / mL.

[0034] In yet another embodiment, the formulation contains AAV5 having a transgene encoding icloRNA, with an AAV5 concentration of 7.5E14 gc / mL. When the volume of administration is limited, stable, high-concentration formulations may be particularly useful for delivering a sufficient number of genomic copies of the drug. One example of such application is when injection into a large intestine (CM, whose average volume is only about 1 ml) is required.

[0035] In some embodiments, the formulation contains AAV9 having a transgene encoding a microRNA, and the concentration of AAV9 is at least 6E14 gc / mL.

[0036] In one example, the formulation contains AAV9 having a transgene encoding microRNA, and the concentration of AAV9 is 3E14 gc / mL.

[0037] In another example, the formulation contains AAV9 having a transgene encoding microRNA, and the concentration of AAV9 is 6E14 gc / mL.

[0038] In yet another example, the formulation contains AAV9 having a transgene encoding microRNA, and the concentration of AAV9 is 7.5E14 gc / mL.

[0039] The formulation may also have a pH value compatible with human cerebrospinal fluid. For example, the formulation may have a pH of about 5 or higher, preferably about 7 or higher. In some embodiments, the formulation may have a pH of 5 to 8, preferably 6 to 8, more preferably 6.5 to 8, and even more preferably 7 to 8, for example, 7.1 to 7.7. In some embodiments of the present invention, the formulation has a pH value of 7.3.

[0040] Tonicity is a measure of the effective osmotic pressure that a liquid formulation may exert, and it depends primarily on the number of dissolved particles in the solution. Osmotic pressure is an important factor affecting effective biological cells. Hypertonicity is the presence of a solution that causes cell contraction. Hypotonicity is the presence of a solution that causes cell swelling. Isotonicity is the presence of a solution that does not cause a change in the volume of the cell. When biological cells are in a hypotonic environment, the inside of the cell stores water, and this water flows through the cell membrane into the cell, where it expands. In the case of mammalian cells, this can cause cell lysis, and therefore tonicity is important when fragile cells such as nerve cells or brain cells are exposed to the composition. Therefore, isotonic agents are added to injectable formulations (preparations) to prevent osmotic shock at the injection site during administration, thereby reducing local irritation or even damage to the CNS. Common isotonic agents are thus excipients used to adjust tonicity and are well known in the art.

[0041] In some embodiments, the isotonic agent comprises a metal chloride of Group 1 or Group 2 of the periodic table, preferably selected from the group consisting of NaCl, KCl, CaCl2, MgCl2, and combinations thereof. Preferably, the isotonic agent comprises NaCl. In one embodiment of the present invention, the isotonic agent comprises a combination of NaCl and KCl. The isotonic agent helps stabilize the formulation. Preferably, the isotonic agent is present in an amount of 5 to 150 mM, preferably 25 to 125 mM, more preferably 75 to 125 mM, for example, 90 mM. For example, the isotonic agent may contain at least 75 mM NaCl. In some embodiments, the isotonic agent is present in an amount of 90 mM. For example, the isotonic agent may contain a combination of NaCl and KCl and may be present in an amount of 90 mM. In some embodiments, the isotonic agent comprises at least 80 mM NaCl and at least 2 mM KCl. For example, the isotonic agent may include 3 mM KCl and 87 mM NaCl or 82 mM NaCl. Therefore, in some embodiments, the isotonic agent is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, and combinations thereof; it is present in an amount of 5 to 150 mM.

[0042] Isotonic agents are primarily used to establish the desired weight osmolality of the formulation. Preferably, the formulation is substantially isotonic with respect to human cerebrospinal fluid. In particular, the formulation may have an osmolality of 250 to 330 milliosmoles / kg. In some preferred embodiments, the formulation has an osmolality of 270 to 320 milliosmoles / kg. For example, the formulation has an osmolality of 280 milliosmoles / kg. Thus, in one embodiment, the formulation has an osmolality of 250 to 330 milliosmoles / kg and a pH of 5 to 8.

[0043] Cryoprotective substances are additives that help minimize the denaturation of polypeptides / biopharmaceuticals when polypeptides are present in a composition. Cryoprotective substances have been found to be particularly advantageous in providing stable pharmaceuticals by eliminating, or substantially eliminating, the formation of aggregates or clumps. In preferred embodiments, the cryoprotective substance is a carbohydrate or polymer. As used herein, the term “carbohydrate” means both natural saccharides and polyols that can be derived from saccharides. Examples of suitable carbohydrates are trehalose, sucrose, glucose, dextran, and combinations thereof. Thus, in some embodiments, the cryoprotective substance is a carbohydrate selected from the group consisting of trehalose, sucrose, glucose, dextran, and combinations thereof. Preferably, the cryoprotective substance is trehalose or dextran. Trehalose is a disaccharide formed by 1,1-glycosidic bonds between α-glucose units, and sucrose is a disaccharide that is O-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside. Optionally, other disaccharides may be used instead. Glucose is D-glucose, and optionally other monosaccharides may be used instead. In a more preferred embodiment of the present invention, the cryoprotective substance is trehalose. In another embodiment of the present invention, the cryoprotective substance is dextran, e.g., dextran 6000. In some embodiments, the cryoprotective substance is present in the formulation in amounts up to 8% (w / v), preferably 2-5% (w / v), e.g., 2-3% (w / v). In some embodiments, the cryoprotective substance is trehalose, present in the formulation in amounts of 2-6% (w / v), e.g., 3% (w / v). In some embodiments, the cryoprotective substance is dextran, e.g., dextran 6000, present in amounts of 1-3% (w / v), e.g., 2-2.5% (w / v). Therefore, in some embodiments, the cryoprotective substance is a carbohydrate selected from the group consisting of trehalose, sucrose, glucose, dextran, and combinations thereof; present in amounts up to 8% (w / v).

[0044] Surfactants and their properties are well known, and surfactants generally contain at least one polar head group and at least one nonpolar or hydrophobic tail. The surfactants used herein preferably contain a single tail and preferably a single head group. They are preferably neutrally charged, meaning that the surfactant has no effective charge under its usage conditions. Alkylated saccharides, in particular polysorbates, and more specifically polysorbate 20, are most preferred for use in the present invention. In some embodiments, the surfactant is a nonionic surfactant. Preferably, the surfactant is a polyethoxylated nonionic surfactant, such as polysorbate. In preferred embodiments of the present invention, the surfactant is polysorbate 20.

[0045] Surfactants have been found to be particularly advantageous for improving stability and reducing the formation of aggregated or clumped particles in the formulation. Optionally, surfactants may be present in amounts of 0.001-0.1% (v / v), preferably 0.03-0.08% (v / v), and more preferably 0.005-0.02% (v / v). In some embodiments, surfactants are present in the formulation in amounts of 0.005-0.015% (v / v). Therefore, in some embodiments, the surfactant is a nonionic surfactant and is present in amounts of 0.0001-0.1% (v / v), preferably 0.03-0.08% (v / v), and more preferably 0.005-0.02% (v / v). In some embodiments, surfactants are present in the formulation in amounts of 0.005-0.015% (v / v), for example, about 0.01% (v / v). For example, the surfactant is polysorbate 20, which is present in an amount of approximately 0.01% (v / v).

[0046] Buffers are known in the art and help maintain the pH of a composition stably within a given range. Buffers are often buffer salts. Buffers can be selected from the group consisting of artificial cerebrospinal fluid (aCSF), phosphate buffer, acetate buffer, citrate buffer, HEPES buffer, Tris buffer, and combinations thereof. In some embodiments, the buffer may be phosphate buffer, for example, phosphate buffer with a pH of 6.5 to 7.5. In some embodiments, the buffer may be acetate buffer, for example, acetate buffer with a pH of 5 to 5.5. In some embodiments, the buffer may be citrate buffer, for example, citrate buffer with a pH of 5.5 to 6.5. In some embodiments, the buffer may be Tris buffer, for example, Tris buffer with a pH of 7.5 to 8. In some embodiments, the buffer may be HEPES buffer, for example, HEPES buffer with a pH of 7 to 8. In some embodiments, the buffer may be aCSF, for example, aCSF with a pH of 7.0 to 7.6. For example, aCSF may have the following final ion concentrations (mM): Na 150; K 3.0; Ca 1.4; Mg 0.8; P 1.0; Cl 155. Preferably, the buffer is phosphate buffer or Tris; for example, the buffer is 10 mM phosphate buffer at pH 7.3.

[0047] Buffers, isotonic agents, cryoprotective substances, and other functionally defined components of a composition, such as surfactants, can be individual substances or mixtures. For example, a buffer may be a single phosphate such as Na2HPO4, but it may also be a mixture of substances, such as a mixture of Na2HPO4 and KH2PO4. Similarly, an isotonic agent may be a single substance such as NaCl, but it may also be a mixture, such as a combination of NaCl and KCl. Throughout this document, when only a single substance is specified with respect to its functional definition, preferably no other substances are present with respect to that same function. The same applies when multiple substances are specified, in which case the specified substances are included with respect to their same function.

[0048] For example, some compounds such as phosphates can act as buffers, but they can also act as isotonic agents. If a compound is present as a functionally defined component of a composition, as used herein, then it is preferably not considered to satisfy any of the conditions of any further functionally defined components. Therefore, if one or more of acetates, citrates, HEPES, TRIS, or phosphates are present, they are preferably considered only as buffers. If one or more of NaCl, KCl, MgCl2, CaCl2, or mixtures thereof are present, they are preferably considered only as isotonic agents. If one or more of trehalose, dextran, sucrose, glucose, or poly(ethylene glycol) are present, they are preferably considered only as cryoprotective substances. If one or more nonionic surfactants are present, they are preferably considered only as surfactants.

[0049] In a particularly preferred embodiment, the formulation is: • Adeno-associated virus vector containing a transgene encoding a microRNA that targets ATXN3 mRNA; • Approximately 5-30 mM phosphate buffer with a pH of 7-8; • At least approximately 75 mM NaCl; ·About 2~5mM KCl; • Trehalose content: approximately 2-4% (w / v); ·PS-20 approx. 0.005~0.015%(v / v) It includes, consists of, or is essentially composed of.

[0050] For example, the formulation is: • Adeno-associated virus vector containing a transgene encoding a microRNA that targets ATXN3 mRNA; • Approximately 9 mM Tris at a pH of approximately 7.3; • Approximately 82 mM NaCl; ·About 3mM KCl; • Trehalose approximately 3% (w / v); • PS-20 (Tween20) approximately 0.01% (v / v) It may include, may consist of, or may essentially consist of.

[0051] For example, the formulation is: • Adeno-associated virus vector containing a transgene encoding a microRNA that targets ATXN3 mRNA; • Approximately 9 mM Tris at a pH of approximately 7.3; • Approximately 87 mM NaCl; ·About 3mM KCl; • Trehalose approximately 3% (w / v); • PS-20 (Tween20) approximately 0.01% (v / v) It may include, may consist of, or may essentially consist of.

[0052] In some embodiments, the formulation is substantially free of visible particles. Thus, as determined and characterized in accordance with the US Pharmaceutical Convention (USP) Chapter 790, “VISIBLE PARTICULATES IN INJECTIONS” guidance of 2014, relating to parenteral drugs “essentially free” of visible particulate matter (USP 790), incorporated herein by reference, this formulation is essentially free of particles visible to the naked human eye. Visible particles may be signs of aggregation, coagulation, and / or disintegration of AAV particles, which may be advantageous in demonstrating a particularly stable formulation free from substantial aggregation or clumping. Preferably, the formulation also includes limited subvisible particles, as determined and characterized in accordance with US Pharmacopeial Convention (USP) Chapter 787, “SUBVISIBLE PARTICULATES MATTER IN THERAPEUTIC PROTEIN INJECTIONS” (USP 787), which is incorporated herein by reference. For example, the number of particles having a diameter of 25 μm or more is 600 or less per vial in the formulation, and the number of particles having a diameter of 10 μm or more is 6000 or less per vial in the formulation. Optionally, the formulation has a Dv90 of less than 50 μm, preferably less than 25 μm, and more preferably less than 10 μm. The Dv90 value represents the percentage (in this case, 90%) of the formulation having a size of less than or equal to the specified value.

[0053] In some embodiments, the formulations of the present invention may be used to treat spinocerebellar ataxia (SCA), for example, spinocerebellar ataxia type 3 (SCA3). In some embodiments, the formulations are administered by intracerebrospinal fluid (CSF) delivery. For example, the formulations are administered by intracerebrospinal fluid delivery, such as intracisional delivery. Optionally, the formulations may be administered by direct injection into the brain, by repeated injections in the cisterna magna, by slow injection in the cisterna magna, or by a combination of direct injection into the brain and injection in the cisterna magna. In some embodiments, the formulations may be administered by injection in the cisterna magna. In some embodiments, the formulations may be administered by a single injection in the cisterna magna. For example, the formulations may be administered by a 5-minute (single) injection into the cisterna magna (optionally under CT guidance).

[0054] The cisterna magna, or cerebellar-medulla oblongata, is one of the three main openings in the subarachnoid space between the arachnoid and pia mater layers of the meninges surrounding the brain. These openings are collectively called cisterns. The cisterna magna is located between the cerebellum and the posterior aspect of the medulla oblongata. Cerebrospinal fluid produced in the fourth ventricle is drained into the cisterna magna through the lateral and median openings.

[0055] In some embodiments, the formulation is administered at a dose of 5 mL / subject with an adeno-associated virus vector concentration of 3E14 gc / mL. In some specific embodiments, the formulation is administered at a dose of 5 mL / injection site with an adeno-associated virus vector concentration of 3E14 gc / mL. In some embodiments, the formulation is administered at a dose of 4E14-5E15 genome copies, preferably 8E14-2E15 genome copies, more preferably 1E15-2E15 genome copies per subject. For example, the formulation is administered at a dose of 1.5E15 genome copies per subject.

[0056] In some embodiments, the formulations of the present invention may be administered with a contrast agent that can be visualized by magnetic resonance imaging (MRI). This may be advantageous when it is desired to monitor the patient's brain by MRI. Alternatively, or in addition as another product of the subject, the nucleotide sequence comprising the transgene encoding the microRNA defined herein further comprises the nucleotide sequence encoding a polypeptide that serves as a select marker protein for evaluating cell transformation and expression. Suitable marker proteins for this purpose include, for example, the fluorescent protein GFP, and the select marker genes HSV thymidine kinase (for selection in HAT medium), bacterial hygromycin B phosphotransferase (for selection in hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection in G418), and dihydrofolate reductase (DHFR) (for selection in methotrexate), CD20, and the low affinity nerve growth factor gene. The raw materials for obtaining these marker genes and methods for using them are provided in Sambrook and Russell, see below.

[0057] Furthermore, the nucleotide sequence comprising the transgene as defined above in this specification may include, where deemed necessary, a further nucleotide sequence encoding a polypeptide that can serve as a fail-safe mechanism enabling the healing of a subject from cells transduced with the AAV vector of the present invention. Such a nucleotide sequence, often referred to as a suicide gene, encodes a protein that can convert the prodrug into a toxic substance capable of killing the transgenerated cells expressing it. Suitable examples of such suicide genes include, for example, the Escherichia coli (E. coli) cytosine deaminase gene, or one of the thymidine kinase genes derived from herpes simplex virus, cytomegalovirus, and varicella-zoster virus, in which case ganciclovir can be used as a prodrug to kill the transgenerated cells in the subject (see, e.g., Clairet al., 1987, Antimicrob. Agents Chemother. 31:844-849).

[0058] In one embodiment, the present invention relates to a method for producing a pharmaceutical product for administration to the central nervous system, i) A step of providing a pharmaceutical product in accordance with the present invention; and ii) The process of ali-coating the formulation into an appropriate dosage form. This provides a method that includes [something].

[0059] The manufactured pharmaceutical product is suitable for administration to the central nervous system, as described elsewhere in this specification, for example. Therefore, this method is suitable for producing formulations used according to the present invention, and preferably, the composition thereof is a composition for intracerebrospinal fluid administration. Accordingly, in one embodiment, the formulation used according to the present invention is administered to the cisterna magna of the patient.

[0060] In yet another embodiment of the present invention, the present invention provides a method for treating SCA, particularly SCA3, by administering the formulation of the present invention to a patient's brain.

[0061] [Definition] In this document and in its claims, the verb “to compris” and its conjunctions are used in a non-restrictive sense to mean that the item following the word is included, but items not specifically mentioned are not excluded. Furthermore, the verb “to consist” may be used instead of “to consist essentially of,” meaning that a combination or composition as defined herein may include additional components other than those specifically identified, and such additional components do not alter the inherent characteristics of the invention. Furthermore, references to elements with the indefinite article “a” or “an” do not exclude the possibility of multiple elements being present unless the context explicitly requires the presence of only one or just one of the elements.

[0062] Therefore, the indefinite article "a" or "an" usually means "at least one".

[0063] Whenever the parameters of a substance are considered in the context of this invention, unless otherwise specified, it is assumed that the parameters are determined, measured, or revealed under physiological conditions. These physiological conditions are known to those skilled in the art and include an aqueous solvent system, atmospheric pressure, a pH value of 6–8, a temperature in the range of room temperature to about 37°C (about 20–about 40°C), and appropriate concentrations of buffer salts or other components.

[0064] In relation to the present invention, a decrease or increase in the parameter being evaluated means a change of at least 5% in the value corresponding to that parameter. More preferably, a decrease or increase in the value means a change of at least 10%, more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In the latter case, there may no longer be a detectable value associated with the parameter.

[0065] When the terms "about" or "approximately" are used in relation to a numerical value (e.g., about 10), it is preferable that the value be ±10% of the given value (10), or optionally ±5%.

[0066] Each embodiment defined herein may be combined with others unless otherwise indicated. The present invention has been described above in relation to many embodiments. Those skilled in the art will be able to conceive of obvious variations of some elements of the embodiments. These are covered within the scope of protection defined in the appended claims. All cited patents and references are incorporated herein by reference in their entirety.

[0067] Furthermore, the following terms are used herein and are defined as follows: • "Capsid" - A protein shell surrounding viral DNA, RNA, or microRNA that helps target genetic material to a specific cell type. • "Gene cassette" - A small fragment of DNA, RNA, or microRNA containing a therapeutic gene and instructions to the cell on how to use the gene. • "Vector" - This can mean the DNA, RNA, or microRNA molecule itself, or a carrier construct containing DNA, RNA, or microRNA that is delivered to a cell. • MicroRNA (also called miRNA or siRNA) - Small, single-stranded, non-coding RNA molecules that typically contain the guide strands mentioned above. • "SCA" - Spinocerebellar ataxia. • "Substantially" - As used herein, the term "substantially" is a broad term and should be given its ordinary and common meaning to those skilled in the art (and not limited to any special or customized meaning), and not necessarily, but meaning that it is largely what is specified. • "PS-20" - Polysorbate 20. • "iPSC" - Induced pluripotent stem cells. • "ITR" - Reverse Iteration Sequence. • "MRI" - Magnetic Resonance Imaging.

[0068] Specific embodiments of the present invention are described in the following paragraphs. 1.Buffer; Adeno-associated virus vectors containing transgenes encoding microRNAs; Isotonic agent; Freeze-protecting substances; and surfactant An isotonic pharmaceutical preparation containing the above. 2. The formulation according to paragraph 1, having an osmolality of 250 to 330 milliosmoles / kg, preferably 260 to 310 milliosmoles / kg, for example, 270 to 300 milliosmoles / kg. 3. The formulation according to paragraph 1 or 2, having a pH value of 5 to 8, preferably 6 to 8, more preferably 6.5 to 8, for example 7 to 8. 4. A formulation described in any one of paragraphs 1 to 3, which substantially does not contain visible particles. 5. A formulation according to any one of paragraphs 1 to 4, wherein an adeno-associated virus vector having a transgene encoding a microRNA targets ATXN mRNA, for example, ATXN3 mRNA. 6. The formulation according to any one of paragraphs 1 to 5, wherein the adeno-associated virus vector comprises AAV2 serotype, AAV5 serotype, AAV9 serotype, hybrid AAV serotype, or a combination thereof, preferably the adeno-associated virus vector comprises AAV5 serotype or AAV9 serotype, for example, AAV5 serotype. 7. The formulation according to any one of paragraphs 1 to 6, wherein the concentration of the adeno-associated virus vector is at least 1E13 gc / ml, for example, at least 3E13 gc / ml. In some embodiments, the concentration of the adeno-associated virus vector is 0.5E13 to 6E13 gc / ml, for example, 1E13 to 5E13 gc / ml. 8. A preparation described in any one of paragraphs 1 to 7, wherein the cryoprotective agent is a sugar. 9. The formulation according to paragraph 8, wherein the cryoprotective substance is a sugar selected from the group consisting of trehalose, sucrose, glucose, dextran, and combinations thereof; for example, the cryoprotective substance is trehalose; or alternatively, the cryoprotective substance is dextran, for example, dextran 6000. 10. A formulation according to any one of paragraphs 1 to 9, wherein the cryoprotective substance is present in an amount up to 8% (w / v), preferably 2-5% (w / v), for example, 2-3% (w / v). 11. A preparation described in any one of paragraphs 1 to 10, wherein the cryoprotective substance is trehalose, present in an amount of 3% (w / v). 12. A preparation described in any one of paragraphs 1 to 11, wherein the isotonic agent is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, and combinations thereof. 13. The formulation described in paragraph 12, wherein the isotonic agent is a combination of NaCl and KCl. 14. The formulation according to any one of paragraphs 1 to 13, wherein the isotonic agent is present in an amount of 5 mM to 150 mM, preferably 25 mM to 125 mM, more preferably 75 mM to 125 mM, for example, 90 mM. 15. The formulation according to paragraph 14, wherein the isotonic agent contains at least 75 mM NaCl. 16. The formulation according to any one of paragraphs 1 to 15, wherein the surfactant is a nonionic surfactant, preferably a polysorbate, such as a polyethoxylated nonionic surfactant like polysorbate 20. 17. A formulation according to any one of paragraphs 1 to 16, wherein the surfactant is present in an amount of 0.001 to 0.1% (v / v), preferably 0.003 to 0.08% (v / v), more preferably 0.005 to 0.02% (v / v), for example, about 0.01% (v / v). 18. A formulation according to any one of paragraphs 1 to 17, wherein the surfactant is polysorbate 20 and is present in an amount of 0.01% (v / v). 19. The formulation according to any one of paragraphs 1 to 18, wherein the buffer is selected from the group consisting of artificial cerebrospinal fluid (aCSF), acetate buffer, citrate buffer, phosphate buffer, HEPES, Tris, and combinations thereof; preferably, the buffer is phosphate buffer or Tris; for example, the buffer is a 9-10 mM phosphate buffer at pH 7.3. 20. A formulation according to any one of paragraphs 1 to 19, which remains stable at room temperature for at least 12 hours, preferably at least 18 hours, for example, at least 24 hours, after melting, without substantially forming aggregates or lumps. 21. A formulation according to any one of paragraphs 1 to 20, used for the treatment of spinocerebellar ataxia (SCA), for example, SCA1, SCA2, or SCA3, preferably SCA3. 22. A formulation for use as described in paragraph 21, which is administered by intracerebrospinal fluid (CSF), preferably by intracisional delivery. 23. A formulation for use as described in paragraph 21 or 22, to be administered to the cisterna magna of the patient. 24. A formulation for use as described in any one of paragraphs 21-23, administered with a contrast agent for MRI to monitor administration. 25. A method for treating spinocerebellar ataxia by administering to a patient any of the preparations described in any one of paragraphs 1 to 24. 26. The method according to paragraph 25, wherein the formulation is administered in a single dose delivered to the patient by intravesical infusion. 27. The method according to paragraph 25 or 26, wherein the preparation is administered together with a contrast agent that can be visualized by MRI.

[0069] Specific embodiments of the present invention will be described as an example with reference to the following accompanying drawings. [Brief explanation of the drawing]

[0070] [Figure 1] The results of Example 1 are shown. Tm (melting temperature of the capsid determined by the iDSF approach) as a function of accelerated stress conditions and duration. [Figure 2] Figure 1 shows protein content and fluorescence as a function of stress conditions and duration for different salt concentrations. NaCl concentrations are shown in different grayscale chromaticities. Different buffers are pooled. UV280 content and Trp_Fluo:280,335 are shown in arbitrary units. For storage at 25°C and 40°C, the scales are 7 and 2 days, respectively, while shaking is 24 hours. [Figure 3] This is a production profiler from DoE experiments based on absorbance and fluorescence response for complete datasets at all time points and for all stress levels. [Figure 4] This is a production profiler from DoE experiments based on absorbance and fluorescence response for complete datasets at all time points and for all stress levels. [Figure 5] The polydispersion index (PDI) measured by dynamic light scattering is shown as a function of the fold concentration. [Figure 6] The z-average measurement due to dynamic light scattering is shown as a function of the enrichment ratio. [Figure 7] The z-mean measurements obtained by dynamic light scattering for enhanced and long-term stability are shown. [Figure 8A] It exhibits subvisible particles with enhanced and long-term stability. [Figure 8B] It exhibits subvisible particles with enhanced and long-term stability. [Figure 9] This is an overview of the visual inspection of AAV9 DP in five different formulation buffers under varying stress conditions. No fill: No visible particles; Diagonal fill: 1-2 visible particles present; Dense diagonal lines: Almost no visible particles. [Figure 10] AAV9(1489) luciferase formulated in different buffers exhibits luciferase activity in cell lysates of transduced Huh7 cells. [Examples]

[0071] Abbreviation AAV is an adeno-associated virus. AC stands for affinity chromatography. aCSF is artificial cerebrospinal fluid. AIM is an Analytical Investigational Method. CSF is cerebrospinal fluid. DLS is dynamic light scattering. DoE is a design of experiments. DP is a pharmaceutical product. DPD is drug development. DS is the active pharmaceutical ingredient. F / T stands for freeze / thaw. Gc is the genome copy number. GMP stands for Good Manufacturing Practice. iDSF is Intrinsic Differential Scanning Fluorescence (DSF). IEX is ion exchange. MW is molecular weight. N / A means not applicable. NF stands for nanofiltration. PD stands for Process Development. PDI is a multi-dispersion index. PEG is polyethylene glycol. RU stands for Arbitrary Unit. SOP stands for Standard Operating Procedure. STR is a stirred tank reactor. Tp is the total number of particles. UF / DF stands for ultrafiltration / diafiltration. VI is a visual inspection.

[0072] Materials and methods The following materials were used in the examples and are available as specified in Table 1 below.

[0073] [Table 1]

[0074] [measurement] The obtained readings were as follows: • Absorbance at 260 nm correlates with DNA concentration. • Absorbance at 280 nm correlates with protein concentration. Absorbance (turbidity) at 350 nm that correlates with protein aggregation and / or particle formation. Absorbance at 900 nm correlates with background signals from the plastic of the wells and seals. • Measure the absorbance of water, and therefore the path length, using the absorbance at 975 nm. • Tryptophan fluorescence at 280 / 335 nm, measured from the top and bottom of the microwell plate. This measurement correlates with protein and / or capsid unfolding and denaturation. The top reading was used in the drawing.

[0075] The protein and DNA concentrations were obtained as follows: According to the Lambert-Beer law, absorbance is proportional to concentration and optical path length. A constant called the absorption coefficient transforms this into an equation: A = cc * L * ε, where A is absorbance, cc is concentration, L is optical path length, and ε is the absorption coefficient.

[0076] The optical path length is measured by the water absorbance (A975) measured at 975 nm and the plastic background (A900) measured at 900 nm: Therefore, L = A975 - A900

[0077] Protein concentration is measured by measuring the absorbance at 280 nm (A280): A280 = cc (protein), L (equivalent to ε (protein)). Background from precipitated and aggregated proteins or turbidity is measured by absorbance at 350 nm (A350). It is assumed to be similar to the contribution of turbidity at 280 nm and is therefore subtracted from the absorbance at 280 nm. Then: cc(protein).ε(protein) = (A280-A350) / (A975-A900)

[0078] Since ε(protein) is a constant, the inventors can conclude that cc(protein).ε(protein) is proportional to the protein concentration and can be calculated by computer from different absorbance values ​​(A280, A350, A975 & A900) measured. A similar reasoning was applied to the DNA concentration at 260 nm.

[0079] Intrinsic differential scanning fluorescence (iDSF) measurement using Nanotemper® technology. The capsid denaturation temperature was tracked using Nanotemper® technology, which involved heating the sample to an increasing temperature and tracking the capsid's fluorescence. As the capsid denatures the fluorescent amino acids and exposes them to the solvent environment, fluorescence decreases as the environment changes from a rigid to a less rigid state within the protein. The ratio of fluorescence at 335 nm to fluorescence at 350 nm is computer-calculated to ensure the measurement is concentration-independent.

[0080] [Example 1 - Formulation Research AAV5] The drug (DP) used in this example was AAV5 with a transgene encoding a microRNA that targeted AAXN3 mRNA at a concentration of 4.7E13 gc / mL. rAAV5-miAAXN3 was buffer-exchanged in a multi-well system using a 96-well filter plate (100kDa cutoff) to produce 30 different formulations with different pH, buffer type, and salt content. After buffer exchange and filtration through a 0.2 μm filter, the formulations were aliquoted into three plates according to the plate layout and buffer composition. The final concentration of AAV5 in the wells was 1.0E13 gc / mL. Each formulation was prepared in double repeats. The outer rows and columns were filled with WFI to eliminate the influence of plate position. The buffers had overlapping pH levels to distinguish between buffering and pH action.

[0081] Each plate was assigned to specific stress conditions: F / T at 80°C, storage at 25°C and 65°C, and shaking at room temperature. Facilitated studies included: • For cycles 1, 3, and 10, freeze at -80°C (for at least 1 hour) and thaw at room temperature (for 1 hour). A table is shown containing experimental data for each F / T cycle. • Shake at room temperature for 1 and 6 hours. Due to deviation, there are two types of shaking measurements in the experiment. In the first shaking experiment, centrifugation was not performed before measurement, therefore a second shaking plate was added, centrifugation was performed, and the samples were shaken at room temperature for 1 and 6 hours. Store at 25°C for 2, 7, and 14 days. Store at 65°C for 2, 7, and 14 days. Under all of the above conditions, microplates were prepared and the initial measurement (T=0) was performed.

[0082] Table 2 shows the T=0 protein content following the absorbance at 280 nm (turbidity and path length corrected in four multi-well plates; arbitrary units). Table 3 shows the T=0 protein content following the fluorescence at 335 nm (top measurement from four multi-well plates; arbitrary units).

[0083] [Table 2]

[0084] [Table 3]

[0085] Table 2 clearly shows that the four multiwell plates have similar, though not identical, values ​​for the expected corrected absorbance (protein concentration) at 280 nm. Furthermore, for the four plates at T=0, the absorbance is high in the presence of 150 mM NaCl for almost all conditions, and therefore, the protein content is high and the degree of aggregation is low. This can be concluded because the measured values ​​include correction for background. Overall, it is confirmed that the effects of the different formulations occur immediately at T=0 during buffer exchange.

[0086] Table 3 confirms that, for most conditions, both 75 mM NaCl and 150 mM NaCl have a stabilizing effect on the salts. Acetates at pH 5 and 5.5 mostly exhibit an destabilizing effect, especially without NaCl. Phosphates at pH 7.5 and citrates at pH 6.5 appear to be favorable for folding. As mentioned above, the fact that the effect is observed at T=0 suggests that these occur during the buffer exchange process.

[0087] The results of the accelerated study are shown in Figure 1. These results highlight the importance of pH and the effect of salt concentration in formulations.

[0088] The overall observation is: • Analytical assay: The effect of all buffers tested (at 20 mM: acetate, citrate, phosphate, and Tris) on the stability of the pharmaceutical product, measured by absorbance and fluorescence, is immediately observed at T=0. The lack of salts and buffers is detrimental to the stability of pharmaceuticals. • The addition of salt (NaCl) at concentrations of 75 mM or higher is particularly beneficial for stabilizing the formulation. • Neutral or slightly basic formulations with a pH of 7.0-7.5 offer better stability for pharmaceuticals. Basic buffering agents such as Tris are more stable compared to buffering agents with a lower pH.

[0089] Formulations containing NaCl in a neutral or slightly basic pH (7.5) buffer solution are used for further testing and optimization.

[0090] [Excipient Screening Study] Based on the results above, a neutral or slightly basic pH and the inclusion of salts aid in capsid stability. Three buffers—phosphate, Tris, and Hepes—were selected to support a pH equal to 7.5. Artificial cerebrospinal fluid (aCSF) was used as a control. 117 formulations were prepared in different buffers with increased concentrations of NaCl and estimated concentrations of excipients; the latter were high enough to affect viscosity but low enough not to significantly affect the osmolality by weight. The different excipients tested, among others, including various surfactants such as PEG, cryoprotective substances such as trehalose, sucrose, glucose, and dextran, and their concentrations are shown in the table below. Polymer excipients were selected to increase the viscosity of the formulations.

[0091] [Table 4]

[0092] In the first excipient screening, four sets of multiwell plates were prepared.

[0093] For a second excipient screening, 18 formulations were prepared in glass vials. These contained the three buffers described above: Tris, HEPES, and phosphate buffer, as well as three proposed excipients: trehalose, low molecular weight (Low MW) dextran, and glucose.

[0094] Plates from the first excipient screening and vials from the second excipient screening were subjected to accelerated studies under different stresses: F / T, shaking, and storage at different temperatures (25°C and 40°C), including the periods / cycles specified in Table 5 below.

[0095] [Table 5]

[0096] Figure 2 shows protein content and fluorescence data as a function of stress conditions and duration. The DoE experiment design was also performed using JMPR software, and the prediction profiler is shown in Figures 3 and 4. The results suggest that higher salt concentrations have a positive effect, and from the effect of the buffer, the most preferred buffers are phosphate and HEPES buffer.

[0097] The overall observation is: The effect of the buffer is immediate, occurring during the buffer exchange process and confirmed at T=0; • A high salt concentration and a slightly alkaline buffer are beneficial for sample stability; Dextran 6000 and trehalose have superior stabilizing effects compared to glucose; HEPES and phosphoric acid are slightly better than Tris.

[0098] The following selections of buffers and excipients will be used for further testing and optimization. • HEPES and phosphoric acid as buffering agents • Dextran 6000 and trehalose are used as sugar excipients.

[0099] [Key points of agglutination research] The four types of buffering agents used in the formulation are as specified in Table 6 below.

[0100] [Table 6]

[0101] These formulations were progressively concentrated by diafiltration. Samples were collected after each concentration step. Table 7 below shows the concentration ratios and expected concentrations.

[0102] [Table 7]

[0103] The measurements following each concentration step were as follows: • Genome copy number / mL (Gc / mL) • Total number of particles / mL (TP / mL) Visual inspection at T=0, as well as after 24 hours at 4°C and after two freeze-thaw (F / T) cycles. Subvisible particles produced by AccuSizer®. • Multidispersion index due to dynamic light scattering.

[0104] Table 8 below shows visual inspection as a function of concentration ratio for four formulations after stress, such as after 24 hours of storage at 4°C or after two F / T cycles. Up to a 6-fold concentration, the phosphate-containing formulation was found to perform better than the HEPES-containing formulation. After the 6-fold concentration process, the trehalose-containing formulation was also found to perform better than the dextran 6000-containing formulation.

[0105] [Table 8]

[0106] Table 9 below confirms that the number of particles increases as different formulations are concentrated. Furthermore, all formulations show very similar results up to the 6-fold concentration step that defines the maximum concentration mentioned above. Only the phosphate-containing formulation is concentrated up to 15-fold, showing the expected increase in particle count.

[0107] [Table 9]

[0108] Information regarding the particle size, uniformity, and morphology of the formulations under study can be obtained from the polydispersity index (PDI) and z-mean measurements obtained by dynamic light scattering. The PDI and z-mean results for the formulations are shown in Figures 5 and 6. PDI represents the distribution of particle size populations within a given sample. PDI values ​​range from 0.0 (samples with perfectly uniform particle size) to 1.0 (highly polydispersible samples with multiple particle size populations). Figure 6 shows that all four formulations reach a plateau after 6-fold concentration, consistent with the GC and TP measurements described above. Furthermore, the presence of trehalose results in smaller aggregates than in the presence of dextran 6000. The same effect is observed for phosphate-containing formulations that induce smaller particles than the HEPES equivalent. The behavior of the z-mean is the same as that of the PDI, as described above.

[0109] Table 10 below shows the results of the stability test.

[0110] [Table 10]

[0111] The overall observations are as follows: • The maximum concentration achievable at 6E14 gc / mL (6-fold concentration) • The formulation in phosphate buffer exhibits a better visually observable particle profile exceeding 6E14 gc / mL. • Phosphate / trehalose exhibits the narrowest particle size distribution.

[0112] [Formulation Confirmation Study] For further testing and optimization, two formulations were proposed to enhance stability: Preparation 1. Phosphate / Trehalose: Phosphate (Na / K) 10mM; NaCl 82mM; KCl 3mM; Trehalose 3% (w / v); pH 7.3. Preparation 2. Phosphate / Dextran: Phosphate (Na / K) 10mM; NaCl 125mM; KCl 3mM; Dextran 6000 2.2% (w / v); pH 7.3.

[0113] Two formulations were filled to a final volume (1.2 mL) in sealed vials (2 mL borosilicate type I vials sealed with silicone stoppers and flip-off seals). These vials were subjected to accelerated stability testing under different stresses: F / T, shaking, and short-term stability at 25°C and 40°C, and 1-year stability (-80°C and 2-8°C), including: • Freeze at -80°C (for at least 1 hour) for 3 cycles (see above in the study's date schedule for actual cycle lengths), then thaw at room temperature (1 hour). Shake at 200 rpm for 24 hours. • Accelerated stability: Store at 25°C for 2 weeks. • Accelerated stability: Store at 40°C for 1 week. Short-term stability study at -20°C for 3 months. Long-term stability studies at -80°C (target DP storage temperature) and 2-8°C for 1, 3, 6, 9, and 12 months. Initial measurements (T=0) were performed under all of the above conditions. For visual inspection, additional time points were added: 1 F / T, 1 week at 25°C, and 2 weeks at 40°C.

[0114] The subsequent measurements were as follows: • Appearance / visible particles (VI) Subvisible particles • Genome copy number (GC); correlates with concentration • tp: Total particles ·ip: infectious particles • gc / ip: (genome copy number / infectious particles); correlates with the number of active particles. • tp / gc (total particles / genome copy number); correlates with purity • PDI calculated using DLS (Polydispersion Index); Since the inventors aimed to obtain a monodispersion system, it is expected to be less than 0.1.

[0115] The complete data is shown in Tables 11-13 below. Further data is illustrated in Figures 7 and 8.

[0116] [Table 11]

[0117] [Table 12]

[0118] [Table 13]

[0119] These results confirm that formulations containing trehalose are preferable to those containing dextran 6000. The strongest data supporting this conclusion is visual inspection analysis, which shows that formulations containing trehalose exhibit significantly fewer particles after F / T cycles or (long-term) storage at 80°C. Furthermore, DLS measurements revealed that the PDI value of the trehalose-containing sample was approximately half that of the dextran 6000-containing sample, indicating that the trehalose-containing sample is more uniform than the dextran 6000-containing sample. The overall conclusion from the subvisible particle data is that trehalose performs considerably better under long-term storage conditions.

[0120] For further optimization, the following formulation is recommended: Phosphate (Na / K) 10mM; NaCl 82mM; KCl 3mM; Trehalose 3% (W / V); pH 7.3. Further research was conducted to select one of the following five different formulations containing different DP concentrations, MgCl2, and / or PS-20 for pharmaceutical use. Based on previous reports of early formulations, a formulation with 10mM phosphate (Na / K); 82mM NaCl; 3mM KCl; 3% (w / v) trehalose; and pH 7.3 is recommended as an initial point for further optimization.

[0121] Different DP materials were formulated as follows: 1. 3E14 gc / mL in trehalose / phosphate buffer; 2. 4E14 gc / mL in trehalose / phosphate buffer; 3. 3E14 gc / mL in trehalose / phosphate buffer + 0.8 mM MgCl2; 4. 3E14 gc / mL in trehalose / phosphate buffer + 0.005% (v / v) PS-20; 5. 3E14 gc / mL in trehalose / phosphate buffer + 0.01% (v / v) PS-20.

[0122] The trehalose / phosphate buffer contains 9 mM phosphate buffer, 82 mM NaCl, 3 mM KCl, and 3% (w / v) trehalose. These formulations were used in stress studies, and the results are shown in Table 14 below.

[0123] [Table 14]

[0124] The results shown above confirm that the addition of PS-20 (especially 0.01% (v / v)) has a positive effect on the stability of the formulation.

[0125] [Conclusion] • Plate-based screening studies highlighted the pH-stabilizing effect of NaCl on neutral to slightly alkaline solutions. • Studies have shown that trehalose and dextran 6k have stabilizing effects in phosphate or HEPES buffer. • Phosphate + trehalose / dextran 6k supports concentration up to 6E14 gc / mL. • Phosphate + trehalose exhibits an excellent profile of visible particles. Optimizing the formulation by adding 0.01% (v / v) PS-20 improves appearance and significantly reduces subvisible particles. • The formulation is optimized for cryopreservation at temperatures below -65°C.

[0126] A particular embodiment of the present invention includes, in addition to an adeno-associated virus vector having a transgene encoding a microRNA, the composition shown in Table 15 below.

[0127] [Table 15]

[0128] [Example 2 - Formulation Research AAV9] The drug (DP) used in this example was AAV9 containing a transgene encoding a luciferase reporter at a concentration of approximately 3E13 gc / mL (see Table 19).

[0129] Table 15 shows the compositions of the five formulation buffers used in this study. All five buffers were prepared in 4 L volumes and filtered using a 0.22 μm PES filter. On the day of the buffer exchange process, poloxamer 188 and PS20 surfactant were newly added to the corresponding formulation buffer.

[0130] Using dialysis, 30 mL of AAV9 preparation was replaced for each buffer condition. Three cycles of buffer exchange (1 L / cycle) were performed at 2-8°C for 16 hours.

[0131] The recovered volume of AAV9 after the buffer exchange process was filtered using a 0.22 μm PVDF syringe filter. AAV9 DP was filled into 2 mL Type I glass vials with a filling volume of 1.2 mL of DP. Table 16 shows the total number of filled DP vials per recovered volume of AAV9 in each formulation buffer.

[0132] [Table 16]

[0133] All filled DP vials were visually inspected. DP vials that passed the visual inspection were placed under the different stress conditions shown in Table 17 and then analyzed as described in Table 18.

[0134] [Table 17]

[0135] [Table 18]

[0136] [result] [Genome copy number] The amount of AAV particles containing vector DNA packaged within them is measured by genome copy number (GC). GC concentration is determined by quantitative PCR. GC assays were performed under stress conditions of T=0 (after buffer exchange), SHK (1 day, 200 rpm), 1FT+7 days, and 1FT+4 weeks at 2-8°C. Table 19 shows the average GC from three repeated measurements (triplicate) for two vials per formulation buffer.

[0137] [Table 19]

[0138] Taking into account a 20% variability in GC assays, the GC of AAV9 was approximately 3E13 gc / ml among all formulations tested. A slightly higher concentration of AAV9, 4E13 gc / ml, was measured with formulation buffer 3. No noticeable decrease in GC was observed in any of the test samples under different stress conditions.

[0139] [pH and gravimetric osmolality] The pH and gravimetric osmolality data were close to the theoretical values ​​for each formulation buffer (see Table 20), indicating that the buffer exchange was performed correctly.

[0140] [Table 20]

[0141] Before buffer exchange, the measured gravimetric osmolality of the AAV9 buffer was 687 mmol / kg and pH 8.5. The buffer composition was 0.1 M glycine, 0.5 M Tris, and pH 8.3. After buffer exchange, all formulations were close to the target pH and theoretical osmolality. Except for buffer 4 and the Zolgensma formulation buffer, the formulations were hypertonic; all other formulations were isotonic.

[0142] [Analysis of visible and subvisible particles] The acceptable standards for visual inspection are as follows: <790> It is defined as conforming to the standard, being a colorless liquid, transparent, and free of visible particles. Visual inspection was performed against white and black backgrounds. All AAV9 DP vials were visually inspected after filling and after stress application. DP vials containing exogenous particles after filling, and vials with insufficient filling volume, were rejected. The results are shown in Table 21 and Figure 9.

[0143] [Table 21]

[0144] Two vials were visually inspected for each formulation buffer and stress condition. AAV9 DP was most stable in formulation buffer 3. In this formulation, AAV9 DP remained particle-free after stirring at room temperature for 1 day, three freeze / thaw cycles, and after 1 week after thawing when stored at room temperature, or 2 weeks after thawing when stored at 2-8°C (Figure 9). Using the light shielding technology AccuSizer FX, two vials for each stress condition and formulation buffer were analyzed for subvisible particles.

[0145] Under all stress conditions, subvisible particles are USP <787> Within acceptable limits: 6000 particles / mL above 10 μm and 600 particles / mL above 25 μm remained. Similar agglutination profiles were observed in the 0.3–1 μm range for all tested formulation buffers. AAV9 agglutination was promoted by freeze-thaw stress, and agitation stress conditions had less effect on agglutination.

[0146] In general, the number of subvisible particles larger than 2 μm in formulation buffer 3 of AAV9 is found to be lower compared to other formulations under all applied stress conditions.

[0147] [Background membrane imaging] (BMI) Using BMI technology and an automated membrane microscopy system in a 96-well format, particles of 2–4 μm were visualized. Consistent with results from visual inspections related to visible and subvisible particle analysis using light shielding technology, AAV9 DP in formulation buffer 4 after 1 FT + 4 weeks at 2–8°C contains the highest number of particles larger than 2 μm compared to other formulations.

[0148] [AAV9 transduction into target cells] The potential effects of different formulations on AAV9 transduction efficiency have been evaluated in vitro using cell assays. AAV9 transduction efficiency was tested in human hepatocellular carcinoma (Huh7) cells under each buffer condition. Huh7 cells were selected as an in vitro model cell line based on data previously published by Pieterszetal., 2021 (Pietersz KL, Plessis FD, Pouw SM, Liefhebber JM, van Deventer SJ, Martens GJM, Konstantinova PS, Blits B.PhP.B. Enhanced Adeno-Associated virus Mediated-Expression Following Systemic Delivery or Direct Brain Administration. Front Bioeng Biotechnol. 2021 Aug 3;9:679483.doi:10.3389 / fbioe.2021.679483.PMID:34414171;PMCID:PMC8370029).

[0149] Huh7 cells were simultaneously transduced with three different AAV9 doses (GC / cell), also known as the multiplicity of infection (MOI), using separately formulated AAV9 DP, wild-type Adeno5, and Ad5ARM adenoviruses. In addition to untreated AAV9 in different formulation buffers, 50 μL of AAV9 for each buffer condition was thermally inactivated by incubation at 95°C for 10 minutes as a negative control for each condition. Readouts were performed using a dual luciferase reporter assay system.

[0150] As shown in Figure 10, luciferase activity in cell lysates of AAV9-transduced Huh7 cells suggests that different buffer formulations did not significantly affect the transduction efficiency of AAV9, regardless of the MOI used.

[0151] [Conclusion] A platform formulation suitability study was conducted to select the most suitable formulation from already developed formulations for cerebral administration, and AAV9-luciferase was proven to be the most suitable under selected stress conditions.

[0152] Overall, AAV9 DP was the most suitable formulation buffer 3 under the applied stress conditions. Compared to other formulations selected under all applied stress conditions, no visible particles or subvisible particles larger than 2 μm were observed in formulation buffer 3. Subvisible particles are USP <787> Within acceptable limits: 6000 particles / mL exceeding 10 μm and 600 particles / mL exceeding 25 μm remained. Other quality characteristics, genome copy number, SEC purity (monomer %), hydrodynamic radius and polydispersity index, and unfolding temperature did not change significantly under the applied stress compared to T=0. Furthermore, AAV9 transduction into target cells was not affected by the formulation buffer, regardless of the infection multiplicity used.

[0153] Therefore, Buffer 3 is considered the most suitable formulation for the storage and quality of the AAV9-luciferase product and should be considered for subsequent studies as well. Furthermore, this formulation is an isotonic formulation with similar totonicity and pH to cerebrospinal fluid: 280-310 milliosmoles / kg and pH 7.3-7.4 (Salvador L, Valero R, Carrero E, Caral L, Fernandez S, Marin JL, Ferrer E, Fabregas N. Cerebrospinal fluid composition modifications after neuroendoscopic procedures. Minim Invasive Neurosurg. 2007 Feb;50(1):51-5. doi:10.1055 / s-2007-973823. PMID:17546545).

[0154] [Table 22]

[0155] Table 23

Claims

1. Buffer; Adeno-associated virus vectors containing transgenes encoding microRNAs; Isotonic agent; Freeze-protecting substances; and surfactant An isotonic pharmaceutical preparation containing [a specific ingredient / method].

2. The formulation according to claim 1, having an osmolality of 250 to 330 milliosmoles / kg and a pH value of 5 to 8.

3. The formulation according to claim 1 or 2, which substantially does not contain visible particles.

4. The formulation according to any one of claims 1 to 3, wherein the adeno-associated virus vector having a transgene encoding a microRNA targets ATXN mRNA, for example, ATXN3 mRNA.

5. The formulation according to any one of claims 1 to 4, wherein the adeno-associated virus vector comprises AAV2 serotype, AAV5 serotype, AAV9 serotype, hybrid AAV serotype, or a combination thereof, preferably the adeno-associated virus vector comprises AAV5 serotype or AAV9 serotype, more preferably AAV5 serotype.

6. The formulation according to any one of claims 1 to 5, wherein the concentration of the adeno-associated virus vector is at least 1E13 gc / ml, for example, at least 3E13 gc / ml.

7. The formulation according to any one of claims 1 to 6, wherein the cryoprotective substance is a carbohydrate selected from the group consisting of trehalose, sucrose, glucose, dextran, and combinations thereof; and is present in an amount up to 8% (w / v).

8. The isotonic agent is NaCl, KCl, MgCl ２ CaCl ２ A formulation according to any one of claims 1 to 7, selected from the group consisting of , and combinations thereof; present in an amount of 5 mM to 150 mM.

9. The formulation according to any one of claims 1 to 8, wherein the surfactant is a nonionic surfactant and is present in an amount of 0.001 to 0.1% (v / v), preferably 0.003 to 0.08% (v / v), more preferably 0.005 to 0.02% (v / v), for example, about 0.01% (v / v).

10. The formulation according to any one of claims 1 to 9, wherein the buffer is selected from the group consisting of artificial cerebrospinal fluid (aCSF), acetate buffer, citrate buffer, phosphate buffer, HEPES, Tris, and combinations thereof.

11. The formulation according to any one of claims 1 to 10, which remains stable at room temperature (15°C to 25°C) for at least 12 hours after melting without substantially forming aggregates or lumps.

12. A formulation according to any one of claims 1 to 11, used for the treatment of spinocerebellar ataxia (SCA).

13. A formulation for use according to claim 12, which is administered by intracerebrospinal fluid (CSF) delivery.

14. A formulation for use according to claim 12 or 13, which is administered to the cisterna magna of a patient.

Images