Pharmaceutical formulations of gene delivery vehicle

Abstract

The present invention relates to formulations comprising miRNAs having improved stability for use in the treatment of diseases, including neurodegenerative diseases, such as spinal cerebellar ataxia type 3.5.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy. In addition, 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 more particularly to pharmaceutical formulations with improved stability for the treatment of diseases including neurodegenerative diseases (such as spinocerebellar ataxia type 3).

[0002] Gene therapy

[0003] The elucidation of DNA as the carrier of genetic information, and therefore the source of inherited diseases, has led to the concept of therapies in which mutated, damaged genes could be replaced or at least silenced. Numerous genes and / or other nucleic acid sequences have been found to play a role in (inherited) diseases. If mutated genes could be replaced by healthy ones, or if genes expressing abnormal (and sometimes toxic) products could be silenced, diseases could be treated at the molecular level and potentially cured. Gene therapy offers a particularly promising concept for diseases caused by single-gene mutations.

[0004] However, delivering the desired nucleic acid to the cells that need to be targeted is not an easy task. A variety of (viral) delivery systems have been studied, all of which have their own advantages and disadvantages. One of the viral delivery vehicles used for gene therapy is adeno-associated virus (AAV).

[0005] AAV

[0006] AAV has a single-stranded DNA genome of approximately 4.8 kilobases (kb). AAV belongs to the Parvoviridae family and relies on co-infection with other viruses (particularly adenoviruses) for replication. The genome contains the Rep (replication) and Cap (capsid) genes. These coding sequences are flanked by inverted terminal repeats (ITRs) required for genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) that replicate the viral genome and facilitate packaging, while Cap expression produces viral capsid proteins (VP; VP1 VP2 VP3), which form the capsid shell.

[0007] For 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 desired nucleic acid (transgene) to be delivered to the target cell. The desired nucleic acid flanks are the ITRs of AAV. Transgenes flanked by the ITRs encoded by rAAV can form circular concatemers that are retained in the nucleus of the transduced cell as free bodies. Since free bodies exist mainly in a free form, if and when the target cell replicates, the expression of the nucleic acid sequence delivered by AAV may be diluted over time. This dilution may not generally be applicable to post-mitotic cells (such as neurons), which are target cells for many neurodegenerative diseases. A review of AAV vectors for gene therapy is provided in Naso et al., Biodrugs [Biological drugs] 2017 (pp. 317-334).

[0008] miRNA

[0009] RNA interference (RNAi) is a naturally occurring mechanism involving sequence-specific downregulation of messenger RNA (mRNA). Downregulation of mRNA results in a reduction in the amount of expressed protein. RNA interference is triggered by double-stranded RNA. One of the chains of the double-stranded RNA is substantially or completely complementary to its target (mRNA). This chain is called a guide strand. The mechanism of RNA interference involves incorporating the guide strand into an RNA-induced silencing complex (RISC). This complex is a multiple turnover complex that can bind to its target mRNA through complementary base pairing of the guide strand. Once bound to its target mRNA, it can cut mRNA or reduce translation efficiency. RNA interference has been widely used to knock down specific target genes since its discovery. The triggering factors used to induce RNA interference that have been used involve the use of small interfering RNA (siRNA) or short hairpin RNA (shRNA). In addition, molecules that can naturally trigger RNAi (so-called microRNA (miRNA)) have been used to make artificial (engineered) miRNAs that mimic their naturally occurring counterparts. What these strategies have in common is that they provide substantially double-stranded RNA molecules designed to target the selected mRNA. RNAi-based therapeutics that exploit the sequence-specific mode of RNAi are under development, and several are currently in clinical trials (see ia Davidson and McCray, Nature Reviews-Genetics, 2011; Vol. 12; 329-340).

[0010] Spinocerebellar ataxia type 3 (SCA3)

[0011] Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is an autosomal dominant, single-gene fatal disorder. The disorder is characterized by progressive degeneration of brain regions caused by a CAG expansion in the human ataxia-3 gene, also known as the ATXN3 gene (OMIM: 607047, reference sequence Homo sapiens ataxia 3 (ATXN3) on chromosome 14, NCBI reference sequence: NG_008198.2). In the 3' region of the gene, there is a cytosine-adenine-guanine (CAG) repeat region. The CAG region is located in frame and produces ataxia-3 protein containing a polyQ region (a repeat sequence of glutamine). Healthy or asymptomatic individuals can have up to 44 CAG repeats in the ATXN3 gene. Affected individuals have expansions, and it has been shown that they may have 52 to 86 or more CAG repeats. Individuals with 45-51 CAG repeats have incomplete penetrance of disease symptoms. This expansion results in an ataxia-3 protein with an extended polyQ region, and the length of the CAG repeats (and therefore the length of the polyQ region within ataxia-3) may correlate with disease progression, i.e., generally, longer regions are associated with more severe disease progression.

[0012] The ataxia-3 protein with amplified poly Q fragments acquires toxic properties (toxic function acquisition), and the formation of neuronal aggregates in the brain is a neuropathological hallmark. Neuropathological studies have detected extensive neuronal loss in various regions of SCA3 patients (including the cerebellum, thalamus, midbrain, pons, medulla oblongata, and spinal cord) (Riess et al., Cerebellum [cerebellum] 2008). Although extensive pathology has been reported, the consensus is that the main pathology is in the cerebellum and brainstem (Eichler et al., AJNR Am J Neuroradiol [American Journal of Neuroradiology], 2011). The disease has complete penetrance, which means that if a person has 52 or more CAG expansions, they will inevitably develop the disease and have a 50% chance of passing it on to their offspring.

[0013] Formulation and administration challenges

[0014] Using AAV vectors to deliver microRNAs directly to the patient's brain to knock down the ATXN3 gene could be a highly innovative and promising approach for treating SCA3.

[0015] However, direct administration to a patient's brain or spine presents particular challenges in the formulation and stability of suitable pharmaceutical formulations, such as the need to avoid the formation of aggregates or agglomerated particles in the formulation that could be harmful to the patient. For example, such formulations can be stored frozen and then thawed before use. Problematic aggregates or agglomerates can form during or after thawing of the formulation, as well as during the period after thawing but before administration of the formulation. Therefore, it would be highly desirable to provide innovative formulations of AAV vectors for the treatment of spinocerebellar ataxia type 3 or other diseases that eliminate or substantially eliminate the formation of aggregates or agglomerates, thereby extending the shelf life of the formulation. Summary of the Invention

[0016] In a first aspect, an isotonic pharmaceutical formulation is provided, comprising:

[0017] Buffer;

[0018] an adeno-associated viral vector with a transgene encoding a microRNA;

[0019] Tonicity agents;

[0020] Cryoprotectants; and

[0021] surfactant.

[0022] In some embodiments, the formulation has an osmotic pressure of 250 to 330 mOsm / kg and a pH of 5 to 8.

[0023] In some embodiments, the formulation is substantially free of visible particles.

[0024] In some embodiments, the adeno-associated viral vector with a transgene encoding a microRNA targets ATXN mRNA, such as ATXN3 mRNA.

[0025] In some embodiments, the adeno-associated viral vector comprises an AAV2 serotype, an AAV5 serotype, an AAV9 serotype, a hybrid AAV serotype, or a combination thereof. Preferably, the adeno-associated viral vector comprises an AAV5 serotype or an AAV9 serotype, more preferably an AAV5 serotype.

[0026] In some embodiments, the concentration of the adeno-associated viral vector is at least 1E13 gc / ml, such as at least 3E13 gc / ml. In some embodiments, the concentration of the adeno-associated viral vector is between 0.5E13 and 6E13 gc / ml, such as between 1E13 and 5E13 gc / ml.

[0027] In some embodiments, the cryoprotectant is a carbohydrate selected from the group consisting of trehalose, sucrose, dextrose, dextran, and combinations thereof; and is present in an amount up to 8% w / v.

[0028] In some embodiments, the tonicity agent is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, and combinations thereof, and is present in an amount of 5 mM to 150 mM. In some embodiments, the surfactant is a nonionic surfactant and is present in an amount of 0.001% v / v to 0.1% v / v, preferably 0.003% v / v to 0.08% v / v, more preferably 0.005% v / v to 0.02% v / v, for example, about 0.01% v / v.

[0029] 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.

[0030] In some embodiments, the formulation remains stable at room temperature (15°C-25°C) and does not substantially form aggregates or agglomerates for at least 12 hours after thawing.

[0031] In a second aspect, there is provided a formulation of the invention for use in the treatment of spinocerebellar ataxia (SCA).

[0032] In some embodiments, the formulation is administered by intra-CSF (cerebrospinal fluid) delivery.

[0033] In some embodiments, the formulation is administered to the cisterna magna of the patient. DETAILED DESCRIPTION

[0034] The present invention is directed to a novel pharmaceutical formulation that reduces the formation of aggregates or agglomerates during processing and is substantially free of visible particles at room temperature for an extended period after thawing. The present invention is also directed to a pharmaceutical formulation comprising one or more excipients that is substantially isotonic with human cerebrospinal fluid (CSF) for improved administration to a patient's brain. The present invention is also directed to a method of treating spinocerebellar ataxia type 3 (SCA3) by administering the formulation of the present invention to the patient's brain.

[0035] In one aspect of the present invention, the present invention provides an isotonic pharmaceutical formulation comprising a buffer, an adeno-associated viral vector having a transgene encoding a microRNA, a tonicity agent, a cryoprotectant, and a surfactant.

[0036] In this way, it has been unexpectedly discovered that the addition of cryoprotectants and surfactants to the formulation helps improve the stability of the drug product, particularly at high concentrations. The formulation can remain stable at a storage temperature of ≤-65°C for at least about 6 months, preferably at least about 12 months, more preferably at least about 24 months (e.g., at least about 36 months), with substantially no aggregates or agglomerates formed. The formulation can also remain stable at room temperature (15°C-25°C) for an extended period of time, preferably at least about 12 hours, more preferably at least about 24 hours, after thawing, with substantially no aggregates or agglomerates formed.

[0037] In some embodiments, an adeno-associated viral vector with a transgene encoding a microRNA can target ATXN mRNA, enabling the formulation to treat spinocerebellar ataxia. For example, the adeno-associated viral vector with a transgene encoding a microRNA can target ATXN3, thereby reducing ATXN3 mRNA and protein levels in the treatment of spinocerebellar ataxia type 3. Thus, in some embodiments, the adeno-associated viral vector with a transgene encoding a microRNA targets ATXN mRNA. In further embodiments, the adeno-associated viral vector with a transgene encoding a microRNA targets ATXN3 mRNA.

[0038] In some embodiments, the adeno-associated viral 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 treating spinocerebellar ataxia type 3. In some embodiments, the adeno-associated viral vector comprises an AAV9 serotype.

[0039] In some embodiments, the adeno-associated viral vector comprises the AAV5 serotype.

[0040] In some embodiments, the adeno-associated viral vector comprises a hybrid AAV serotype. For example, the hybrid AAV serotype can be a hybrid AAV2 / AAV5 serotype, an AAV2 / AAV9 serotype, or an AAV5 / AAV9 serotype.

[0041] Examples of methods and means for delivering miRNA to target cells are provided in WO 2020 / 104469 A1, which is incorporated herein by reference.

[0042] In some embodiments, the concentration of the adeno-associated viral 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 viral vector is at least 6E14 gc / mL. In some embodiments, the concentration of the adeno-associated viral vector is between 4E14 gc / mL and 5E14 gc / mL.

[0043] In some embodiments, the formulation comprises AAV5 with a transgene encoding a microRNA, and the concentration of AAV5 is at least 6E14 gc / mL.

[0044] In one example, the formulation comprises AAV5 with a transgene encoding a microRNA, and the concentration of AAV5 is 3E14 gc / mL.

[0045] In another example, the formulation comprises AAV5 with a transgene encoding a microRNA, and the concentration of AAV5 is 6E14 gc / mL.

[0046] In yet another example, the formulation comprises AAV5 with a transgene encoding a microRNA, and the concentration of AAV5 is 7.5E14 gc / mL.

[0047] Stable, high-concentration formulations are particularly useful for delivering sufficient genomic copies of a drug product when dose volume is limited. An example of such an application is when injection into the cisterna magna (CM, which has an average volume of only approximately 1 ml) is required.

[0048] In some embodiments, the formulation comprises AAV9 with a transgene encoding a microRNA, and the concentration of AAV9 is at least 6E14 gc / mL.

[0049] In one example, the formulation comprises AAV9 with a transgene encoding a microRNA, and the concentration of AAV9 is 3E14 gc / mL.

[0050] In another example, the formulation comprises AAV9 with a transgene encoding a microRNA, and the concentration of AAV9 is 6E14 gc / mL.

[0051] In yet another example, the formulation comprises AAV9 with a transgene encoding a microRNA, and the concentration of AAV9 is 7.5E14 gc / mL.

[0052] The formulation may also have a pH that is 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, even more preferably 7 to 8, such as 7.1 to 7.7. In some embodiments of the invention, the formulation has a pH of 7.3.

[0053] Tonicity is a measure of the effective osmotic pressure that a liquid formulation can exert, and is primarily determined by the number of dissolved particles in the solution. Osmotic pressure is an important factor affecting biological cells. Hypertonicity refers to the presence of a solution that causes cell contraction. Hypotonicity refers to the presence of a solution that causes cell expansion. Isotonicity refers to the presence of a solution that does not produce a change in cell volume. When a biological cell is in a hypotonic environment, water accumulates inside the cell, and the water flows through the cell membrane into the cell, causing the cell to swell. For mammalian cells, this may lead to cell lysis, and therefore, when fragile cells (such as nerve cells or brain cells) are exposed to the composition, tonicity is important. Therefore, tonicity agents are added to injectable formulations to prevent osmotic shock at the injection site during administration, thereby reducing local irritation or even damage to the CNS. Therefore, typical tonicity agents are excipients for tonicity regulation and are known in the art.

[0054] In some embodiments, the tonicity agent comprises a metal salt from Group 1 or Group 2 of the Periodic Table of the Elements, preferably a metal chloride salt from Group 1 or Group 2 of the Periodic Table of the Elements, preferably selected from the group consisting of NaCl, KCl, CaCl2, MgCl2, and combinations thereof. Preferably, the tonicity agent comprises NaCl. In one embodiment of the present invention, the tonicity agent comprises a combination of NaCl and KCl. The tonicity agent helps stabilize the formulation. Preferably, the tonicity 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. For example, the tonicity agent may comprise at least 75 mM NaCl. In some embodiments, the tonicity agent is present in an amount of 90 mM. For example, the tonicity agent may comprise a combination of NaCl and KCl and be present in an amount of 90 mM. In some embodiments, the tonicity agent comprises at least 80 mM NaCl and at least 2 mM KCl. For example, the tonicity agent may comprise 3 mM KCl and 87 mM NaCl or 82 mM NaCl. Thus, in some embodiments, the tonicity agent is selected from the group consisting of: NaCl, KCl, MgCl2, CaCl2, and combinations thereof; and is present in an amount of 5 mM to 150 mM.

[0055] Tonicity agents are primarily used to establish the desired osmotic pressure of the formulation. Preferably, the formulation is substantially isotonic with human cerebrospinal fluid. In particular, the formulation can have an osmotic pressure of 250 to 330 mOsm / kg. In some preferred embodiments, the formulation has an osmotic pressure of 270 to 320 mOsm / kg. For example, the formulation has an osmotic pressure of 280 mOsm / kg. Thus, in one embodiment, the formulation has an osmotic pressure of 250 to 330 mOsm / kg and a pH of 5 to 8.

[0056] Cryoprotectants are additives that help minimize polypeptide / biologic denaturation when a polypeptide is present in the composition. Cryoprotectants have been found to be particularly advantageous in providing a stable drug product that eliminates or substantially eliminates the formation of aggregates or coagulations. In preferred embodiments, the cryoprotectant is a carbohydrate or polymer. As used herein, the term "carbohydrate" refers to naturally occurring sugars and polyols that can be derived from sugars. Examples of suitable carbohydrates are trehalose, sucrose, dextrose, dextran, and combinations thereof. Thus, in some embodiments, the cryoprotectant is a carbohydrate selected from the group consisting of trehalose, sucrose, dextrose, dextran, and combinations thereof. Preferably, the cryoprotectant is trehalose or dextran. Trehalose is a disaccharide formed from two α-glucose units via a 1,1-glycosidic bond, and sucrose is a disaccharide that is O-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside. Optionally, other disaccharides may be used instead. Dextrose is D-glucose, and optionally, other monosaccharides may be used instead. In a more preferred embodiment of the present invention, the cryoprotectant is trehalose. In another embodiment of the present invention, the cryoprotectant is dextran, such as dextran 6000. In some embodiments, the cryoprotectant is present in the formulation in an amount of up to 8% w / v, preferably 2% w / v to 5% w / v, such as 2% w / v to 3% w / v. In some embodiments, the cryoprotectant is trehalose and is present in the formulation in an amount of 2% w / v to 6% w / v, such as 3% w / v. In some embodiments, the cryoprotectant is dextran, such as dextran 6000, and is present in an amount of 1% w / v to 3% w / v, such as 2% w / v to 2.5% w / v. Thus, in some embodiments, the cryoprotectant is a carbohydrate selected from the group consisting of trehalose, sucrose, dextrose, dextran, and combinations thereof; and is present in an amount of up to 8% w / v.

[0057] Surfactants and their characteristics are well known, and surfactants generally comprise at least one polar head group and at least one non-polar or hydrophobic tail. Surfactants used herein preferably comprise a single tail and preferably a single head group. Surfactants are preferably charge neutral, meaning that they carry no net charge under the conditions of their use. Alkylated sugars are most preferably used in the present invention, particularly polysorbates, more particularly polysorbate 20. In some embodiments, the surfactant is a nonionic surfactant. Preferably, the surfactant is a polyethoxylated nonionic surfactant, such as a polysorbate. In a preferred embodiment of the present invention, the surfactant is polysorbate-20.

[0058] Surfactants have been found to be particularly advantageous in improving stability and reducing the formation of aggregated or agglomerated particles in the formulation. Optionally, the surfactant can be present in an amount of 0.001% v / v to 0.1% v / v, preferably 0.03% v / v to 0.08% v / v, more preferably 0.005% v / v to 0.02% v / v. In some embodiments, the surfactant is present in the formulation in an amount of 0.005% to 0.015% v / v. Therefore, in some embodiments, the surfactant is a nonionic surfactant and is present in an amount of 0.0001% v / v to 0.1% v / v, preferably 0.03% v / v to 0.08% v / v, more preferably 0.005% v / v to 0.02% v / v. In some embodiments, the surfactant is present in the formulation in an amount of 0.005% to 0.015% v / v, for example, about 0.01% v / v. For example, the surfactant is polysorbate-20 and is present in an amount of about 0.01% v / v.

[0059] Buffers are known in the art and help maintain the pH of the composition within a given range. The buffer is typically a buffer salt. The buffer 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.

[0060] In some embodiments, the buffer may be a phosphate buffer, such as a phosphate buffer having a pH of 6.5 to 7.5. In some embodiments, the buffer may be an acetate buffer, such as an acetate buffer having a pH of 5 to 5.5. In some embodiments, the buffer may be a citrate buffer, such as a citrate buffer having a pH of 5.5 to 6.5. In some embodiments, the buffer may be a Tris buffer, such as a Tris buffer having a pH of 7.5 to 8. In some embodiments, the buffer may be a HEPES buffer, such as a HEPES buffer having a pH of 7 to 8. In some embodiments, the buffer may be aCSF, such as aCSF having a pH of 7.0 to 7.6. For example, the aCSF may have the following final ion concentrations (in mM): Na 150; K 3.0; Ca 1.4; Mg 0.8; P 1.0; Cl 155. Preferably, the buffer is a phosphate buffer or TRIS; for example, the buffer is a 10 mM phosphate buffer at a pH of 7.3.

[0061] Buffers, tonicity agents, cryoprotectants, and any other functionally defined components of the composition (such as surfactants) can be separate substances, but can also be mixtures. For example, a buffer can be a single phosphate (such as Na2HPO4), but can also be a mixture of substances (such as a mixture of Na2HPO4 and KH2PO4). Similarly, a tonicity agent can be a single substance (such as NaCl), but can also be a mixture (such as a combination of NaCl and KCl). Throughout this document, when only a single substance is specified for a functional definition, preferably no other substances are present for that same function. The same applies to situations where multiple substances are specified, in which case only the specified substances are included to achieve that same function.

[0062] Some compounds (such as phosphates) can act as buffers, but can also act as tonicity agents. As used herein, when a compound exists as a functionally defined component of a composition, then the compound is preferably considered to not meet any requirements of other functionally defined components. Thus, when one or more of acetate, citrate, HEPES, TRIS or phosphate exist, these are preferably considered only as buffers. When one or more of NaCl, KCl, MgCl , CaCl , or a mixture thereof exist, these are preferably considered only as tonicity agents. When one or more of trehalose, dextran, sucrose, dextrose or poly (ethylene glycol) exist, these are preferably considered only as cryoprotectants. When one or more nonionic surfactants exist, these are preferably considered only as surfactants.

[0063] In particularly preferred embodiments, the formulation comprises, consists of, or consists essentially of:

[0064] An adeno-associated viral vector with a transgene encoding a microRNA targeting ATXN3 mRNA;

[0065] About 5-30 mM phosphate buffer at a pH of 7 to 8;

[0066] at least about 75 mM NaCl;

[0067] About 2-5 mM KCl;

[0068] About 2% w / v to 4% w / v trehalose;

[0069] • About 0.005% v / v to 0.015% v / v PS-20.

[0070] For example, a formulation may comprise, consist of, or consist essentially of:

[0071] An adeno-associated viral vector with a transgene encoding a microRNA targeting ATXN3 mRNA;

[0072] About 9 mM Tris at a pH of about 7.3;

[0073] About 82 mM NaCl

[0074] About 3 mM KCl

[0075] About 3% w / v trehalose;

[0076] • About 0.01% v / v PS-20 (Tween 20).

[0077] For example, a formulation may comprise, consist of, or consist essentially of:

[0078] An adeno-associated viral vector with a transgene encoding a microRNA targeting ATXN3 mRNA;

[0079] About 9 mM Tris at a pH of about 7.3;

[0080] About 87 mM NaCl

[0081] About 3 mM KCl

[0082] About 3% w / v trehalose;

[0083] • About 0.01% v / v PS-20 (Tween 20).

[0084] In some embodiments, the formulation is substantially free of visible particles. Thus, the formulation is substantially free of particles visible to the naked eye, as determined and characterized according to the 2014 United States Pharmacopeial Convention (USP) Chapter 790, "VISIBLE PARTICULATES IN INJECTIONS" guidance for parenteral medicinal products ("substantially free" of visible particulate matter) (USP 790), which is incorporated herein by reference. Visible particles may be a sign of AAV particle aggregation, agglomeration and / or degradation, and thus the absence of visible particles may be a useful indicator that the formulation is particularly stable and does not have significant aggregation or agglomeration. Preferably, the formulation also contains limited subvisible particles, as determined and characterized according to the United States Pharmacopeial Convention (USP) Chapter 787, "SUBVISIBLE PARTICULATE MATTER IN THERAPEUTIC PROTEIN INJECTIONS" (USP 787), which is incorporated herein by reference. For example, in the formulation, the number of particles with a diameter of ≥25 μm does not exceed 600 per vial, and the number of particles with a diameter of ≥10 μm does not exceed 6000 per vial. Optionally, the formulation has a Dv90 of less than 50 μm, preferably less than 25 μm, more preferably less than 10 μm. The Dv90 value represents the percentage of the formulation (in this case, 90%) that is no larger than the specified value.

[0085] In certain embodiments, the preparation of the present invention can be used to treat spinocerebellar ataxia (SCA), such as spinocerebellar ataxia type 3 (SCA3). In certain embodiments, the preparation is administered by (cerebrospinal fluid) delivery in CSF. For example, the preparation is administered by delivery in CSF (such as intracisternal delivery). Optionally, the preparation can be administered by direct injection into the brain, or by repeated injection in the cerebral cisternae, or by slow infusion in the cerebral cisternae, or by direct injection into the brain and by a combination of one or more injections in the cerebral cisternae. In certain embodiments, the preparation can be administered by injection at the cerebral cisternae. In certain embodiments, the preparation can be administered by a single injection at the cerebral cisternae. For example, the preparation can be administered by (single) injection (optionally CT guided) into the cerebral cisternae within 5 minutes.

[0086] The cisterna magna, also known as the cerebellomedullary cistern, is one of three major openings in the subarachnoid space between the arachnoid and pia mater layers of the meninges surrounding the brain. These openings are collectively referred to as cisterna magna. The cisterna magna is located between the dorsal aspect of the cerebellum and medulla oblongata. Cerebrospinal fluid produced in the fourth ventricle flows into the cisterna magna through the lateral and median foramina.

[0087] In some embodiments, the formulation is administered at a dose of 5 mL per subject, wherein the concentration of the adeno-associated viral vector is 3E14 gc / mL. In some specific embodiments, the formulation is administered at a dose of 5 mL per injection site, wherein the concentration of the adeno-associated viral vector is 3E14 gc / mL. In some embodiments, the formulation is administered at a dose of 4E14 to 5E15 genome copies per subject, preferably 8E14 to 2E15 genome copies, more preferably 1E15 to 2E15 genome copies per subject. For example, the formulation is administered at a dose of 1.5E15 genome copies per subject.

[0088] 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.

[0089] Alternatively, or in addition as another kind of product of interest, the transgenic nucleotide sequence comprising coding as defined above MicroRNA can further comprise the nucleotide sequence of coded polypeptide, and this polypeptide serves as selection marker protein to assess cell transformation and expression.Suitable marker protein for this purpose is such as fluorescent protein GFP and selective marker gene HSV thymidine kinase (for selecting on HAT culture medium), bacterium hygromycin B phosphotransferase (for selecting on hygromycin B), Tn5 aminoglycoside phosphotransferase (for selecting on G418) and dihydrofolate reductase (DHFR) (for selecting on methotrexate), CD20, low-affinity nerve growth factor gene.Sambrook and Russel provide the source and the method for using thereof of obtaining these marker genes, see below.

[0090] In addition, the nucleotide sequence comprising the transgenic as defined above can include the other nucleotide sequence of coded polypeptide, which can serve as a fail-safe mechanism, and if it is considered necessary, the fail-safe mechanism makes the subject free from the influence of the cell transduced with the AAV vector of the present invention.Such nucleotide sequence (commonly referred to as suicide gene) encoding can convert prodrug into the protein of toxic substance, and this toxic substance can kill the transgenic cell in which protein is expressed.Suitable examples of such suicide gene include such as Escherichia coli (E.coli) cytosine deaminase gene or one of the thymidine kinase genes from herpes simplex virus, cytomegalovirus and varicella-zoster virus, in which case ganciclovir can be used as a prodrug to kill the transgenic cell of the subject (see, for example, Clair et al., 1987, Antimicrob.Agents Chemother [antimicrobial agent and chemotherapy] 31:844-849).

[0091] In one aspect, the present invention provides a method for preparing a pharmaceutical product for administration to the central nervous system, the method comprising the steps of:

[0092] i) providing a formulation according to the invention; and

[0093] ii) Dividing the formulation into suitable dosage forms.

[0094] The prepared drug product is suitable for administration to the central nervous system, for example as described elsewhere herein. Thus, the method is suitable for preparing a formulation for use according to the present invention, preferably wherein the composition is for administration into the cerebrospinal fluid. Thus, in one embodiment, the formulation for use according to the present invention is administered to the cisterna magna of a patient.

[0095] In yet another aspect of the invention, the present invention provides a method of treating SCA, particularly SCA3, by administering the formulation of the present invention to the brain of a patient.

[0096] definition

[0097] In this document and its claims, the verb "to comprise" and its conjugations are used in its non-limiting sense to mean including the items following the word, but not excluding items not specifically mentioned. Furthermore, the verb "to consist of" can be replaced by "consisting essentially of," meaning that a combination or composition as defined herein may include one or more additional components in addition to those specifically identified, provided that the one or more additional components do not alter the unique characteristics of the invention. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the presence of more than one of the element, unless the context clearly requires that one and only one of the element be present.

[0098] Thus, the indefinite article "a" or "an" usually means "at least one".

[0099] Whenever a parameter of a substance is discussed in the context of the present invention, it is assumed that the parameter is determined, measured, or displayed under physiological conditions unless otherwise specified. Physiological conditions are known to those skilled in the art and include an aqueous solvent system, atmospheric pressure, a pH between 6 and 8, a temperature from room temperature to about 37° C. (about 20° C. to about 40° C.), and appropriate concentrations of buffer salts or other components.

[0100] In the context of the present invention, a decrease or increase in a parameter to be evaluated means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase in that value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it may be the case that there is no longer a detectable value associated with this parameter.

[0101] The word "about" or "approximately" when used in conjunction with a numerical value (eg, about 10) preferably means that the value may be plus or minus 10%, optionally plus or minus 5%, of the given value (10).

[0102] Unless otherwise indicated, each of the embodiments described herein may be combined. The invention has been described above with reference to a number of embodiments. Those skilled in the art will appreciate minor variations in some of the elements of the embodiments. These variations are encompassed by the scope of protection defined in the appended claims. All patents and references cited are hereby incorporated by reference in their entirety.

[0103] Additionally, the following terms are used herein and are defined as follows.

[0104] “Capsid” – a protein shell surrounding viral DNA, RNA, or microRNA that helps target the genetic material to specific cell types.

[0105] A “gene cassette” – a small piece of DNA, RNA, or microRNA that contains a therapeutic gene and instructions for how cells should use that gene.

[0106] • "Vector" - this can refer to the DNA, RNA or microRNA molecule itself or a vector construct containing the DNA, RNA or microRNA to be delivered to the cell.

[0107] • "MicroRNA" (also called "miRNA" or "siRNA") - a single-stranded non-coding small RNA molecule, typically containing a guide strand as described above.

[0108] “SCA” – Spinocerebellar ataxia.

[0109] “Substantially” – The term “substantially” as used herein is a broad term and is to be given its ordinary and customary meaning to those skilled in the art (and not limited to a special or customary meaning), and, without limitation, means largely (but not necessarily entirely) as specified.

[0110] “PS-20” – Polysorbate 20.

[0111] “iPSC” – induced pluripotent stem cells.

[0112] • “ITR” – Inverted terminal repeat.

[0113] “MRI” – magnetic resonance imaging.

[0114] Specific embodiments of the invention are described in the following paragraphs.

[0115] 1. An isotonic pharmaceutical formulation comprising:

[0116] Buffer;

[0117] an adeno-associated viral vector with a transgene encoding a microRNA;

[0118] Tonicity agents;

[0119] Cryoprotectants; and

[0120] surfactant.

[0121] 2. The formulation of paragraph 1, wherein the formulation has an osmotic pressure of 250 to 330 mOsm / kg, preferably 260 to 310 mOsm / kg, for example 270 to 300 mOsm / kg.

[0122] 3. The formulation of paragraph 1 or 2, wherein the formulation has a pH of 5 to 8, preferably 6 to 8, more preferably 6.5 to 8, for example 7 to 8.

[0123] 4. The formulation of any of the preceding paragraphs, wherein the formulation is substantially free of visible particles.

[0124] 5. The formulation of any of the preceding paragraphs, wherein the adeno-associated viral vector with a transgene encoding a microRNA targets ATXN mRNA, such as ATXN3 mRNA.

[0125] 6. The formulation of any of the preceding paragraphs, wherein the adeno-associated viral vector comprises an AAV2 serotype, an AAV5 serotype, an AAV9 serotype, a hybrid AAV serotype, or a combination thereof, preferably the adeno-associated viral vector comprises an AAV5 serotype or an AAV9 serotype, such as an AAV5 serotype.

[0126] 7. The formulation of any of the preceding paragraphs, wherein the concentration of the adeno-associated viral vector is at least 1E13 gc / ml, such as at least 3E13 gc / ml. In some embodiments, the concentration of the adeno-associated viral vector is between 0.5E13 and 6E13 gc / ml, such as between 1E13 and 5E13 gc / ml.

[0127] 8. The formulation of any of the preceding paragraphs, wherein the cryoprotectant is a sugar.

[0128] 9. The formulation of paragraph 8, wherein the cryoprotectant is a sugar selected from the group consisting of trehalose, sucrose, dextrose, dextran, and combinations thereof; for example, the cryoprotectant is trehalose; alternatively, the cryoprotectant is dextran, such as dextran 6000.

[0129] 10. The formulation of any of the preceding paragraphs, wherein the cryoprotectant is present in an amount of up to 8% w / v, preferably 2% w / v to 5% w / v, such as 2% w / v to 3% w / v.

[0130] 11. The formulation of any of the preceding paragraphs, wherein the cryoprotectant is trehalose and is present in an amount of 3% w / v.

[0131] 12. The formulation of any of the preceding paragraphs, wherein the tonicity agent is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, and combinations thereof.

[0132] 13. The formulation of paragraph 12, wherein the tonicity agent is a combination of NaCl and KCl.

[0133] 14. The formulation of any of the preceding paragraphs, wherein the tonicity agent is present in an amount of 5mM to 150mM, preferably 25mM to 125mM, more preferably 75mM to 125mM, for example 90mM.

[0134] 15. The formulation of paragraph 14, wherein the tonicity agent comprises at least 75 mM NaCl.

[0135] 16. The formulation of any of the preceding paragraphs, wherein the surfactant is a nonionic surfactant, preferably a polyethoxylated nonionic surfactant, such as a polysorbate, for example polysorbate-20.

[0136] 17. The formulation of any of the preceding paragraphs, wherein the surfactant is present in an amount of 0.001% v / v to 0.1% v / v, preferably 0.003% v / v to 0.08% v / v, more preferably 0.005% v / v to 0.02% v / v, for example about 0.01% v / v.

[0137] 18. The formulation of any of the preceding paragraphs, wherein the surfactant is polysorbate 20 and is present in an amount of 0.01% v / v.

[0138] 19. The formulation of any of the preceding paragraphs, 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 9-10 mM phosphate buffer at pH 7.3.

[0139] 20. The formulation of any of the preceding paragraphs, wherein the formulation remains stable at room temperature and substantially does not form aggregates or agglomerates for at least 12 hours, preferably at least 18 hours, such as at least 24 hours after thawing.

[0140] 21. The formulation of any of the preceding paragraphs, for use in the treatment of spinocerebellar ataxia (SCA), such as SCA Type 1, SCA Type 2, or SCA Type 3, preferably SCA Type 3.

[0141] 22. The formulation for use as described in paragraph 21, wherein the formulation is administered by intra-CSF (cerebrospinal fluid) delivery; preferably, the formulation is administered by intracisternal delivery.

[0142] 23. The formulation for use as described in paragraph 21 or 22, wherein the formulation is administered to the cisterna magna of the patient.

[0143] 24. The formulation for use as described in any one of paragraphs 21 to 23, wherein the formulation is administered with a contrast agent for MRI monitoring of the administration.

[0144] 25. A method of treating spinocerebellar ataxia by administering to a patient the formulation of any of the preceding paragraphs.

[0145] 26. The method of paragraph 25, wherein the formulation is administered to the patient as a single dose delivered by intracisternal injection.

[0146] 27. The method of paragraph 25 or 26, wherein the formulation is administered with a contrast agent for visualization by MRI. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] Specific embodiments of the present invention will be described by way of example with reference to the accompanying drawings, in which:

[0148] Figure 1 Shown are the results of Example 1. The Tm (melting temperature of the capsid as determined by the iDSF method) changes with accelerated stress conditions and duration.

[0149] Figure 2The changes in protein content and fluorescence as a function of stress conditions and duration are shown for different salt concentrations in Example 1. NaCl concentrations are shaded gray. Different buffers were combined. UV280 content and Trp_Fluo:280,335 are in arbitrary units. The time scales are 7 days and 2 days for storage at 25°C and 40°C, respectively, and 24 hours for shaking.

[0150] Figure 3 The production curve is obtained from a DoE experiment based on the absorbance and fluorescence responses over the complete data set for all time points and all stresses.

[0151] Figure 4 The production curve is obtained from a DoE experiment based on the absorbance and fluorescence responses over the complete data set for all time points and all stresses.

[0152] Figure 5 Shown are the results of polydispersity index (PDI) measurements by dynamic light scattering as a function of concentration factor.

[0153] Figure 6 Shown are the z-average measurements by dynamic light scattering as a function of the concentration factor.

[0154] Figure 7 Shown are z-average measurements of accelerated stability and long-term stability as measured by dynamic light scattering.

[0155] Figure 8A / B shows subvisible particle analysis of accelerated stability and long-term stability.

[0156] Figure 9 The figure shows a summary of visual inspection of AAV9 DP in five formulation buffers under different stress conditions: No filling: no visible particles; Diagonal filling: one to two visible particles; Dense diagonal filling: a few visible particles.

[0157] Figure 10 Shown are luciferase activities in cell lysates of Huh-7 cells transduced with AAV9(1489)-luciferase formulated in different buffers.

[0158] Examples

[0159] abbreviation

[0160] AAV is adeno-associated virus; AC is affinity chromatography; aCSF is artificial cerebrospinal fluid; AIM is analytical research method; CSF is cerebrospinal fluid; DLS is dynamic light scattering; DoE is experimental design; DP is drug product; DPD is drug product development; DS is drug substance; F / T is freeze / thaw; Gc is genome copies; GMP is good manufacturing practice; iDSF is endogenous differential scanning fluorimetry; IEX is ion exchange; MW is molecular weight; N / A is not applicable; NF is nanofiltration; PD is process development; PDI is polydispersity index; PEG is polyethylene glycol; RU is arbitrary unit; SOP is standard operating procedure; STR is stirred tank reactor; Tp is total particle size; UF / DF is ultrafiltration / diafiltration; VI is visual inspection

[0161] Materials and Methods The following materials were used in the Examples, their sources are specified in Table 1 below.

[0162] Table 1: Materials used in the examples and their suppliers

[0163] raw materials supplier Acetic acid Merck glacial acetic acid 100%, citric acid Merck Dextran 20000 Sigma Dextran 55000 Sigma Dextran 6000 Sigma Glucan 70000 Sigma dextrose Sigma Ethanol 96% Fisher Chemical Preparation buffer PS-20 JTBaker HCl (37%) Merck Hepes Sigma Hepes sodium salt Sigma <![CDATA[K2HPO4 (salt)]]> MP Biomedical KCl Merck <![CDATA[KH2PO4 (acid)]]> Merck L-histidine Sigma <![CDATA[MgCl2-6H2O]]> Sigma NaCl Merck PEG 200 Sigma PEG 20000 Sigma PEG 35000 Sigma PEG 4000 Sigma Polysorbate 20 (PS-20) JTBaker Sodium acetate Merck Trisodium citrate dihydrate Sigma-Aldrich Disodium hydrogen phosphate Merck sucrose Merck Trehalose Sigma Tris Merck

[0164] Measurement results

[0165] The readings obtained are as follows:

[0166] • Absorbance at 260 nm, which is related to DNA concentration.

[0167] • Absorbance at 280 nm, which is related to protein concentration.

[0168] • Absorbance at 350 nm (turbidity), which is associated with protein aggregation and / or particle formation.

[0169] • Absorbance at 900 nm, which is related to the background signal from the plastic of the wells and seals.

[0170] Absorbance at 975 nm measures the absorbance of water and thus the optical path length.

[0171] Tryptophan fluorescence at 280 / 335 nm, measured from the top and bottom of the microplate. This measurement correlates with protein and / or capsid unfolding and denaturation. The top reading is used in the figures.

[0172] Protein and DNA concentrations were obtained as follows:

[0173] According to the Lambert-Beer law, absorbance is proportional to concentration and path length. A constant called the extinction coefficient converts this into an equation.

[0174] A = cc.L.ε, where A is the absorbance, cc is the concentration, L is the optical path length, and ε is the extinction coefficient.

[0175] The optical path length is measured by subtracting the plastic background measured at 900 nm (A900) from the absorbance of water measured at 975 nm (A975), thus:

[0176] L=A975-A900

[0177] Protein concentration was determined by measuring absorbance at 280 nm (A280), thus:

[0178] A280=cc(protein).L.ε(protein)

[0179] The background or turbidity of precipitated and aggregated proteins is measured by absorbance at 350 nm (A350). It is assumed to have a similar contribution to the turbidity at 280 nm and is therefore subtracted from the absorbance at 280 nm. Then:

[0180] cc(protein).ε(protein)=(A280–A350) / (A975-A900)

[0181] Since ε(protein) is a constant, we can conclude that cc(protein).ε(protein) is proportional to the protein concentration, which is calculated from the different absorbance values ​​measured (A280, A350, A975, and A900). Similar reasoning applies to the DNA concentration at 260 nm.

[0182] use Intrinsic Differential Scanning Fluorescence (iDSF) measurement

[0183] use The denaturation temperature of the capsid is measured using a technique that heats the sample at elevated temperatures and measures the capsid's fluorescence. As the capsid denatures and exposes the fluorescent amino acids to the solvent environment, the fluorescence decreases as the environment changes from the rigid interior of the protein to a less rigid one. The ratio between the fluorescence at 335 nm and 350 nm is calculated, making the measurement independent of concentration.

[0184] Example 1 - Formulation Studies AAV5

[0185] The drug product (DP) used in this example was AAV5 with a transgene encoding a microRNA targeting AAXN3 mRNA at a concentration of 4.7E13 gc / mL.

[0186] rAAV5-miAAXN3 was buffer-exchanged into thirty different formulations containing different pH, buffer types, and salt contents in a multiwell system using a 96-well filter plate (molecular weight cutoff of 100 kDa). After buffer exchange and filtration through a 0.2 μm filter, the formulations were aliquoted into four plates according to the plate layout and buffer formulation composition. The final concentration of AAV5 in the wells was 1,0E13 gc / mL. Each formulation was prepared in duplicate. The outer rows and columns were filled with WFI to exclude plate position effects. The buffers had overlapping pH to distinguish between buffer and pH effects.

[0187] Each plate was assigned to a specific stress condition: F / T at 80°C, storage at 25°C and 65°C and shaking at RT. The accelerated study included the following:

[0188] • Freezing at -80°C (at least 1 h) and thawing at RT (1 hour) for 1, 3 and 10 cycles. The table with the experimental dates shows when each F / T cycle was performed.

[0189] Shake at RT for 1 and 6 hours. Due to deviations, there were two shake measurements in the experiment. The first shake experiment was not centrifuged before measurement, so a second shaker plate was added, which was centrifuged and shaken at RT for 1 and 6 hours.

[0190] • Storage at 25°C for 2, 7 and 14 days.

[0191] • Storage at 65°C for 2, 7 and 14 days.

[0192] • For all conditions described above, prepare the microplate and perform an initial measurement (T=0).

[0193] The protein content at T = 0, measured by absorbance at 280 nm (corrected for turbidity and optical path length in four multiwell plates; arbitrary units), is shown in Table 2. The protein content at T = 0, measured by fluorescence at 335 nm (measurements from the top of four multiwell plates; arbitrary units), is shown in Table 3.

[0194] Table 2: Protein content at T=0 measured by absorbance at 280 nm (A: F / T at -80°C; B: shaking; C: 25°C; D: 65°C)

[0195]

[0196] Table 3: Protein content at T=0 measured by fluorescence at 335 nm (A: F / T at -80°C; B: shaking; C: 25°C; D: 65°C)

[0197]

[0198] Table 2 clearly shows that the corrected absorbance values ​​(protein concentration) at 280 nm for the four multiwell plates are similar but not identical, as expected. In addition, for the four plates at T = 0, the absorbance in the presence of 150 mM NaCl is higher for almost all conditions, thus indicating a higher protein content and a lower degree of aggregation. This can be concluded because the measured values ​​include background correction. Overall, it was observed that the effect of the different formulations occurred immediately at T = 0 during the buffer exchange.

[0199] In Table 3, stabilizing salt effects were observed for most conditions at both 75 mM and 150 mM NaCl. Acetate at pH 5 and 5.5 produced mostly destabilizing effects, especially in the absence of NaCl. Phosphate at pH 7.5 and citrate at pH 6.5 appeared to favor the folded conformation. As noted above, the fact that effects were observed at T = 0 suggests that these effects occurred during the buffer exchange step.

[0200] The results of the accelerated study were shown in Figure 1 These results demonstrate the importance of pH in the formulation and the influence of salt concentration.

[0201] The overall observations are:

[0202] • Effect of all tested buffers (20 mM: acetate, citrate, phosphate and Tris) on drug product stability measured by analytical assay: absorbance and fluorescence are seen immediately at T=0.

[0203] • The lack of salts and buffers is detrimental to the stability of the drug product.

[0204] • The addition of salt (NaCl), especially at concentration levels of 75 mM or higher, is beneficial for formulation stabilization effects.

[0205] • Neutral or slightly alkaline formulation pH (7.0-7.5) is better for drug product stability.

[0206] • Formulations with alkaline buffering capacity (such as Tris) are more stable when compared to buffered formulations with lower pH.

[0207] Formulations with NaCl in buffer at neutral or slightly alkaline pH (7.5) were used for further testing and optimization.

[0208] Excipient screening studies

[0209] According to the above results, neutral to slightly alkaline pH and the inclusion of salts support the stability of the capsid. Three buffers were selected: phosphate, Tris and Hepes to support a pH equal to 7.5. As a control, artificial cerebrospinal fluid (aCSF) was used. 117 formulations in different buffers were prepared with increasing concentrations of NaCl and defined concentrations of putative excipients; the concentrations of the latter were set to be sufficient to affect viscosity, but low enough not to have a significant effect on molar osmotic pressure concentration. The different excipients tested and their concentrations are shown in Table 4 below, including surfactants (such as various PEGs) and cryoprotectants (such as trehalose, sucrose, dextrose and dextran). Polymeric excipients were selected to increase the viscosity of the formulations.

[0210] Table 4: Excipients and their corresponding concentrations

[0211]

[0212]

[0213] In the first excipient screen, four sets of multiwell plates were prepared.

[0214] For the second excipient screen, 18 formulations were prepared in glass vial containers. These contained three buffers, Tris, HEPES, and phosphate, as described previously, and three putative excipients: trehalose, low MW dextran, and dextrose.

[0215] The plates of the first excipient screen and the vials of the second excipient screen were subjected to accelerated studies under different stresses: F / T, shaking and storage at two different temperatures (25°C and 40°C) with the following durations / cycles as specified in Table 5 below:

[0216] Table 5: Accelerated stress test conditions used

[0217]

[0218] The changes in protein content and fluorescence data as a function of stress conditions and duration are shown in Figure 2 Also used The software performs DoE experimental design and the prediction curve is shown in Figure 3 and Figure 4 The results showed that higher salt content had a positive effect, while the effect of buffer showed that the most favorable buffers were phosphate and HEPES.

[0219] The overall observations are:

[0220] The effect of the buffer is rapid, occurring during the buffer exchange step and observed at T = 0;

[0221] High salt concentration is beneficial to sample stability, as is slightly alkaline buffer;

[0222] Dextran 6000 and trehalose have better stabilizing effects than dextrose

[0223] HEPES and phosphate are slightly better than Tris.

[0224] The following buffers and excipients were selected for further testing and optimization.

[0225] HEPES and phosphate as buffers

[0226] Dextran 6000 and trehalose as sugar excipients.

[0227] Aggregate point research

[0228] The four buffers used in the formulations are specified in Table 6 below;

[0229] Table 6: Composition of buffer used

[0230]

[0231] These formulations were gradually concentrated by diafiltration. Samples were collected after each concentration step. Table 7 below shows the concentration factor and expected concentration.

[0232] Table 7: Sequential concentration steps applied to different formulations

[0233]

[0234] The following measurements were taken after each concentration step:

[0235] Genome copies / mL (Gc / mL)

[0236] Total particles / mL (TP / mL)

[0237] • Visual inspection was performed at T=0 and after 24 hours at 4°C and after two freeze and thaw (F / T) cycles.

[0238] ·pass Measured sub-visible particles.

[0239] • Polydispersity index by dynamic light scattering.

[0240] Table 8 below shows a visual inspection of the four formulations as a function of concentration factor, also after stress (e.g., storage at 4°C for 24 hours or after two F / T cycles). It was observed that the formulations containing phosphate performed better than the formulations containing HEPES up to six-fold concentration. It was also observed that the formulations containing trehalose performed better than the formulations containing dextran 6000 after the six-fold concentration step.

[0241] Table 8: Inspection of visible particles as a function of concentration fold after storage at 4°C and F / T cycles.

[0242]

[0243] In Table 9 below, it is observed that the number of particles increases as the different formulations are concentrated. Furthermore, all formulations show very similar results before the six-fold concentration step, which defines the maximum possible concentration as described above. Only the formulations containing phosphate were subjected to a concentration step up to 15-fold, which showed an increase in the number of particles as expected.

[0244] Table 9: Changes of subvisible particles with concentration factor

[0245]

[0246] The polydispersity index (PDI) and z-average measurements by dynamic light scattering give information about the particle size, uniformity and shape of the formulations studied. The results of the PDI and z-average for these formulations are shown in Figure 5 and Figure 6 The PDI represents the distribution of particle size populations within a given sample. PDI values ​​range from 0.0 (for a sample that is completely uniform in particle size) to 1.0 (for a highly polydisperse sample with multiple particle size populations). Figure 6 All four formulations showed a plateau after sixfold concentration, consistent with the GC and TP measurements described above. Furthermore, the presence of trehalose resulted in smaller aggregates than those in the presence of dextran 6000. The same effect was observed for the phosphate-containing formulations, which induced smaller particles than their HEPES counterparts. The z-average behavior mirrored that of the PDI, as described above.

[0247] Table 10 below shows the results of the stability test.

[0248] Table 10: Stability test with concentration multiples.

[0249]

[0250] C = clear; Co = cracked; T = turbid.

[0251] The overall observations are:

[0252] The highest achievable concentration is 6E14 gc / mL (6-fold concentration)

[0253] The formulation in phosphate buffer showed a better visible particle profile exceeding 6E14 gc / mL

[0254] Phosphate / trehalose showed the narrowest particle size distribution

[0255] Formulation Qualification Studies

[0256] For further testing and optimization, two formulations with enhanced stability were proposed:

[0257] Formulation 1. Phosphate / Trehalose: Phosphate (Na / K) 10 mM, NaCl 82 mM, KCl 3 mM, Trehalose 3% (w / v), pH 7.3.

[0258] Formulation 2. Phosphate / Dextran: Phosphate (Na / K) 10 mM, NaCl 125 mM, KCl 3 mM, Dextran 6000 2.2% (w / v), pH 7.3.

[0259] Both formulations were filled to a final volume (1.2 mL) and packaged into container capped vials (2 ml Type I borosilicate glass vials sealed with siliconized stoppers and flip-off seals). These vials were subjected to accelerated studies under the following stresses: F / T, shaking, and short-term development stability at 25°C and 40°C, and one-year development stability (-80°C and 2°C-8°C), and these stress conditions included the following:

[0260] • Freeze at -80°C (at least 1 h) and thaw at RT (1 hour) for 3 cycles (see upper portion of study date schedule for actual cycle lengths).

[0261] Shake at 200 rpm for 24 hours.

[0262] Accelerated stability: 2 weeks at 25°C

[0263] Accelerated stability: Store at 40°C for 1 week.

[0264] A short-term developmental stability study at -20°C for 3 months

[0265] Long-term developmental stability studies at -80°C (target DP storage temperature) and 2°C-8°C for 1, 3, 6, 9, and 12 months

[0266] • For all the above conditions, an initial measurement (T=0) was performed.

[0267] Additional time points were added for appearance and visual inspection after 1 F / T, after 1 week at 25°C and after 2 weeks at 40°C

[0268] The subsequent measurements are as follows:

[0269] Appearance / visible particles (VI)

[0270] Subvisible particles

[0271] Genome copies (gc); this is related to concentration

[0272] tp: total particles

[0273] ip: infectious particles

[0274] gc / ip: (genome copies / infectious particles); this is related to the number of active particles

[0275] tp / gc (total particles / genomic copies); this is related to purity

[0276] PDI (polydispersity index) measured by DLS; it is expected to be lower than 0.1 since we aim to obtain a monodisperse system

[0277] Complete data are shown in Tables 11 to 13 below. Figure 7 Additional data are shown in Figures 2 and 3.

[0278] Table 11: Complete data set used for the accelerated stress condition study.

[0279]

[0280] Table 12: Complete data set for long-term development stability studies at 2°C-8°C, -80°C and -20°C.

[0281]

[0282] Table 13: Visual inspection results of stress test

[0283]

[0284] C = clear; Co = cracked; T = turbid.

[0285] The results confirmed that the formulation containing trehalose was superior to the formulation containing Dextran 6000. The strongest data supporting this conclusion came from visual inspection analysis, which showed that the formulation containing trehalose produced significantly fewer particles after F / T cycling or (long-term) storage at -80°C. Furthermore, DLS measurements showed that the PDI values ​​of the samples containing trehalose were almost half of those of the samples containing Dextran 6000, meaning that the samples containing trehalose were more uniform than those containing Dextran 6000. The overall conclusion drawn from the subvisible particle data was that trehalose showed significantly better results under long-term storage conditions.

[0286] This formulation: Phosphate (Na / K) 10 mM, NaCl 82 mM, KCl 3 mM, Trehalose 3% (W / V), and pH 7.3 is recommended for further optimization.

[0287] Further studies were conducted to select one of the following five different formulations containing different DP concentrations, MgCl2 and / or PS-20 for the drug product. Previous early formulation reports indicated that the formulation with phosphate (Na / K) 10 mM, NaCl 82 mM, KCl 3 mM, trehalose 3% w / v, pH 7.3 was recommended as a starting point for further optimization.

[0288] Different DP materials are formulated as follows:

[0289] 1. 3E14 gc / mL in trehalose / phosphate buffer;

[0290] 2. 4E14 gc / mL in trehalose / phosphate buffer;

[0291] 3. 3E14 gc / mL in trehalose / phosphate buffer + 0.8 mM MgCl2;

[0292] 4. 3E14 gc / mL + 0.005% v / v PS-20 in trehalose / phosphate buffer;

[0293] 5. 3E14 gc / mL + 0.01% v / v PS-20 in trehalose / phosphate buffer.

[0294] The trehalose / phosphate buffer contained 9 mM phosphate buffer, 82 mM NaCl, 3 mM KCl and 3% w / v trehalose. Stress studies were performed on these formulations and the results are shown in Table 14 below.

[0295] Table 14: Results of stress tests C = clear; Co = cracked; T = turbid.

[0296]

[0297] The results shown above demonstrate that the addition of PS-20, especially at 0.01% v / v, has a positive effect on the stability of the formulation.

[0298] in conclusion

[0299] Plate-based screening studies highlight the stabilizing effects of NaCl and neutral to slightly alkaline pH

[0300] Studies have shown that trehalose and dextran 6k have a stabilizing effect in phosphate buffer or HEPES buffer

[0301] Phosphate + Trehalose / Dextran 6k supports concentrations up to 6E14 gc / mL

[0302] Phosphate + Trehalose shows excellent visible particle curve

[0303] Formulation optimization by adding 0.01% v / v PS-20 improved visual appearance and significantly reduced subvisible particles

[0304] The formulation has been optimized for frozen storage at ≤-65°C

[0305] In addition to the adeno-associated viral vector having a transgene encoding a microRNA, a specific embodiment of the present invention further comprises the compositions shown in Table 15 below.

[0306] Table 15: Assay buffers

[0307]

[0308]

[0309] Example 2 - Formulation Studies AAV9

[0310] The drug product (DP) used in this example was AAV9 with a transgene encoding a luciferase reporter gene at a concentration of approximately 3E13 gc / mL (see Table 19).

[0311] The compositions of the five formulation buffers used in this study are listed in Table 15. All five buffers were prepared in a volume of 4 L and filtered using a 0.22 μm PES filter. Poloxamer 188 and PS20 surfactants were freshly added to the corresponding formulation buffers on the day of the buffer exchange process.

[0312] Dialysis was used to exchange 30 mL of AAV9 formulation for each buffer condition. Three buffer exchange cycles (1 L per cycle) were performed at 2°C-8°C for 16 hours.

[0313] The AAV9 volume collected after the buffer exchange process was filtered using a 0.22 μm PVDF syringe filter. AAV9 DP was filled in 2 mL type I glass vials with a fill volume of 1.2 mL DP. Table 16 shows the total number of DP-filled vials per collected AAV9 volume in each formulation buffer.

[0314] Table 16: Number of vials filled with AAV9 DP in each formulation buffer

[0315]

[0316]

[0317] All DP filled vials were visually inspected. DP vials that passed the visual inspection were subjected to different stress conditions as listed in Table 17 and analyzed as described in Table 18.

[0318] Table 17: Number of AAV9 DP vials per stress condition

[0319]

[0320] Table 18: Overview of analytical methods

[0321]

[0322]

[0323] result

[0324] Genome copies

[0325] Genome copies (GC) measure the amount of AAV particles that are packaged with vector DNA. GC concentration was determined by quantitative PCR. GC assays were performed under 2°C-8°C stress conditions for T=0 (after buffer exchange), SHK (1 day, 200 rpm), 1FT+7 days, and 1FT+4W. The average of three GC assays of two vials of each formulation buffer at T=0 is shown in Table 19.

[0326] Table 19: Genome copies of AAV9 DP in different formulation buffers after DP filling

[0327] describe Genome copies (gc / mL) AAV9 Buffer 1 3.17E+13 AAV9 Buffer 2 2.98E+13 AAV9 Buffer 3 4.00E+13 AAV9 Buffer 4 3.27E+13 AAV9 Buffer 5 2.67E+13

[0328] Taking into account the 20% GC assay variation, AAV9 GC was approximately 3E13 gc / ml in all formulations tested. In formulation buffer 3, the measured AAV9 concentration was slightly higher, at 4E13 gc / ml. No observable GC loss was detected in any of the samples tested under the different stress conditions.

[0329] pH and osmolarity

[0330] The pH and osmolarity data were close to the theoretical values ​​for each formulation buffer (see Table 20), indicating that the buffer exchange was performed correctly.

[0331] Table 20: pH and osmolarity of AAV9 DP in different formulation buffers

[0332]

[0333] Before buffer exchange, the measured osmolarity of the AAV9 buffer was 687 mOsm / kg and the pH was 8.5, with a buffer composition of 0.1 M glycine and 0.5 M Tris at a pH of 8.3. After buffer exchange, all formulations were close to the target pH and theoretical osmolarity. With the exception of buffer 4, Zoltgensma's formulation buffer, and the hypertonic formulation, all other formulations were isotonic.

[0334] Visual and subvisible particle analysis

[0335] Visual Inspection Acceptance Criteria and USP <790> The AAV9 DP vials were visually inspected after filling and after stress application. DP vials containing extraneous particles after filling and vials with low fill volumes were excluded. The results are shown in Tables 21 and Figure 9 middle.

[0336] Table 21. Visual inspection results at T=0 after DP filling and completion

[0337]

[0338] 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 was free of particles after one day of agitation at RT, after three freeze / thaw cycles, and after one week of thawing when stored at RT, or two weeks of thawing when stored at 2°C-8°C. Figure 9 ). Two vials under each stress condition and formulation buffer were analyzed for subvisible particles using light obscuration technology AccuSizer FX.

[0339] Subvisible particles maintained at USP under all stress conditions <787> The results were within the acceptance criteria of 6000 particles / mL for particles larger than 10 μm and 600 particles / mL for particles larger than 25 μm. Similar aggregation profiles were observed for all formulation buffers tested in the 0.3-1 μm region. AAV9 aggregation was accelerated under freeze-thaw stress, while agitation stress conditions had little effect on aggregation.

[0340] It was generally observed that under all applied stress conditions, the number of subvisible particles of AAV9 larger than 2 μm was lower in formulation buffer 3 when compared to the other formulations.

[0341] Background membrane imaging (BMI)

[0342] Particles sized 2 to 4 μm were visualized using the Aura system, which uses BMI technology, an automated membrane microscopy technique that can count particles and determine size and morphology in a 96-well format. After 1FT+ for 4 weeks at 2°C-8°C, AAV9 DP in formulation buffer 4 contained the highest number of particles >2 μm compared to other formulations, which was consistent with visual inspection of visible and subvisible particle analysis using light obscuration techniques.

[0343] AAV9 transduction into target cells

[0344] The potential effects of different formulations on AAV9 transduction efficiency have been evaluated in vitro in a cell-based assay. For each buffer condition, AAV9 transduction efficiency was tested in human hepatocellular carcinoma (Huh7) cells. Huh7 cells were selected as an in vitro model cell line based on previously published data from Pietersz et al., 2021 (Pietersz KL, Plessis FD, Pouw SM, Liefhebber JM, van Deventer SJ, Martens GJM, Konstantinova PS, Blits B. PhP. BEnhanced Adeno-Associated Virus Mediated-expression after Systemic Deliveryor Direct Brain Administration. Front Bioeng Biotechnol. 2021 Aug 3;9:679483. doi:10.3389 / fbioe.2021.679483. PMID:34414171; PMCID:PMC8370029).

[0345] Differentially formulated AAV9 DP and wild-type Adeno5 and Ad5 ARM adenovirus were co-transduced into Huh7 cells at three different AAV9 doses (GC / cell), also known as the multiplicity of infection (MOI). In addition to untreated AAV9 in the different formulation buffers, 50 μL of AAV9 was heat-inactivated by incubation at 95°C for 10 minutes in each buffer condition as a negative control for each condition. Reading was performed using a dual-luciferase reporter gene assay system.

[0346] like Figure 10 As shown, from the luciferase activity in cell lysates of Huh-7 cells transduced with AAV9, it can be concluded that different buffer formulations do not significantly affect the transduction efficiency of AAV9, regardless of the MOI used.

[0347] in conclusion

[0348] A platform formulation fitting study was performed to select the most suitable formulation for brain administration from among those already developed, where AAV9-luciferase proved to be the most stable under the selected stress conditions.

[0349] Overall, AAV9 DP was most stable in Formulation Buffer 3 under stress conditions. No visible particles were observed in Formulation Buffer 3 under all stress conditions, and fewer subvisible particles larger than 2 μm were observed compared to the other selected formulations. Subvisible particles remained within the USP <787> The results were within the acceptance criteria of 6000 particles / mL for particles larger than 10 μm and 600 particles / mL for particles larger than 25 μm. Other quality attributes, genome copies, SEC purity (% monomer), hydrodynamic radius and polydispersity index, and unfolding temperature, did not show significant changes in response to applied stress when compared to T=0. Furthermore, AAV9 transduction into target cells was not affected by the formulation buffer, regardless of the multiplicity of infection used.

[0350] Therefore, buffer 3 was considered the most suitable formulation for maintaining the quality of the AAV9-luciferase product and should be considered for subsequent studies. In addition, this formulation is isotonic, with a tonicity and pH similar to cerebrospinal fluid: 280-310 mOsm / kg and pH 7.3-7.4 (Salvador L, Valero R, Carrero E, Caral L, Fernández S, Marín JL, Ferrer E, Fábregas N., Cerebrospinal fluid composition modifications after neuroendoscopic procedures. MinimInvasive Neurosurg. 2007 Feb;50(1):51-5. doi:10.1055 / s-2007-973823. PMID:17546545).

[0351] Table 22: Formulation of a specific embodiment of the present invention (excluding adeno-associated viral vector)

[0352]

[0353] Table 23: Formulation of a specific embodiment of the present invention (excluding adeno-associated viral vector)

[0354]

Claims

1. An isotonic pharmaceutical formulation comprising: Buffer; an adeno-associated viral vector with a transgene encoding a microRNA; Tonicity agents; Cryoprotectants; and surfactant.

2. The formulation of claim 1, wherein the formulation has an osmotic pressure of 250 to 330 mOsm / kg and a pH of 5 to 8.

3. The formulation of any preceding claim, wherein the formulation is substantially free of visible particles.

4. The formulation of any preceding claim, wherein the adeno-associated viral vector with a transgene encoding a microRNA targets ATXN mRNA, such as ATXN3 mRNA.

5. The formulation of any preceding claim, wherein the adeno-associated viral vector comprises an AAV2 serotype, an AAV5 serotype, an AAV9 serotype, a hybrid AAV serotype, or a combination thereof, preferably the adeno-associated viral vector comprises an AAV5 serotype or an AAV9 serotype, more preferably an AAV5 serotype.

6. The formulation of any preceding claim, wherein the concentration of the adeno-associated viral vector is at least 1E13 gc / ml, such as at least 3E13 gc / ml.

7. The formulation of any preceding claim, wherein the cryoprotectant is a carbohydrate selected from the group consisting of trehalose, sucrose, dextrose, dextran, and combinations thereof; and is present in an amount of up to 8% w / v.

8. The formulation of any preceding claim, wherein the tonicity agent is selected from the group consisting of: NaCl, KCl, MgCl2, CaCl2, and combinations thereof; and is present in an amount of 5 mM to 150 mM.

9. A formulation as claimed in any preceding claim, wherein the surfactant is a nonionic surfactant; and is present in an amount of 0.001% v / v to 0.1% v / v, preferably 0.003% v / v to 0.08% v / v, more preferably 0.005% v / v to 0.02% v / v, for example about 0.01% v / v.

10. The formulation of any preceding claim, 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 of any preceding claim, wherein the formulation remains stable at room temperature (15-25°C) and forms substantially no aggregates or agglomerates for at least 12 hours after thawing.

12. A formulation as claimed in any preceding claim for use in the treatment of spinocerebellar ataxia (SCA).

13. The formulation for use according to claim 12, wherein the formulation is administered by intra-CSF (cerebrospinal fluid) delivery.

14. A formulation for use as claimed in claim 12 or claim 13, wherein the formulation is administered to the cisterna magna of a patient.

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