formulations for antiviral drug products

Abstract

This invention relates to the field of gene therapy. Furthermore, it relates to the field of diseases caused by defective or deficient proteins. In particular, this invention relates to gene therapy for enzyme replacement, and more particularly to pharmaceutical formulations with improved stability for treating diseases that can benefit from enzyme replacement, including lysosomal storage disorders.

Description

Technical Field

[0001] This invention relates to the field of gene therapy. Furthermore, this invention relates to the field of diseases caused by gene defects that result in partial or complete protein defects (non-functionality) or lack thereof, or both. In particular, this invention relates to gene therapies for protein replacement, and more particularly to pharmaceutical formulations of those gene therapies with improved stability for treating diseases that can benefit from such gene therapies for protein replacement, especially enzyme replacement, including lysosomal storage disorders. Background Technology

[0002] The understanding that DNA is the carrier of genetic information and therefore the source of hereditary diseases has spurred the development of therapies in which mutated, damaged genes can be replaced or silenced. Many genes or other nucleic acids have been found to play a role in (hereditary) diseases. If one or more mutated genes can be replaced by healthy genes, or if genes expressing abnormal (sometimes toxic) products can be silenced, then the disease can be treated at the molecular level and potentially cured. Gene therapy offers a promising concept, particularly for diseases caused by mutations in a single gene, where such mutations result in reduced expression or function, or both (i.e., partial or complete absence of the protein and / or partial or complete non-functionality). However, delivering the desired nucleic acid into cells is not an easy task. Various (viral) delivery systems have been investigated, each with its own advantages and disadvantages. One viral delivery medium used in gene therapy is recombinant adeno-associated virus (rAAV).

[0003] Wild-type 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 Rep (replication) and Cap (capsid) genes. These coding sequences are flanked by inverted terminal repeats (ITRs) that help with genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) that replicate the viral genome and facilitate packaging, while the Cap gene is expressed to produce viral capsid proteins (VPs; namely VP1, VP2, and VP3), which form the capsid outer shell. For gene therapy, the viral DNA of wild-type AAV is almost completely removed. Recombinant AAV (rAAV) used for gene therapy consists of a protein capsid containing the desired nucleic acid (transgenic) to be delivered to target cells. The desired nucleic acid is flanked by the AAV's ITRs.

[0004] Protein replacement therapy is a medical treatment designed to replace or supplement specific protein deficiencies caused by mutations in the affected patient, resulting in a lack of or non-functional protein. For example, enzyme replacement therapy (ERT) replaces a defective or deficient enzyme in the body. This is done by administering an intravenous infusion of a composition containing the enzyme to the patient. This therapy is used for some lysosomal storage disorders: Gaucher disease, Fabry disease, MPS I, MPS II (Hunter syndrome), MPS VI, and Pompe disease. Enzyme replacement therapy cannot correct the underlying genetic defect. Therefore, treatment requires lifelong intravenous infusion of the therapeutic enzyme. This procedure is expensive; in the United States, it can cost more than [amount missing] per patient per year. 200,000. Furthermore, the biodistribution of these infusions is uneven; certain areas of the body (such as bones, lungs, and brain) receive less of the enzyme. For this reason, many symptoms of diseases requiring ERT remain untreated, especially neurological symptoms. Additionally, the efficacy of ERT is often reduced due to unwanted immune responses against the enzyme, which inhibit metabolic function. Moreover, enzyme levels typically do not remain at the same level during ERT administration but decrease after administration. This results in peaks and troughs in enzyme activity. During the troughs, any harmful symptoms or damage caused by the disease can still occur. The use of rAAV gene therapy vectors allows cells to produce the curative enzyme themselves, thus allowing for a longer-term solution with improved and stable biodistribution. Furthermore, treatment costs are reduced because gene therapy is expected to be given only once in a lifetime, which is not only cost-effective but also improves the patient's quality of life.

[0005] Recipes intended for storage and subsequent administration of rAAV to patients present particular challenges in terms of stability, such as the need to avoid the formation of undesirable aggregates or agglomerates in the recipes, which could be harmful to the therapeutic compound and / or the patient. In particular, such recipes containing rAAV may be frozen and then thawed before use. Aggregates or agglomerates may adversely form during or after thawing the rAAV-containing recipes, and during the time following thawing but before administration, as well as during administration. In some cases, where administration exceeds several hours, a consistently aggregate-free recipe is required. Therefore, there is a strong need to provide innovative recipes for rAAV carriers that reduce or eliminate aggregate formation, thereby extending shelf life. Summary of the Invention

[0006] In one aspect, an isotensile article is provided, the isotensile article comprising: Buffer solution; Recombinant adeno-associated virus vector containing a transgene encoding a therapeutic protein; and Cyclodextrin or its derivatives.

[0007] On the other hand, isotonic formulations, as disclosed herein, are provided for use in pharmaceuticals. Detailed Implementation

[0008] This invention aims to provide a novel pharmaceutical formulation comprising rAAV that reduces aggregate or agglomerate formation during processing and is substantially free of visible particles for an extended period at room temperature after thawing of the formulated pharmaceutical product. Such aggregates may include, but are not limited to, aggregates comprising rAAV particles, salt crystal aggregates, and combinations thereof. This invention also seeks to provide a method for treating diseases related to defective enzymes (such as lysosomal storage disorders) by administering to a patient a formulation of the present invention comprising rAAV. Therefore, this invention provides an isotonic formulation comprising: Buffer solution; Recombinant adeno-associated virus vector containing a transgene encoding a therapeutic protein; and Cyclodextrin or its derivatives.

[0009] It has been unexpectedly discovered that the presence of cyclodextrin or its derivatives in the isotonic formulations of the present invention contributes to improving the stability of pharmaceutical products. As used herein, the term "pharmaceutical product" refers to rAAV pharmaceutical products. Pharmaceutical products contained in isotonic formulations as described herein can remain stable, for example, at storage temperatures 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 36 months), without substantially forming aggregates or clusters. Pharmaceutical products contained in isotonic formulations as described herein can also remain stable at room temperature (15°C-25°C) for, for example, an extended time after thawing from a frozen state, preferably at least about 12 hours, more preferably at least about 24 hours, without substantially forming aggregates or clusters. Therefore, in some embodiments, pharmaceutical products contained in isotonic formulations as described herein remain stable at room temperature (15°C-25°C) for at least 12 hours after thawing from a frozen state, without substantially forming aggregates or clusters. As presented in the examples included in this article, other formulations containing rAAV pharmaceutical products do indeed form aggregates or clusters within 12 hours of thawing from a frozen state.

[0010] Preferably, the formulation of the present invention is substantially isotonic with human blood. Tonicity is a measure of the effective osmotic pressure that a liquid formulation can exert, and it depends primarily on 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 swelling. Isotonicity refers to the presence of a solution that does not produce changes in cell volume. When biological cells are in a hypotonic environment, water accumulates inside the cell, flowing across the cell membrane into the cell, causing cell swelling. For mammalian cells, this can lead to cell lysis, and therefore tonicity is important when vulnerable cells are exposed to the composition. Therefore, tonic agents can be added to formulations (such as injectable formulations) to prevent osmotic pressure shocks at the injection site during administration, thereby reducing local irritation or even damage to tissues or blood cells.

[0011] Preferably, the isotonic formulations described herein are substantially isotonic with human blood, having a tonic or osmotic concentration of about 290 mOsm / kg. In particular, the formulations of the present invention can have an osmolality (also called tonic or osmotic concentration) of 250 to 330 mOsm / kg (e.g., 260 to 310 mOsm / kg). In some preferred embodiments, the isotonic formulations described herein have an osmolality of 260 to 320 mOsm / kg, more preferably 270 to 315 mOsm / kg, and most preferably 274 to 310 mOsm / kg. For example, the isotonic formulations described herein can have an osmolality of 290 mOsm / kg.

[0012] Typical tension modifiers are excipients used for tension regulation and are known in the art. Tension modifiers may include dextran, glycerol, mannitol, and metal salts. Metal salts are preferred, and preferably pharmaceutically acceptable metal salts. Thus, in preferred embodiments, isotonic formulations further comprise a pharmaceutically acceptable salt at a concentration of at least 50 mM, wherein the salt is preferably NaCl, KCl, CaCl2, MgCl2, or a combination thereof.

[0013] Pharmaceutically acceptable metal salts may include metal salts from Group 1 or Group 2 of the periodic table, preferably metal chloride salts from Group 1 or Group 2 of the periodic table, and preferably selected from the group consisting of NaCl, KCl, CaCl2, MgCl2, and combinations thereof. NaCl is particularly preferred.

[0014] The isotonic formulations described herein preferably contain a pharmaceutically acceptable metal salt at a concentration greater than about 55, 60, or 65 mM (particularly 75 mM), which has been found to be beneficial to the stability of the pharmaceutical product, as measured by absorbance and visual inspection according to the examples disclosed herein. Preferably, the concentration of the pharmaceutically acceptable salt can be from about 75 mM to about 200 mM, preferably from about 80 mM to about 175 mM, more preferably from about 85 mM to about 160 mM, more preferably from about 90 to about 155 mM, more preferably from about 95 to about 150 mM, more preferably from about 100 to about 145 mM, more preferably from about 105 to about 140 mM, more preferably from about 115 to about 135 mM, more preferably from about 120 to about 130 mM, such as most preferably from about 125 mM. Therefore, in some embodiments, the isotonic formulation of the present invention comprises a pharmaceutically acceptable salt selected from the group consisting of NaCl, KCl, CaCl2, MgCl2 and combinations thereof, at a concentration of about 100 mM to about 150 mM, preferably about 115 mM to about 135 mM, more preferably about 120 to about 130 mM (such as most preferably about 125 mM).

[0015] In some specific embodiments, the pharmaceutically acceptable salt comprises NaCl, which is present in the preparation at a concentration of about 75 mM or higher, preferably about 75 mM to about 150 mM, more preferably about 100 mM to about 150 mM, preferably about 115 mM to about 135 mM, and most preferably about 125 mM.

[0016] The isotonic formulation of the present invention can have a pH value compatible with human blood. For example, the isotonic formulation can have a pH of about 6.5 or higher, preferably about 7 or higher. In some embodiments, the formulation can have a pH value of 6.5 to 8.5, preferably 7 to 8, more preferably 7.3 to 7.7. In some embodiments of the present invention, the isotonic formulation has a pH value of 7.5. In some embodiments, the isotonic formulation has a pH value of 6.5 to 8.5, preferably 7.5 to 8, most preferably 7.5. In preferred embodiments, the isotonic formulation has a pH value of 7.2 to 7.8, most preferably 7.3 to 7.5.

[0017] In one embodiment, the isotonic formulation as described herein comprises a buffer solution. Buffers are known in the art and help maintain the pH of the composition stable within a given range. Buffers are typically buffer salts. Therefore, the isotonic formulation of the present invention may comprise a buffer solution selected from acetates, citrates, phosphates, Tris (tris(hydroxymethyl)aminomethane or tromethamine) and derivatives (e.g., Tris hydrochloride) and combinations thereof, including combinations of tromethamine and Tris hydrochloride. In a preferred embodiment, Tris is tromethamine. The isotonic buffer solution may be a Tris buffer solution with a pH of about 7.5 to about 8.0, a citrate buffer solution with a pH of about 5.5 to about 6.5, or a phosphate buffer solution with a pH of about 7.0 to about 7.5. Preferably, the buffer solution is a Tris buffer solution. In some embodiments, the isotonic formulation comprises a Tris buffer solution with a pH of about 7.5 to about 8.0. In some specific embodiments, the buffer solution is a Tris buffer solution with a pH of 7.5. The buffer is preferably present in the form of about 5 to about 50 mM, more preferably about 10 to about 40 mM, still more preferably about 12 to about 35 mM, still more preferably about 14 to about 30 mM, and most preferably about 15 to about 25 mM. The buffer may also be present in the form of about 16 to about 24 mM, more preferably about 17 to about 23 mM, still more preferably about 18 to about 22 mM, and most preferably about 19 to 21 mM (e.g., 20 mM). In some preferred embodiments, the buffer is a 20 mM Tris buffer at pH 7.5.

[0018] The isotonic formulations of the present invention comprise cyclodextrin or derivatives thereof. The inventors have unexpectedly discovered that cyclodextrin can help improve the preservation of the stability of recombinant adeno-associated virus and / or is particularly advantageous in providing stable pharmaceutical products with significantly reduced aggregate or cluster formation. Cyclodextrin is a family of cyclic oligosaccharides composed of a macrocycle of glucose subunits linked by α-1,4 glycosidic bonds. Cyclodextrin can be α-cyclodextrin having 6 glucose subunits, β-cyclodextrin having 7 glucose subunits, or γ-cyclodextrin having 8 glucose subunits. The preferred cyclodextrin used in the present invention is β-cyclodextrin. Combinations of cyclodextrins may also be used.

[0019] Cyclodextrins can be substituted or unsubstituted. Preferably, the cyclodextrin is an unsubstituted or substituted β-cyclodextrin. Substituted cyclodextrins are typically modified at their hydroxyl moieties, preferably at all their hydroxyl moieties, and preferably have the same modification at all their hydroxyl moieties. Examples of substitution are methylation, acetylation, and hydroxypropylation, such as 2-hydroxypropylation (e.g., having a hydroxyl moieties derived using propylene oxide). Preferred cyclodextrins are substituted cyclodextrins, particularly substituted β-cyclodextrins. Preferred substituted cyclodextrins are hydroxypropyl-cyclodextrins, and 2-hydroxypropyl-β-cyclodextrin is particularly preferred (CAS No. 128446-35-5).

[0020] In some embodiments, cyclodextrin is present in isotropic formulations in amounts less than about 4% w / v (weight / volume percentage), preferably less than about 3% w / v, preferably about 0.05% w / v to about 4% w / v, preferably about 0.1% w / v to about 3.5% w / v, preferably about 0.5% w / v to about 3.2% w / v, more preferably about 1% w / v to about 3.1% w / v, more preferably about 1.2% w / v to about 3% w / v, more preferably about 1.4% w / v to about 2.8% w / v, more preferably about 1.6% w / v to about 2.6% w / v, more preferably about 1.8% w / v to about 2.4% w / v, and most preferably about 1.9% w / v to about 2.2% w / v (e.g., about 2% w / v). In some specific embodiments of the isotropic formulation, the cyclodextrin is a substituted β-cyclodextrin at a concentration of 1.5% w / v to 2.5% w / v.

[0021] In some embodiments, the isotropic formulation further comprises a sugar or sugar alcohol, preferably a monosaccharide, disaccharide, or sugar alcohol. Preferably, the sugar or sugar alcohol may be selected from the group consisting of trehalose, sucrose, maltose, mannitol, and derivatives thereof and combinations thereof. For example, the sugar or sugar alcohol is mannitol. It has been found that sugars or sugar alcohols can act as cryoprotectants and can be particularly advantageous in providing stable pharmaceutical formulations. In preferred embodiments, the formulation does not further comprise a sugar or sugar alcohol.

[0022] In some embodiments, when present, the sugar or sugar alcohol is present in the isotonic formulation in an amount of about 0.01% w / v to about 4% w / v, preferably about 0.05% w / v to about 2% w / v, and preferably about 0.1% w / v to about 1% w / v. Therefore, in some embodiments, the isotonic formulation of the present invention comprises a sugar or sugar alcohol at a concentration of about 0.05% w / v to about 2% w / v. In some specific embodiments, the isotonic formulation comprises a sugar or sugar alcohol at a concentration of about 0.05% w / v to about 2% w / v, wherein the sugar or sugar alcohol is selected from the group consisting of trehalose, sucrose, maltose, mannitol, and derivatives thereof and combinations thereof.

[0023] When the sugar or sugar alcohol is mannitol, it may be present in the isotonic formulation in an amount of less than about 2% w / v, preferably about 1% w / v or less, preferably about 0.05% w / v to about 1% w / v, preferably about 0.1% w / v. In some embodiments, the isotonic formulation comprises mannitol at a concentration of about 0.1% w / v.

[0024] In some embodiments, the isotropic formulation comprises a combination of 2-hydroxypropyl-β-cyclodextrin and mannitol, preferably in an amount of about 0.1 w / v% of 2-hydroxypropyl-β-cyclodextrin and an amount of about 0.1 w / v% of mannitol.

[0025] In some embodiments, isotonic formulations further comprise amino acids, which have been found to benefit the stability of the pharmaceutical product. Amino acids are natural permeabilizers and can stabilize proteins when in solution. For example, amino acids may be selected from cysteine, arginine, histidine, glycine, and their derivatives and combinations thereof. Preferably, the amino acid may comprise histidine and / or glycine. When the amino acid is histidine and / or glycine, it may be present at a concentration of about 2 mM to about 3 mM, preferably about 2.5 mM. In preferred embodiments, isotonic formulations do not further comprise amino acids.

[0026] In some embodiments, when present, the isotonic formulation comprises an amino acid at a concentration of about 2 mM to about 3 mM, selected from the group consisting of cysteine, arginine, histidine, glycine, and derivatives and combinations thereof. In some specific embodiments, the isotonic formulation comprises histidine or a derivative thereof at a concentration of about 2.5 mM.

[0027] Preferably, the isotonic formulation does not contain a surfactant. Surfactants and their characteristics are well known; surfactants generally contain at least one polar head group and at least one nonpolar or hydrophobic tail, and are preferably charge-neutral, i.e., the surfactant does not carry a net charge under its conditions of use. For example, the isotonic formulation does not contain polysorbate 20, polysorbate 80, or poloxamer 188. The inventors have discovered that the general effects of surfactants can be achieved by using formulations as disclosed, and therefore it is not necessary to expose the subject to the surfactant.

[0028] In one embodiment, the transgene contained in the recombinant adeno-associated virus vector in the isotonic formulation described herein is a transgene encoding a therapeutic protein. Examples of suitable therapeutic proteins include proteins for treating X-linked recessive disorders (preferably hemophilia A, hemophilia B, or glucose-6-phosphate dehydrogenase deficiency). X-linked recessive inheritance is a pattern of inheritance in which a gene mutation on the X chromosome results in the expression of a phenotype that is consistently present in males (who are necessarily homozygous for the gene mutation because they possess one X chromosome and one Y chromosome) and females homozygous for the gene mutation. A female possessing one copy of the mutated gene is a carrier. In some embodiments, the transgene encoding the therapeutic protein encodes an enzyme. Examples of suitable enzymes include aspartic glucosidase, α-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine ​​transporter, acidic ceramidinase, acidic α-L-fucosidase, protective protein / cathepsin A, acidic β-glucosidase or glucocerebrosidase, acidic β-galactosidase, iduronate-2-sulfatase, α-L-iduronase, galactocerebrosidase, acidic α-mannosidase, acidic β-mannosidase, arylsulfatase B, arylsulfatase A, and N-acetylgalactosidase. The enzymes include glycosamine-6-sulfatate sulfatase, acidic β-galactosidase, N-acetylglucosamine-1-phosphotransferase, acidic sphingomyelinase, NPC intracellular cholesterol transporter 1, acidic α-glucosidase, β-aminohexosidase B, heparin N-sulfatase, α-N-acetylglucosidase, acetyl-CoA:α-glucosinolate N-acetyltransferase, N-acetylglucosamine-6-sulfatate sulfatase, α-N-acetylglucosidase, α-ceramidinase, β-glucuronide glycoside, β-aminohexosidase A, and acidic lipase. Preferred enzymes are galactosidases, particularly α-galactosidases such as α-galactosidase A. In preferred embodiments, the transgene does not encode microRNA. Additionally, in preferred embodiments, the therapeutic protein is not a biomarker or encoded by a suicide gene.

[0029] In a particularly preferred embodiment, the transgene in the recombinant adeno-associated virus vector contained in the isotonic formulation described herein encodes α-galactosidase A.

[0030] In a preferred embodiment, the transgene in the recombinant adeno-associated virus vector contained in the isotonic preparation described herein comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 01 or a variant thereof.

[0031] In another embodiment, the transgenic encoding amino acid sequence in the recombinant adeno-associated virus vector contained in the isotonic preparation described herein comprises or consists of SEQ ID NO: 02 or a variant thereof.

[0032] In some embodiments, a recombinant adeno-associated virus vector having a transgene encoding an enzyme can encode an enzyme associated with lysosomal storage disorders. A group of metabolic disorders known as lysosomal storage disorders or diseases (LSDs) includes more than forty genetic disorders, many of which involve genetic defects in various lysosomal enzymes, such as hydrolases. Representative lysosomal storage disorders and associated defective enzymes are listed in the table below. In a preferred embodiment, the formulation is used to treat the disorders in the left column, and the transgene encoding the enzyme encodes the associated enzyme in the right column. In a preferred embodiment, the isotonic formulation described herein is used to treat the disorders in the left column, and the transgene encoding the enzyme encodes the associated enzyme in the right column.

[0033] In one embodiment, the isotonic formulation comprising a recombinant adeno-associated virus vector described herein is used to treat Fabry disease.

[0034] In a preferred embodiment, the isotonic formulation for treating Fabry disease comprises an adeno-associated virus vector containing a transgene of SEQ ID NO: 01 or a variant thereof, and / or encoding a transgene of an amino acid sequence comprising SEQ ID NO: 02 or a variant thereof or consisting of SEQ ID NO: 02 or a variant thereof.

[0035] In one embodiment, a method for treating Fabry disease by applying an isotonic formulation as described herein is provided.

[0036] In a preferred embodiment, a method for treating Fabry disease by administering an isotonic formulation as described herein is provided, wherein the recombinant adeno-associated virus vector comprises a transgene containing SEQ ID NO: 01 or a variant thereof, and / or a transgene encoding an amino acid sequence comprising SEQ ID NO: 02 or a variant thereof or consisting of SEQ ID NO: 02 or a variant thereof.

[0037] In some embodiments, the recombinant adeno-associated virus (AAV) vector comprises a capsid protein of the AAV2 serotype, the AAV5 serotype, or a combination thereof. These serotypes have been found to be particularly preferred for treating diseases or conditions associated with defective enzymes, such as lysosomal storage disorders. In some embodiments, the recombinant AAV vector comprises the AAV5 serotype. In one instance, the recombinant AAV vector comprises an AAV5 variant. In some embodiments, the recombinant AAV vector comprises a combination of capsid proteins from different serotypes, forming a heterozygous AAV serotype. For example, a heterozygous AAV serotype could be a heterozygous AAV2 / AAV5 serotype. Preferably, the viral vector contains only a single serotype. Most preferably, the viral vector is of both the AAV5 and AAV2 serotypes, which may also be referred to as the AAV2 / 5 serotype. In this document, the first 136 residues of the AAV5 VP1 protein are replaced by the first 137 residues of the AAV2 VP1 protein.

[0038] In a preferred embodiment, the isotonic formulation described herein comprises a recombinant adeno-associated virus vector, wherein the recombinant adeno-associated virus vector is of serotype AAV5, and wherein the first 136 residues of the AAV5 VP1 protein are replaced by the first 137 residues of the AAV2 VP1 protein.

[0039] Some recombinant adeno-associated virus (rAAV) vectors (such as AAV5 serotype (rAAV5)) have been shown to guide stable gene transfer and expression in hepatocytes, exhibiting increased hepatic transduction compared to other rAAVs. Therefore, in some embodiments, isotonic formulations as described herein are used to administer viral vectors, preferably transducible to the liver, as described herein. Isotonic formulations as disclosed herein have particular applications as formulations suitable for treating diseases that can be treated with rAAV-based therapies exhibiting increased hepatic transduction, such as LSD described above or coagulation disorders, such as hemophilia A or hemophilia B. It is also conceivable, in another embodiment, that isotonic formulations as disclosed herein are suitable for storing pharmaceutical products for treating liver-related diseases. Liver diseases and liver-related diseases include, but are not limited to: viral diseases (such as hepatitis A, hepatitis B, and hepatitis C); diseases caused by drugs or toxins (such as alcohol) that lead to fatty liver disease and cirrhosis; autoimmune hepatitis; liver cancer; hereditary diseases such as hemochromatosis, alpha-1 antitrypsin deficiency (AATD), and Wilson's disease; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis (NASH); and biliary atresia. In this context, the transgene encodes the associated therapeutic protein. Therefore, in some embodiments, a recombinant adeno-associated virus vector containing a transgene encoding an enzyme can encode an enzyme associated with liver storage diseases or metabolic liver diseases.

[0040] In some embodiments, the concentration of the recombinant adeno-associated virus vector in the isotonic formulation described herein is as high as 5E15 gc / ml (genome copies / mL), preferably 1E15 gc / ml, more preferably 5E14 gc / ml (genome copies / mL), and still more preferably as high as 1E14 gc / ml. Preferably, the concentration of the recombinant adeno-associated virus vector is 1E10 to 5E14 gc / ml, preferably 5E10 to 2E14 gc / ml, more preferably 1E11 to 1E14 gc / ml, more preferably 5E11 to 8E13 gc / ml, more preferably 1E12 to 6E13 gc / ml, and more preferably 5E12 to 5E13 gc / ml. In other embodiments, the concentration of the recombinant adeno-associated virus vector is 1E13 to 1E15 gc / ml, preferably 5E13 to 5E14 gc / ml, and more preferably 6E13 to 2E14 gc / ml. In one example, the isotonic formulation comprises rAAV5 having a transgene encoding a therapeutic protein (preferably an enzyme, such as α-galactosidase A), and the concentration of rAAV5 is 5E13 gc / ml or 6E13 gc / ml, preferably 5E13. In another example, the isotonic formulation comprises rAAV5 having a transgene encoding a therapeutic protein (preferably an enzyme, such as α-galactosidase A), and the concentration of rAAV5 is 1E14 gc / ml or 2E14 gc / ml, preferably 2E14 gc / ml. In another embodiment, the isotonic formulation as described herein comprises an rAAV5 vector containing a transgene encoding a therapeutic protein, wherein the concentration of the rAAV5 vector in the formulation is from 3E13 gc / ml to 7E13 gc / ml, preferably 5E13 to 6.5E13. In a preferred embodiment, the therapeutic protein is an enzyme. In another preferred embodiment, the enzyme is α-galactosidase A. The present invention has particular applications in situations where high doses of rAAV are required and longer administration times may be necessary (e.g., administration exceeding 1 hour or more), where a continuous, aggregate-free formulation is required during the duration of administration.

[0041] In one embodiment, the use of isotonic formulations as described herein in the manufacture of a medicament for treating Fabry disease is provided.

[0042] In a preferred embodiment, the use of an isotonic formulation as described herein in the manufacture of a medicament for treating Fabry disease is provided, wherein the formulation comprises a recombinant adeno-associated virus vector containing a transgene of SEQ ID NO: 01 or a variant thereof, and / or wherein the transgene encodes an amino acid sequence comprising SEQ ID NO: 02 or a variant thereof or consisting of SEQ ID NO: 02 or a variant thereof, preferably wherein the concentration of the recombinant adeno-associated virus vector in the isotonic formulation is from 3E13 gc / ml to 7E13 gc / ml, more preferably from 5E13 to 6.5E13.

[0043] A recombinant adeno-associated virus vector containing a transgene encoding a therapeutic protein refers to a vector comprising one or more target polynucleotide sequences, a target gene, or a “transgene” flanked by an AAV inverted terminal repeat (ITR). rAAV preferably comprises a nucleic acid construct containing a target gene encoding a therapeutic protein, i.e., a transgene, flanked by at least one ITR. In another preferred embodiment, the rAAV vector comprises an expression cassette for the transgene, wherein i) the transgene is operatively linked to a promoter, preferably a liver-specific promoter (such as the LP1 promoter, more preferably a promoter selected from liver-specific promoters described in WO2020 / 104424 (which is incorporated herein by reference), and / or ii) the transgene is operatively linked to a polyadenylation site, preferably an SV40-derived polyA site. In a preferred embodiment, the liver-specific promoter in i) comprises or consists of SEQ ID NO: 03. In another preferred embodiment, the expression cassette is flanked by at least one ITR. In one embodiment, the expression cassette is flanked by two AAV ITR nucleotide sequences, whereby the expression cassette is located between the two AAV ITR nucleotide sequences. In another embodiment, the expression cassette is flanked by an ITR engineered with two D regions, wherein the expression cassette is located on either side of the engineered ITR. In a preferred embodiment, at least one ITR is derived from AAV1, AAV2, AAV4, and / or AAV7. In one embodiment, the expression cassette is located between two AAV2 ITR nucleotide sequences.

[0044] In some embodiments, isotonic formulations as disclosed herein have particular use as formulations suitable for storing pharmaceutical products for treating diseases treated with hepatotropic AAV-based therapies. In some embodiments, isotonic formulations as disclosed herein have particular use as formulations suitable for storing pharmaceutical products for treating diseases treated via the liver. Therefore, isotonic formulations as described herein are used as medicines. The medicine is preferably used to treat disorders associated with defective or deficient proteins, preferably enzymes, more preferably lysosomal storage disorders. This is because the isotonic formulation contains a recombinant adeno-associated virus vector having a transgene encoding a protein that can be readily used to replace or supplement the defective or impaired enzyme. Examples of suitable encoded enzymes and combinations with the condition to be treated have been provided elsewhere herein. Suitable subjects are subjects in need of treatment. Isotonic formulations containing a recombinant adeno-associated virus vector, as disclosed herein, can be readily used in treatment methods, the recombinant adeno-associated virus vector containing a transgene encoding a therapeutic protein (i.e., one or more products). These products can also be used themselves to manufacture medicines, preferably in which the medicine is used to treat the indicated condition. The isotonic formulation according to the invention has been found to be suitable for treating lysosomal storage disorders and therefore can be beneficial for administering treatments to subjects, such as treatments intended for the liver of subjects via intravenous injection. In all embodiments, the isotonic formulation is used for intravenous administration. In a preferred embodiment, the isotonic formulation is not used for administration to the central nervous system. In a preferred embodiment, the isotonic formulation is not used for administration to the brain.

[0045] This invention provides a method for preparing a pharmaceutical product for administration to the liver, the method comprising the following steps: i) Providing an isotensile article according to the invention; and ii) Divide the prepared products into appropriate dosage forms.

[0046] The prepared pharmaceutical product is suitable for administration to the liver. Therefore, this method is suitable for preparing isotonic formulations for use according to the invention, preferably wherein the formulation is for intravenous administration.

[0047] Optionally, the isotonic formulation of the present invention is administered at a dosage regimen of 1E12 to 1E15 genome copies / kg (gc / kg), for example, 6E12 to 6E14 genome copies / kg. For example, when the isotonic formulation is administered intravenously, the injection volume may be 100-800 mL, and the concentration (of the recombinant adeno-associated virus vector) may be 2E12 or 2E13 gc / mL.

[0048] In a preferred embodiment, the isotonic formulation of the present invention is administered at a dosage regimen of 1E13 to 1E15 gc / kg, preferably 5E13 to 5E14 gc / kg, more preferably 6E13 to 3E14 gc / kg, and most preferably 6.0E13 gc / kg or 3.0E14 gc / kg.

[0049] In a preferred embodiment, the isotonic formulation as described herein, comprising a recombinant adeno-associated virus vector, is administered at a dose of 1E13 to 1E15 gc / kg, preferably 4E13 to 8E14 gc / kg, more preferably 6E13 to 5E14 gc / kg, and most preferably 6.0E13 gc / kg or 3.0E14 gc / kg.

[0050] The range of 1E13 to 1E15 gc / kg as described above includes, but is not limited to, doses of 4E13 gc / kg, 6E13 gc / kg, 2E14 gc / kg, 3E14 gc / kg, 5E14 gc / kg, and 7.3E14 gc / kg.

[0051] The isotonic formulations according to the invention have been found to have excellent stability and are not prone to forming aggregates or precipitates. In preferred embodiments, the formulations are substantially free of visible particles. In other words, the isotonic formulations are preferably substantially free of particles visible to the naked eye, as identified and characterized according to the United States Pharmacopeia (USP) Guideline 790, "VISIBLE PARTICULATES IN INJECTIONS," concerning parenteral medicine products ("substantially free" of visible particulate matter) (USP 790). Visible particles can be a marker of rAAV particle aggregation, agglomeration, and / or degradation; therefore, the absence of visible particles may be advantageous in indicating that the formulation is particularly stable and does not exhibit significant aggregation or agglomeration. Preferably, the isotonic formulation also contains a limited number of subvisible particles, as determined and characterized according to United States Pharmacopeia (USP) Chapter 787, “Subvisible Particular Matter in Therapeutic Protein Injections” (USP 787), which is incorporated herein by reference. For example, the number of particles ≥ 25 µm in diameter in the formulation does not exceed 600 particles / vial, and the number of particles ≥ 10 µm in diameter does not exceed 6000 particles / vial. Optionally, the isotonic 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 whose size is not greater than a specified value (in this case, 90%). As used herein, the term Dv90 defines a point in the size distribution where 90% of the total volume of the formulation is of a certain size or smaller.

[0052] The following are some preferred embodiments of the present invention: • Isotonic formulations with a weight osmolality of 250 to 330 mOsm / kg and a pH of 6.5 to 8.

[0053] • It is an isotonic formulation that is substantially isotonic with human blood. For example, the formulation has a weight osmolality of 260 to 310 mOsm / kg and a pH of 7.1 to 7.7.

[0054] • Includes the following isotropic products Approximately 15-25 mM buffer solution; A transgenic recombinant adeno-associated virus vector encoding a therapeutic protein; Approximately 110-140 mM pharmaceutically acceptable salts; Approximately 1.5%-2.5% (w / v) cyclodextrin or its derivatives; The formulation has a pH of approximately 7 to 8.

[0055] • Includes the following isotropic products Approximately 15-25 mM Tris; Recombinant adeno-associated virus vector containing the AAV5 serotype; Approximately 110-140 mM NaCl; Approximately 1.5%-2.5% (w / v) hydroxypropyl-β-cyclodextrin; The formulation has a pH of approximately 7 to 8.

[0056] • Includes the following isotropic products Approximately 18-22 mM buffer solution; A transgenic recombinant adeno-associated virus vector encoding a therapeutic protein; Approximately 120-130 mM pharmaceutically acceptable salt; Approximately 1.8%-2.2% (w / v) cyclodextrin or its derivatives; The formulation has a pH of approximately 7.2 to 8.

[0057] • Includes the following isotropic products Approximately 20 mM buffer solution; A transgenic recombinant adeno-associated virus vector encoding a therapeutic protein; Approximately 125 mM pharmaceutically acceptable salt; Approximately 2% (w / v) cyclodextrin or its derivatives; The formulation has a pH of approximately 7 to 8, such as 7.5 to 8, such as 7.5.

[0058] • Includes the following isotropic products Approximately 18-22 mM Tris; Recombinant adeno-associated virus vector containing the AAV5 serotype; Approximately 120-130 mM NaCl; Approximately 1.8%-2.2% (w / v) hydroxypropyl-β-cyclodextrin; The formulation has a pH of approximately 7.2 to 8.

[0059] • Includes the following isotropic products Approximately 20 mM Tris; Recombinant adeno-associated virus vector containing the AAV5 serotype; Approximately 125 mM NaCl; Approximately 2% (w / v) hydroxypropyl-β-cyclodextrin; The formulation has a pH of approximately 7 to 8, such as 7.5 to 8, such as 7.5.

[0060] Methods for manufacturing recombinant adeno-associated virus (rAAV) vectors suitable for incorporation into formulations according to the invention are known in the art. Generally, suitable methods and means (such as expression constructs for expressing AAV rep proteins) for generating rAAV vectors as described herein in mammalian or insect host cells are described in the following literature for mammalian cells: Clark et al. (Hum. Gene Ther. [Human Gene Therapy] 1995, 6, 1329-134), Gao et al. (Hum. Gene Ther. [Human Gene Therapy] 1998, 9, 2353-2362), Inoue and Russell (J. Virol. [Journal of Virology] 1998, 72, 7024-7031), Grimm et al. (Hum. Gene Ther. [Human Gene Therapy] 1998, 9, 2745-2760), Xiao et al. (J. Virol. [Journal of Virology] 1998, 72, 2224-2232), and Judd et al. (Mol Ther Nucleic Acids). [Molecular Therapy Nucleic Acids] 2012; 1: e54), and for insect cells: Urabe et al. (Hum. Gene Ther. [Human Gene Therapy] 2002, 13:1935-1943), WO2007 / 046703, WO2007 / 148971, WO2009 / 014445, WO2009 / 104964, WO2011 / 122950, ​​WO2013 / 036118, WO2015 / 137802, WO2019 / 016349, WO2021 / 198508, WO2021 / 198510 and WO2022 / 253955, all of which are incorporated herein by reference in their full text.

[0061] AAV Rep and ITR sequences are particularly conserved across most serotypes. The Rep78 protein of various AAV serotypes, for example, exhibits over 89% identity, and the total nucleotide sequence identity at the genomic level is approximately 82% among AAV2, AAV3A, AAV3B, and AAV6 (Bantel-Schaal et al., J. Virol. [Journal of Virology], 1999, 73(2):939-947). Furthermore, it is known that the Rep sequences and ITRs of many AAV serotypes are effectively cross-complementary (i.e., functionally substituted) with corresponding sequences from other serotypes when generating AAV particles in mammalian cells. US2003148506 reports that AAV Rep and ITR sequences are also effectively cross-complementary with other AAV Rep and ITR sequences in insect cells. Modified “AAV” sequences can also be used in this context, for example, for generating rAAV vectors in insect cells. Such modified sequences (e.g., sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or higher nucleotide and / or amino acid sequence identity with AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13 ITR or Rep) may replace wild-type AAV ITR or Rep sequences.

[0062] In one embodiment, the mammalian cells used to generate the rAAV vector as described herein are selected from cell lines of any mammalian species, including but not limited to: A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK 293 cells, Saos, C2C12, L cells, HT1080, HepG2, and primary fibroblasts, hepatocytes, and myoblasts derived from mammals (including humans, monkeys, mice, rats, rabbits, and hamsters). The choice of mammalian species providing the cells is not a limitation of this disclosure; nor is the type of mammalian cell (i.e., fibroblasts, hepatocytes, tumor cells) a limitation of the invention. The mammalian cell lines used to generate the rAAV vector particularly include a broad range of HEK293 cell lines, with the HEK293T cell line being preferred.

[0063] The insect cell line used to generate the rAAV vector as described herein can be any cell line suitable for generating heterologous proteins. Preferably, the insect cells allow for the replication of the baculovirus vector and can be maintained in a culture, more preferably in a suspension culture. In a preferred embodiment, the insect cells allow for the replication of recombinant parvovirus vectors (including rAAV vectors). For example, the cell line used can be derived from the fall armyworm (… Spodoptera frugiperda ), fruit flies ( Drosophila ) or mosquitoes, including Aedes albopictus ( Aedes albopictus In a preferred embodiment, the insect cells or cell lines are derived from insect species susceptible to baculovirus infection, including but not limited to: S2 (CRL-1963, ATCC), Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, Ha2302, Hz2E5, High Five (Invitrogen, California, USA) and expresSF+® (US6,103,526; Protein Sciences Corp., Connecticut, USA).

[0064] The rAAV vector released into the supernatant of mammalian or insect cell cultures can be recovered and / or purified using suitable techniques known to those skilled in the art. In one embodiment, purification and concentration are performed using a monolithic column (e.g., in ion exchange, affinity, or IMAC mode), chromatography (e.g., capture chromatography, immobilization chromatography, and expanded bed chromatography), centrifugation, filtration, and / or precipitation. These methods can be used alone or in combination. In one embodiment, capture chromatography (including column-based or membrane-based systems) is used in combination with filtration and precipitation. Those skilled in the art can readily select suitable precipitation methods, including but not limited to chromatographic methods using polyethylene glycol (PEG) 8000 and NH3SO4. Subsequently, the precipitate can be treated with enzymes, including but not limited to totipotent nucleases, and purified using suitable techniques. Furthermore, recovery preferably includes the step of affinity purification of the rAAV vector using an anti-AAV antibody (preferably an immobilized antibody). The anti-AAV antibody is preferably a monoclonal antibody. Particularly suitable antibodies are single-chain camelid antibodies or fragments thereof, such as those obtained from camels or llamas (see, for example, Muyldermans, Biotechnol. [Biotechnology] 2001, 74: 277-302). Antibodies used for affinity purification of rAAV vectors are preferably antibodies that specifically bind to epitopes on rAAV capsid proteins, whereby the epitopes are preferably epitopes present on capsid proteins of more than one AAV serotype. For example, antibodies can be generated or selected based on specific binding to the AAV6 capsid, but can also specifically bind to the rAAV5 capsid at the same time.

[0065] In a preferred embodiment, a recombinant adeno-associated virus (rAAV) vector as described herein is obtained using a method comprising the following steps: I) culturing host cells under conditions that produce an rAAV vector, the host cells comprising: i) an expression vector for expressing the rAAV vector; and ii) an expression vector encoding an expression cassette flanked by at least one AAV inverted terminal repeat (ITR), the expression cassette comprising a nucleic acid molecule encoding at least one gene product; the host cells preferably further comprising a nucleotide sequence encoding a parvovirus replication (Rep) protein; and II) recovering the rAAV vector, preferably wherein the recovery of the rAAV vector comprises at least one of the following: affinity purification of the rAAV vector using an immobilized anti-AAV antibody, preferably a single-chain camelid antibody or a fragment thereof, or filtration using a filter with a nominal pore size of 30-70 nm.

[0066] General definition In this document and its claims, the verb "comprising" and its variations are used in a non-limiting sense to mean including the items following the word, but not excluding items not specifically mentioned. Furthermore, the verb "constituting" can be replaced with "constituting substantially of," meaning that a combination or composition as defined herein may contain one or more additional components besides those specifically specified, which do not alter the distinctive features of the invention. Moreover, reference to an element by the indefinite article "a / an" does not preclude the possibility of more than one such element, unless the context explicitly requires the presence of one and only one such element. Therefore, the indefinite article "a / an" generally means "at least one / a."

[0067] Whenever parameters of a substance are discussed in the context of this invention, it is assumed that, unless otherwise specified, the parameters are determined, measured, or displayed under physiological conditions. Physiological conditions are known to those skilled in the art and include aqueous solvent systems, atmospheric pressure, pH values ​​between 6 and 8, temperatures from room temperature to about 37°C (about 20°C to about 40°C), and suitable concentrations of buffer salts or other components. The word “about” or “approximately” when used in conjunction with a numerical value (e.g., about 10) preferably means that the value can be a given value (10) plus or minus 10%, optionally plus or minus 5%. In the context of this invention, a decrease or increase in the parameter to be evaluated preferably means a change corresponding to at least 5% of the value of the parameter. More preferably, the decrease or increase in the value means a change of at least 10%, or even 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, it may be that there is no longer a detectable value associated with this parameter. "Substantially" – as used herein, the term "substantially" is a broad term and will be given its common and customary meaning to those skilled in the art (and not limited to a particular or customary meaning), and without limitation, refers to the meaning that is substantially (but not necessarily entirely) what is specified.

[0068] Unless otherwise indicated, each embodiment described herein may be combined together. The invention has been described above with reference to numerous embodiments. Minor variations of some elements of the embodiments will be conceived by those skilled in the art. These are all included within the scope of protection defined in the appended claims. All patents and references cited are hereby incorporated in their entirety by reference.

[0069] A “nucleic acid construct” is defined as a nucleic acid molecule isolated from a naturally occurring gene, or a nucleic acid molecule modified to contain nucleic acid segments combined or juxtaposed in a manner not normally found in nature. Nucleic acid molecules are represented by nucleotide sequences. Optionally, the nucleotide sequences present in the nucleic acid construct are operatively linked to one or more control sequences that direct the production or expression of the peptide or polypeptide in a cell or subject.

[0070] The “variants” of the nucleic acid sequences mentioned herein have at least 70%, at least 75%, at least 80%, at least 85%, preferably at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98%, or 99% to 99,999% nucleic acid sequence identity with the listed nucleic acid sequences.

[0071] The “variants” of the amino acid sequences mentioned herein have at least 70%, at least 75%, at least 80%, at least 85%, preferably at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98%, or 99% to 99,999% amino acid sequence identity with the listed amino acid sequences.

[0072] The term "homologous" when used to indicate the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell should be understood to mean that, in nature, the nucleic acid or polypeptide molecule is produced by host cells or organisms of the same species. The term "heterologous" can be used to indicate that the nucleic acid or polypeptide molecule is produced in nature by host cells or organisms of different species.

[0073] "Expression control sequence" refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked. An expression control sequence is "operably linked" to a nucleotide sequence when it controls and regulates transcription and / or translation of the nucleotide sequence. Therefore, an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, start codons preceding protein-coding genes, intron splicing signals, and stop codons. The term "expression control sequence" is intended to include at least a sequence whose presence is designed to influence expression, and may also include additional advantageous components. For example, leader sequences and fusion coupler sequences are expression control sequences. The term may also include nucleic acid sequence designs that remove unwanted potential start codons, both inside and outside the frame. It may also include nucleic acid sequence designs that remove unwanted potential splicing sites. It includes sequences that guide the addition of multi-A tails or multi-adenosylated sequences (pA), i.e., a string of adenine residues at the 3' end of mRNA, which may be referred to as a multi-A sequence. It may also be designed to enhance mRNA stability. Expression control sequences (e.g., promoters) that affect transcriptional and translational stability and sequences that affect translation (e.g., Kozak sequences) suitable for use in insect cells are well known to those skilled in the art. Expression control sequences can have properties that allow regulation of the nucleotide sequences operatively linked to them, resulting in either lower or higher expression levels.

[0074] As used herein, the term "promoter" or "transcriptional regulatory sequence" refers to a nucleic acid fragment that controls the transcription of one or more coding sequences, is upstream of the transcription start site relative to the coding sequence, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art to directly or indirectly regulate the amount of transcription from a promoter, including, for example, attenuators or enhancers and silencers. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated (e.g., by the application of a chemical inducer). A "tissue-specific" promoter is active only in a specific type of tissue or cell.

[0075] A “3' UTR” or “3' untranslated sequence” (often also called a 3' untranslated region or 3' end) refers to a nucleic acid sequence found downstream of a gene coding sequence that contains, for example, a transcription termination site and (in most, but not all, eukaryotic mRNAs) a polyadenylation signal (e.g., AAUAAA or its variants). After transcription termination, the mRNA transcript can be cleaved downstream of the polyadenylation signal, and a poly(A) tail can be added, which participates in the transport of mRNA to the cytoplasm, where translation occurs.

[0076] A "vector" is a nucleic acid molecule (typically DNA or RNA) used to transfer a nucleic acid sequence (i.e., DNA or RNA) into a host cell. Three common types of vectors include plasmids, bacteriophages, and viruses. Preferably, the vector is a virus. Vectors containing both a promoter and a cloning site are well known in the art, to which a polynucleotide can be operatively linked. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech (Madison, Wisconsin). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate additional, potentially inappropriate alternative translation start codons or other sequences that may interfere with or reduce expression at the transcriptional or translational level. Alternatively, a shared ribosome binding site may be inserted immediately adjacent to the start codon at the 5' end to enhance expression.

[0077] “Viral vector” refers to a vector containing some or all of the following: a viral gene encoding a gene product, a control sequence, and a viral packaging sequence. “Parvoviral vector” is defined as a recombinant parvovirus or parvovirus particle containing a polynucleotide to be delivered in vivo, in vitro, or in vitro to host cells. Examples of parvoviral vectors include, for example, adeno-associated virus vectors. In this document, a parvoviral vector construct refers to a polynucleotide containing a viral genome or a portion thereof and a transgene. As used herein, “recombinant adeno-associated virus vector” is defined as a recombinant adeno-associated virus vector or recombinant adeno-associated virus particle containing a polynucleotide or expression cassette to be delivered in vivo, in vitro, or in vitro to host cells.

[0078] As used herein, the term "promoter" or "transcriptional regulatory sequence" refers to a nucleic acid fragment that controls the transcription of one or more coding sequences, is upstream of the transcription start site relative to the coding sequence, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art to directly or indirectly regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated (e.g., by the application of a chemical inducer). A "tissue-specific" promoter is active only in specific types of tissues or cells.

[0079] A promoter can be any suitable promoter sequence that exhibits transcriptional activity in the cell, including mutant, truncated, and heterozygous promoters, and can be obtained from genes that encode extracellular or intracellular polypeptides that are homologous (natural) or heterologous (exogenous) to the cell.

[0080] Regarding flanking sequences of one or more other elements herein, the term "flanking" indicates the presence of one or more flanking elements upstream and / or downstream (i.e., 5' and / or 3') relative to the sequence. The term "flanking" is not intended to indicate that the sequence must be continuous. For example, intercalation sequences may exist between the nucleic acid encoding the transgene and the flanking element. "Flanking" for a sequence of two other elements (e.g., an ITR) indicates that one element is located at the 5' of the sequence and the other at the 3' of the sequence; however, intercalation sequences may exist between them. In a preferred embodiment, the nucleotide sequence of (i) is flanked by a parvovirus inverted terminal repeat nucleotide sequence on either side.

[0081] The terms “polynucleotide” and “nucleic acid”, used interchangeably herein, refer to polymeric forms of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, this term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or equivalently modified, non-natural, or derived nucleotide bases. “Oligonucleotide” generally refers to a polynucleotide of about 5 to about 100 nucleotides in single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also called oligomers or oligos and can be isolated from genes or chemically synthesized by methods known in the art.

[0082] As used herein, the terms “treatment,” “treating,” etc., refer to achieving a desired pharmacological and / or physiological effect. This effect can be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects attributable to the disease. As used herein, “treatment” encompasses any treatment of diseases in mammals, particularly humans, and includes: (a) Prevention of disease in subjects who may be susceptible to the disease but have not yet been diagnosed with the disease; (b) Suppress the disease, that is, prevent its development; and (c) Relieve disease, that is, cause the disease to subside.

[0083] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Global alignment algorithms (e.g., Needleman-Wunsch) are preferred for aligning sequences of similar length, as these algorithms optimally align sequences across their entire length; while local alignment algorithms (e.g., Smith-Waterman) are preferred for aligning sequences of significantly different lengths. Sequences (when optimally aligned using default parameters via, for example, programs GAP or BESTFIT) can be described as "substantially identical" or "commonly similar" when they have at least a minimum percentage of sequence identity (as defined below). GAP uses Needleman and Wunsch global alignment algorithms to align two sequences across their entire length (full length), maximizing the number of matches and minimizing the number of gaps. Global alignment is suitable for determining sequence identity when two sequences have similar lengths. Typically, the default GAP parameters are used, where the vacancy creation penalty = 50 (nucleotides) / 8 (proteins) and the vacancy extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff and Henikoff, 1992, PNAS [Proceedings of the National Academy of Sciences] 89, 915-919). Sequence alignment and sequence identity percentage scores can be determined as follows: using a computer program, such as GCG Wisconsin software package version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, 92121-3752, USA; or using open-source software, such as the programs "needle" (using the global Needman-Onsch algorithm) or "water" (using the local Smith-Waltman algorithm) in EmbossWIN version 2.10.0, using the same parameters as GAP above, or using the default settings (both for "needle" and "water," and both for protein and DNA alignments, the default gap opening penalty is 10.0, and the default gap extension penalty is 0.5; for protein, the default score matrix is ​​Blossum62; and for DNA, the default score matrix is ​​DNAFull). When sequences have significantly different total lengths, local alignment, such as local alignment using the Smith-Waltman algorithm, is preferred. Alternatively, the similarity or identity percentage can be determined by searching public databases using algorithms such as FASTA, BLAST, etc.

[0084] When this article mentions a dosage such as 1E14 gc / kg, it can also be referred to as 1x10.14 gc / kg or 1.0 x 10 14 The dosage is gc / kg.

[0085] For the avoidance of ambiguity, the terms “preparation as described herein”, “preparation of the present invention”, “isotropic preparation”, “isotropic preparation of the present invention”, “isotropic preparation as disclosed herein” and “isotropic preparation as described herein” are used interchangeably. Attached Figure Description

[0086] Figure 1 - Absorbance measurements at 280 nm and fluorescence measurements at 335 nm under different salt concentrations vary with acceleration stress conditions and duration. Different pH values ​​are also considered.

[0087] Figure 2 - Absorbance measurements at 280 nm and fluorescence measurements at 335 nm vary with accelerated stress conditions and duration. Different pH values ​​are shown as a gradient from dark (pH 8.0) to light (pH 5.0). Different salt concentrations are combined.

[0088] Figure 3A - Genome copies of various formulations after accelerated stability studies at 25°C.

[0089] Figure 3B - Similar to Figure 3A However, it shows the ratio of genome copies to infectious particles.

[0090] Figure 3C - Similar to Figure 3A However, sub-visible particles >10 µm were observed.

[0091] Figure 3D - Similar to Figure 3A However, visible particles are shown.

[0092] Figure 4A - The ratio of genome copies to infectious particles in various formulations after a 4-week degradation study at 40°C.

[0093] Figure 4B - Similar to Figure 4A However, sub-visible particles >10 µm were observed.

[0094] Figure 4C - Similar to Figure 4A However, it exhibits polydispersity.

[0095] Figure 5A - Genome copies of various formulations after long-term stability studies at -80°C.

[0096] Figure 5B - Similar to Figure 5A However, sub-visible particles >10 µm were observed.

[0097] Figure 5C - Similar to Figure 5A However, it shows the ratio of total particles to genome copies.

[0098] Figure 6A - Genome copies of various formulations after long-term stability studies at 2℃-8℃.

[0099] Figure 6B - Similar to Figure 6A However, visible particles are shown.

[0100] Figure 6C - Similar to Figure 6A However, sub-visible particles >10 µm were observed.

[0101] Figure 6D - Similar to Figure 6A However, sub-visible particles >0.3 µm were observed.

[0102] Figure 7 Liver transduction and GLA activity – Liver samples were collected from non-human primates (NHPs) at 3 and 6 months post-dose, and vector DNA (A), transgenic α-galactosidase A (GLA) mRNA (B), and GLA activity (C) in these liver samples were analyzed. Solid symbols represent samples collected at 3 months post-dose, and hollow symbols represent samples collected at 6-month follow-up. Squares depict male animals, and circles depict female animals. Horizontal lines represent the mean + / - SD for each group. Group means and SDs for vector DNA and transgenic GLA mRNA were calculated using the mean values ​​from different hepatic lobes for each animal. The lower limit of quantification (LLOQ) for vector DNA was 50 copies / µg DNA, and for transgenic GLA mRNA, the LLOQ was 5,000 copies / µg RNA. For all analyzed samples, total GLA activity in the liver was within the reportable range as determined.

[0103] Figure 8 Plasma GLA activity – Plasma samples were collected at different time points before and after administration, and the GLA activity of these plasma samples was analyzed. Values ​​are plotted as mean + SEM for each group. (A) Plasma GLA activity in all animals up to 3 months after administration. (B) Plasma GLA activity in animals followed up to 6 months after administration (n = 4 animals / group). For all analyzed samples, plasma GLA activity was within the measured dynamic range.

[0104] Figure 9 Vector DNA levels in the liver of NHP patients at 3 and 6 months post-IV infusion of the drug product – (A) shows vector DNA levels (mean ± SD) in the liver of NHP patients treated with different doses of the drug product at 3 months post-administration. (B) Vector DNA levels in the liver of NHP patients treated with 2.0 × 10⁻⁶ doses at 3 and 6 months post-administration. 14 and 7.3 × 10 14 Vector DNA in the liver of NHP treated with gc / kg (mean ± SD). Each symbol represents one animal, bars depict the group mean, and whisker lines depict the SD. Group mean and SD were calculated using the mean of different liver lobes for each animal. LLOQ is 50 copies of vector DNA / µg DNA.

[0105] Figure 10 Biodistribution of vector DNA in NHP treated with the drug product at 3 months post-dose – The diagram shows vector DNA (mean ± SD) from tissues of NHP treated with different doses of the drug product at 3 months post-dose. LLOQ is 50 vector DNA copies / µg DNA (dashed line). The limit of detection (LOD) is 12.5 vector DNA copies / µg DNA. For some samples from the epididymis, heart, muscle, sciatic nerve, dorsal root ganglion, thyroid gland, administration site, spinal cord, aorta, vena cava, abdominal skin, and fingertip skin, less than 1 µg DNA can be extracted, and depending on the amount of DNA input per reaction, the LOD is up to 117 copies / µg DNA and the LLOQ is up to 467 copies / µg DNA. To calculate the group mean and standard deviation, samples with results between LLOQ and LOD were assigned a value of 18.75 (=(LLOQ-LOD) / 2), and samples with results below LOD were assigned a value of 6.25 (=LOD / 2).

[0106] Figure 11 Vector DNA Levels in Blood and Plasma from NHP Treated with the Drug Product – The table shows vector DNA levels (mean ± SD) in blood (A) and plasma (B) from NHP treated with different doses of the drug product up to 3 months post-dose. LLOQ is 50 copies of vector DNA / µg DNA in blood. For some time points, less than 1 µg of DNA may be extracted from blood samples, and depending on the amount of DNA input for each reaction, the LLOQ is a maximum of 6.485 x 10⁻⁶. 3 One copy / µg DNA. The LLOQ of plasma was not determined because DNA was extracted from these samples using vector nucleic acids, which hindered the assessment of sample DNA concentration.

[0107] Figure 12 Drug-product-derived GLA mRNA expression in selected tissues of NHP treated with the drug product at 3 months post-dose – The drug-product-derived GLA mRNA (mean ± SD) is shown in tissues of NHP treated with different doses of the drug product at 3 months post-dose. LLOQ is 50 mRNA copies / reaction, corresponding to 5000 copies / µg RNA (dashed line). LOD is 3.125 mRNA copies / reaction (312.5 copies / µg RNA). To calculate the group mean and standard deviation, samples with results between LLOQ and LOD were assigned a size of 23.44 x 10⁻⁶. 3 The value of (=(LLOQ-LOD) / 2) is assigned, and samples with results below LOD are assigned the value 1.5625 (=LOD / 2).

[0108] Example Material The following materials are used in the examples, and their sources are specified in Table 1 below.

[0109] Table 1: Materials used in the examples and their suppliers Data points The stress conditions and data collection time points for each instance are specified in Table 2 below.

[0110] Table 2: Stress conditions and data collection time points for the examples RT: Room temperature NA: Unavailable Measurement results 1. The readings obtained are as follows: • Absorbance at 260 nm, which is related to DNA concentration.

[0111] • Absorbance at 280 nm, which is related to protein concentration.

[0112] • Absorbance (turbidity) at 350 nm, which is associated with protein aggregation, or agglomeration and / or particle formation.

[0113] • Absorbance at 900 nm, which is correlated with the background signal from the plastic of the pores and seals.

[0114] • Absorbance at 975 nm: The absorbance of water is measured, thereby measuring the optical path length.

[0115] • Tryptophan fluorescence at 280 / 335 nm, measured from the top and bottom of the microplate. This measurement is correlated with protein and / or capsid unfolding and denaturation. Bottom readings are used in the accompanying figures.

[0116] 2. Visual inspection Conduct visual inspections in a generally accepted manner.

[0117] 3. Data Processing Protein and DNA concentrations are obtained as follows: According to the Lambert-Beer law, absorbance is proportional to concentration and optical path length. A constant called the extinction coefficient transforms this into the equation A = cc.L.ε, where A is absorbance, cc is concentration, L is optical path length, and ε is the extinction coefficient. The optical path length is measured by subtracting the plastic background (A900) measured at 900 nm from the absorbance of water (A975) measured at 975 nm; therefore: L = A975 - A900 Protein concentration is measured by measuring absorbance at 280 nm (A280), therefore: A280 = cc （蛋白质） .L.ɛ （蛋白质） 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 turbidity at 280 nm, and therefore subtracted from the absorbance at 280 nm. Then: cc （蛋白质） .ɛ （蛋白质） = (A) 280 –A 350 ) / (A 975 –A 900 ) Since α (protein) is a constant, we can conclude that cc (protein).α (protein) is proportional to the protein concentration, and the protein concentration is determined by the different absorbance values ​​measured (A). 280 A 350 A 975 and A 900 The calculation is performed. Similar reasoning applies to DNA concentrations at 260 nm.

[0118] Example 1: Pre-mixed The initial solution used in Example 1 was a transgenic recombinant adeno-associated virus vector serotype 5 (rAAV5) at a concentration of 4E13 gc / mL in PBS - / - (NaCl 137 mM; KCl 2.7 mM; Na2HPO4 10 mM and KH2PO4 1.8 mM), 5% w / v sucrose, and rAAV5-protein (which is used to treat Fabry disease) α-galactosidase A.

[0119] The initial solution containing rAAV5 was buffer-displaced in a multi-well system using 96-well plates (100 kDa molecular weight cutoff) to create thirty different formulations with varying pH values, buffer types, and salt concentrations. After buffer displacement and filtration through a 0.2 µm filter, the formulations were aliquoted into the plates according to the plate layout and buffer formulation compositions specified in Table 3 below. Each formulation was prepared in duplicate. Outer rows and columns were filled with WFI to eliminate plate position effects. The buffers had overlapping pH values ​​to distinguish between buffer and pH effects.

[0120] Table 3: Buffer plate layout and buffer preparation composition in Example 1 WFI is water for injection. Absorbance (at 280 nm), fluorescence (at 335 nm), and turbidity (at 350 nm) of different formulations were monitored. Absorbance was corrected for background turbidity and for optical path length. As specified in Table 2 above, three plates were prepared for each stress condition, and measurements were performed in duplicate for each condition (or each well), with the average of the replicates taken.

[0121] The results obtained at T = 0 are presented in tabular form in Table 4. Table 4 shows that the corrected absorbance values ​​(protein concentration) of the three well plates at 280 nm are similar, as expected. Furthermore, for the three plates at T = 0, the absorbance is higher in the presence of salt, particularly in the presence of 150 mM NaCl, thus indicating higher protein content and lower aggregation or agglomeration. This can be concluded because the measured values ​​include background correction as explained in the previous section. The presence of NaCl shows a positive effect. Buffers with a pH of 6.5 to 8 (especially with added salt) are preferred because higher absorbance (protein concentration) is observed.

[0122] Table 4: Protein content at T = 0, measured by absorbance at 280 nm (corrected for turbidity and optical path length in three multi-well plates; arbitrary units). A: F / T at -80°C; B: 28°C and C: 40°C.

[0123] Results from fluorescence measurements (protein and / or capsid unfolding and denaturation) are shown in Table 5. Lower fluorescence indicates greater protein unfolding. These results suggest that the optimal buffers for stabilizing the viral capsid are citrate at pH 6.5, phosphate at pH 7.0 and 7.5, and Tris at pH 7.5 and 8.0 with 75 mM and 150 mM NaCl, respectively, with the 150 mM NaCl concentration showing improvement compared to the 75 mM NaCl concentration.

[0124] Table 5: Fluorescence at T = 0 measured by fluorescence at 335 nm (measurements from the bottom of three multi-well plates; arbitrary units). A: F / T at -80 °C; B: 28 °C and C: 40 °C.

[0125] Accelerated studies were conducted under various conditions. Various measurements were performed on samples at 28°C and 40°C after two and seven days; and on samples under F / T stress after 1, 3, and 10 cycles.

[0126] Figure 1 The absorbance at 280 nm and fluorescence (Trp) at 335 nm for different formulations are shown in the accelerated stress study. It can be seen that higher salt concentrations are associated with higher absorbance at 280 nm and higher fluorescence at 335 nm. This observation is consistent with that observed at T = 0, suggesting that higher salt concentrations are beneficial to the formulation in terms of protein concentration.

[0127] exist Figure 2 In the figure, absorbance at 280 nm and fluorescence at 335 nm vary with different applied stresses. Three subplots exist for different salt concentrations: the left subplot for 0 mM, the middle subplot for 75 mM, and the right subplot for 150 mM. As observed in the previous figure, the effect of salt on absorbance and fluorescence is clear: higher salt concentrations result in higher absorbance and fluorescence, demonstrating the beneficial effect of salt. The effect of pH is also visible: higher pH leads to higher absorbance and fluorescence. This effect is particularly observed in fluorescence (Trp), although its magnitude is smaller than that observed in the salt effect.

[0128] Therefore, Example 1 concludes that salt concentration (above 75 mM) and pH (above 7.0) have a beneficial effect on enhancing capsid stability. Tris pH 7.5 and 8.0, as well as pH 7.0 phosphate buffer, were selected for further testing and optimization.

[0129] Example 2: Excipient Screening Accelerated studies were conducted under various conditions. Samples were tested at 28°C and 40°C after one and two days; samples under F / T stress after 1 and 3 cycles; and samples were tested after shaking at room temperature for 1 hour. Compared to control conditions (using buffer alone), the different excipients cysteine, arginine, and MgCl2 showed no significant or measurable effect on product stability at any test pH: 7.0, 7.5, or 8.0.

[0130] A second excipient screening experiment was conducted to evaluate the effects of two carbohydrates on the stability of the drug product: mannitol and cyclodextrin. The primary variables in this example were the concentrations of mannitol and cyclodextrin individually or in combination. The concentrations of mannitol and cyclodextrin ranged from low (0.05% w / v) to high (4% w / v).

[0131] Various buffer formulations were further tested and optimized to assess the effects of adding mannitol and / or cyclodextrin (hydroxypropyl-β-cyclodextrin was used here). Several multiwell plates were prepared as specified in Table 2 above and assigned to different stress conditions. The alkaline buffer (referred to as TH+) was Tris 20 mM buffer at pH 7.5. The buffer further contained tonicants to set the buffer at the desired osmolality (290 mOsm / kg) to simulate physiological conditions (here: 5 mM histidine, 2.5 mM MgCl2, 2.5 mM KCl, 2.5 mM CaCl2, and NaCl). The pharmaceutical product used in Example 2 was the same rAAV5 used in Example 1. Different percentages of mannitol or cyclodextrin were added to the TH+ buffer (see Table 6). As controls, PBS buffer supplemented with 5% sucrose at pH 7 (Control 1) and TH+ buffer alone (Control 2) were used.

[0132] The formulation matrices tested for Example 2 are shown in Table 6. Each formulation was prepared in triplicate. The outer rows and columns were filled with WFI to exclude plate position effects.

[0133] Table 6: Buffer plate layout and buffer preparation composition in Example 3 Table 7 shows the results of the formulation validation experiment. It displays the absorbance of the plate at 280 nm, as measured at T = 0. Generally, formulations containing cyclodextrin always exhibit better formulation characteristics.

[0134] Table 7: Protein content at T = 0 as measured by absorbance at 280 nm (corrected for turbidity and optical path length in four-well plates; arbitrary units). A: F / T at -80°C; B: 28°C; C: 40°C; and D: oscillation.

[0135] Protein content was measured under different stress conditions for varying durations. Stress conditions included incubation at 28°C and 40°C for 0, 3, or 7 days; F / T cycles of 2 and 5; and shaking for 2 hours (SHK). Different concentrations of mannitol or cyclodextrin as stability-enhancing excipients did not show significant differences beyond T = 0 under different stress conditions or durations (data not shown). This may indicate that the stabilizing effects of different formulations achieved at T = 0 remain effective under applied stress.

[0136] Conclusions based on Examples 1 and 2: The conclusions of this study are as follows: • Cyclodextrins exhibit beneficial stabilizing effects.

[0137] • Salts (such as NaCl) also help stabilize pharmaceutical products. Adding salt at a concentration level of 75 mM or higher is beneficial.

[0138] • Neutral or slightly alkaline formulations with a pH (7.0-8.0) improve the stability of pharmaceutical products.

[0139] Example 3: Testing the stability of rAAV5 in the formulation.

[0140] Therefore, based on the above two examples, the following formulations of the present invention are specifically illustrated: • Transgenic rAAV5 encoding therapeutic proteins • 20 mM Tris at pH 7.5 • 125 mM sodium chloride, and • 2% w / v HP-β-cyclodextrin This buffer is referred to as FB in this document. The rAAV5 used in this example has a transgene encoding a therapeutic protein.

[0141] Table 8: Materials Visual inspection No visible particles of rAAV5- protein were observed in FB under 1 F / T+ at room temperature (15℃–25℃) for 24 h, 1 D of agitation at room temperature, and 3 F / T stress conditions (Table 9). Results showed small particles at 37℃ (1 W and 2 W) and 25℃ (2 W and 4 W). In 1–2 of 3 vials, particles were observed in FB at 2℃–8℃ for 1 M. However, no visible particles were observed in FB at 2℃–8℃ for 3 M and 6 M time points. Therefore, particles observed at 2℃–8℃ (1 M) can be disregarded. No visible particles were also observed at 1 M in a 500 L batch (with a concentration of 5E13 gc / ml instead of 1E14 gc / ml as in this study) at 2℃–8℃.

[0142] Table 9: Overview of visual inspection results of the platform formulation fit study of rAAV5-protein in FB with a target gc of 1E14 gc / mL. Subvisible particles Subvisible particle content was analyzed from two replicates at each time point / condition. For each particle size range, the data from the two replicates were averaged. Table 10 summarizes the cumulative subvisible particle counts / vials for sizes >10 µm and >25 µm. Under all stress conditions, the subvisible particle count of the rAAV5-enzyme in FB remained within the Acceptance Standard (USP).

[0143] Table 10. Cumulative subvisible particle counts of rAAV5- protein in FB at a target gc of 1E14 gc / ml / vial (production batch 1).

[0144] Genome copy, total particles and infectivity For the platform formulation fitting study, the gc concentration of rAAV5- protein in FB remained at approximately 1E14 gc / ml under different conditions, within the range of measurement variation (% CV < 25%, genomic copy number of rAAV5- enzyme test samples determined by quantitative PCR). Therefore, it can be concluded that under the test conditions, F / T cycling, temperature stress, and 1D agitation had no effect on the genomic copy number of rAAV5- protein in FB.

[0145] The total particle content of rAAV5- protein in FB remained stable between 1.1E+14 total particles (tp) / mL and 1.3E+14 tp / mL, except under stress conditions of 37°C (1 W and 2 W) and 25°C (2 W), under which the total particle content decreased from approximately 1.2E14 tp / mL to 7E13 tp / mL, which may indicate the presence of rAAV5 aggregates, and is relevant to the conclusions of visual examination. Under all conditions, the tp / gc ratio of rAAV5- protein in FB remained below 2.

[0146] Infectivity, expressed as infectious particles (ip) / mL, shows a variable dataset, primarily due to the variability of the method (CV% < 80%, based on replication-based infectious vector titer determination). Typically, the infectivity of rAAV5- protein in FB decreases at 37°C and 25°C. Compared to the control at T0, the infectivity of rAAV5- protein in FB did not show significantly different results after 3 F / T cycles and 1 D agitation.

[0147] Dynamic light scattering (DLS) DLS measurements yielded a polydispersity index (PDI) and a z-mean. For the rAAV5-enzyme in FB, the PDI value was below 0.1 under various conditions, indicating monodispersity, where the size remained at approximately 25 nm.

[0148] Example 4: Evaluating stability at a batch size 10 times larger The stability results for rAAV5- protein obtained in FB were from a 50 L production batch. Comparability studies were conducted between batch sizes ranging from 50 L to 500 L to compare the stability of rAAV5- protein produced from different batch sizes with comparable titers of 1E14 gc / mL. This was performed under four different stress conditions: one F / T + at room temperature (15℃–25℃) for 24 h, three F / T cycles, one D agitation, and one W at 37℃.

[0149] Under the four different stress conditions described above, data from the 50 L batch (production batch 1) were compared with those from the 500 L batch of rAAV5-protein. The results are summarized in Table 11. All attributes monitored by visual inspection, subvisible particles, genome copy concentration, total particles, infectious vector titer, monomer particle purity and size distribution, gc / ip ratio, and tp / gc ratio remained within the rAAV5-protein drug product specifications (MS-07014), and there were no significant differences between the 50 L batch (production batch 1, at 1E14 gc / mL) and the 500 L batch (production batch 2, at 1E14 gc / mL). Therefore, based on the overall collected data, the rAAV5-protein from the 50 L (production batch 1, at 1E14 gc / mL) and 500 L (production batch 2, at 1E14 gc / mL) batches are comparable.

[0150] Table 11: Overview data of rAAV5-protein from 50 L production batch 1 in FB compared with rAAV5-enzyme (at 1E14 g c / mL) from 500 L production batch 2 under 4 different stress conditions.

[0151] Example 5: Further comparison of FB with other buffers Using the materials and methods described for the previous examples, the following buffers were compared with the rAAV5 protein (where the therapeutic protein is factor VIII) in the presence of a formulation containing approximately 4E13-5E13 genomic copies: 1. PBS + 5% sucrose + 0.02% PS20; 2. FB (20 mM Tris pH: 7.5 + 125 mM NaCl + 2% HP-β-cyclodextrin); 3. 20 mM citrate / phosphate pH: 7.5 + 125 mM NaCl + 0.1% PS80; and 4. 20 mM Tris pH: 7,5 + 125 mM NaCl + 0,1% PS80.

[0152] Accelerated developmental stability studies were conducted on the formulations at 25°C for up to three months. The four formulations showed similar performance. Figure 3A Except for preparation 3, the GC / IP of the product exceeded the acceptance criteria at 3 months. Figure 3B For formulations 3 and 4, an increase in the number of subvisible particles was observed. Figure 3CEven after 3 months, no visible particles were found in any of the preparations. Figure 3D ).

[0153] Degradation studies were also conducted at 40°C for a period of 4 weeks. All formulations showed an increase in GC / IP, with formulations 2 and 4 exhibiting the best performance. Figure 4A The subvisible particle count also increased in all formulations except for formulation 2. Figure 4B After 2 weeks at 40°C, formulations 1 and 3 showed increased polydispersity. Figure 4C ).

[0154] The four formulations were placed at -80°C for long-term developmental stability studies. Nearly parallel curves of genome copy number showed no significant differences in this parameter among the four formulations. Figure 5A Formulation 1 showed visible particles after 6 months. Formulation 3 showed an increase in subvisible particles, and all formulations except formulation 2 showed an increase in >10 µm particles. Figure 5B Furthermore, preparations 3 and 4 showed a higher total particle to genome copy ratio (tp / gc) than other samples, implying more impurities after 1 month. Figure 5C Furthermore, in the first few months of testing, formulations 3 and 4 showed an increase in polydispersity index.

[0155] The four formulations were also placed in a long-term developmental stability study at 2℃–8℃. The nearly parallel curves of genome copy number again showed that this parameter did not differ significantly among the four formulations. Figure 6A Furthermore, formulations 3 and 4 were less stable, showing visible particles after 6 and 12 months, respectively. Figure 6B ), and an increase in subvisible particles was observed after 6 months ( Figure 6C All formulations except formulation 2 showed an increase in subvisible particles >0.3 µm. Figure 6D ).

[0156] Example 6: Determination of the stability of prepared drug products 2℃-8℃, for up to 12 months ≤ -65℃, up to 24 months For 5 x 10 13 gc / mL 500 L scale, non-GMP (Good Manufacturing Practice) certified The rAAV5 vector contains a transgene encoding α-galactosidase A.

[0157] In a 2 mL glass vial containing 20 mM Tris, 125 mM NaCl, and 2% HP-β-CD (w / v) (pH 7.5), the solution was prepared at 5 × 10⁻⁶ ppm. 13 Pharmaceutical products (DPs) were prepared at a concentration of (gc) / mL and filled to a volume of 1.2 mL. DP vials were stored upright under storage conditions (≤ -65°C) for up to 24 months and under accelerated storage conditions (2°C–8°C) for up to 12 months. The stored DPs were sampled at six predetermined time points (0, 1, 3, 6, 12, and 24 months) to determine quality attributes (QA).

[0158] a Based on a single duplicate value, gc = genome copy, ip = infectious particles, tp = total particles. a Based on a single duplicate value, gc = genome copy, ip = infectious particles, tp = total particles. Subvisible particles The number of subvisible particles was determined using the accusizer method. Particles ≥ 10 µm and ≥ 25 µm are both shown in Tables 12 and 13 (particles / 10.7 mL DP). Acceptance criteria conform to USP. <787> Small-volume parenteral specifications: ≤ 6000 particles / vial (≥ 10 µm) and ≤ 600 particles / vial (≥ 25 µm). Subvisible particle counts for all test time points and corresponding storage conditions were within pharmacopoeia specifications and well below acceptance criteria. The results shown (Tables 12 and 13) were extrapolated to 10.7 mL, as this is the DP volume per clinical vial.

[0159] Genome copy, total particles and infectivity Under two storage conditions lasting up to 24 M, the GC concentration remained at the DP specification (3.1 × 10⁻⁶) of the GMP material. 13 - 6.8×10 13 Within (Tables 12 and 13). Variation was observed between T0 and different time points, but at all test time points, the variation was within the range determined under both storage conditions (2℃–8℃ and ≤ -65℃). Under both storage conditions, at DP 5 × 10 13Size exclusion chromatography (SEC) at all time points in gc / mL was performed with 100% purity. During the entire storage period of up to 24 M at 2℃–8℃ and ≤ -65℃, the variation in total particulate matter (TP) content of DP (5 × 10¹³ gc / mL) was slightly greater than the variation measured (relative standard deviation (RSD) % ≤ 8%). However, no significant trend was observed between these time points, and no change was observed between 12 M and 24 M at ≤ -65℃. The tp / gc ratio remained within the DP product specification (≤ 2.0 tp / gc) during the entire storage period of up to 24 M at both 2℃–8℃ and ≤ -65℃.

[0160] The infectious titer of DP varied within the measured range (coefficient of variation < 67%). Therefore, it was concluded that no difference in infectious titer was observed. (Using 1 × 10⁻⁶) 14 Similar observations were made for DPs with gc / mL. For DPs stored for up to 12 or 24 M at 2℃–8℃ and ≤-65℃, the genome copy to infectious particle (gc / ip) ratio remained equal to or less than 48 gc / ip (Tables 12 and 13). DP gc / ip specifications ≤ 70, and therefore the DPs studied (5 × 10⁻⁶) were... 13 (gc / mL) conforms to the specifications set for GMP batches.

[0161] Dynamic light scattering (DLS) DLS was performed according to standard procedures. The Z-mean and polydispersity index (PdI) of DP varied only slightly between time points under both storage conditions starting from T0 (Z-mean range: 26.8–27.1 nm, and PdI range: 0.036–0.060). At T0, both the Z-mean and PdI were higher, likely due to sample or measurement artifacts (e.g., dust, bubbles), as all other time points showed comparable lower values. Both the Z-mean and PdI were consistent with those used in long-term stability studies using 1 × 10⁻⁶. 14 The observations for gc / mL DP were consistent (data not shown).

[0162] result Based on the 12 M and 24 M results of the long-term stability study (500 L) of DP, the following conclusions are drawn: At 2℃-8℃, 5 × 10 13 All quality attributes (QA) of DP at gc / mL (500 L) remained comparable to T0, except for visible particles. Visible particles were observed at 6 and 12 months. At ≤ -65°C, 5 × 10 13The QA of DP at gc / mL (500 L) was comparable to T0, except for visible particles. Visible particles were observed only at the 12-month time point.

[0163] However, when stored at ≤ -65℃, 1 × 10 14 DP (data not shown) at gc / mL (50 L) was free of visible particles for up to 24 M. Under both storage conditions, at 1 × 10⁻⁶ g / mL (50 L), the DP was also free of visible particles. 14 In DP at gc / mL (50 L), no difference compared to T0 was observed in any of the other QAs for up to 24 M.

[0164] In summary, DP QA demonstrates that the formulation can accommodate high concentrations of rAAV carriers while maintaining the advantageous characteristics that allow for patient administration, and that these characteristics are maintained for at least 24 months when stored at ≤ -65°C and for at least 12 months when stored at 2°C–8°C.

[0165] Example 7: Determination of the stability of GMP-grade prepared drug products Table 14 provides stability data for clinical GMP-grade pharmaceutical products (DP) batches under long-term storage conditions of ≤ -65°C. Values ​​were obtained as described above. Weight osmolality was determined using standard procedures.

[0166] Similar to the conclusions drawn from non-GMP grade DP, the above data show that prepared drug products can be stored for extended periods at temperatures ≤-65°C.

[0167] Example 8: Dose evaluation of a 26-week single intravenous administration biodistribution and toxicity study (GLP) in cynomolgus monkeys. The aim of this study was to evaluate the biodistribution, α-galactosidase A (GLA) pharmacodynamic markers, and toxicity (see Table 15) of a drug product formulated in 20 mM Tris (pH 7.5), 125 mM NaCl, and 2% HP-β-cyclodextrin in male and female NHPs following a single IV administration, followed by 3-month and 6-month post-dosing observation periods. GLA pharmacodynamic markers are described below. Detailed evaluations of pharmacokinetic parameters, vector DNA biodistribution, and transgenic mRNA expression, as well as a detailed description of all safety evaluations, are also provided below.

[0168] Table 15: Design of Single-Dose Toxicity Studies of Pre-Prepared Drug Products in NHP aMedium: Buffer solution: 20 mM Tris (pH 7.5), 125 mM NaCl, 2% HP-β-cyclodextrin Pharmacology To determine whether durable and functional expression of hepatic drug-derived GLA proteins and their transport to plasma could be obtained in large animal models, GLA activity in the liver (3 and 6 months post-drug administration) and plasma was measured during the study. Values ​​obtained in the liver and plasma of animals treated with the drug product were compared with endogenous NHP GLA activity found in NHP treated with the vehicle. In addition, GLA activity was measured in selected extrahepatic tissues; adrenal glands, brain, dorsal root ganglia, heart, kidneys, and spleen.

[0169] Methods for testing pharmacodynamic endpoints GLA activity was measured using the same methods commonly used in the clinical diagnosis and follow-up of Fabry disease patients. The suitability of this assay for analyzing tissue and plasma samples from NHP was confirmed, and the assay was deemed qualified for use. In addition to GLA activity measurements, hepatic transduction was assessed as an additional pharmacokinetic / pharmacodynamic parameter by evaluating the expression of vector DNA and vector-derived GLA mRNA. Further information regarding the development and validation of the qPCR method for detecting vector DNA and vector-derived GLA mRNA is presented below.

[0170] Blood and tissue sampling procedures for pharmacodynamic endpoints Blood samples for pharmacodynamic evaluation were collected periodically before and during the observation period after administration. Blood was collected from the cephalic vein or saphenous vein in the forearm. For plasma separation, venous blood was collected into tubes containing K2EDTA to prevent coagulation and held on wet ice until centrifugation to harvest the plasma. For serum, venous blood was collected into serum tubes and allowed to coagulate at room temperature for >30 min, then centrifuged to separate the serum. Plasma and serum samples were stored frozen (<-65°C) until use.

[0171] Measurement of antibodies against drug-derived GLA in NHP Antibodies against GLA protein in NHP serum samples were determined using a semi-quantitative MesoScale Discovery (MSD) assay. Since no calibrators were available, results are expressed in relative electrochemiluminescence units. While this method does not produce titers, the resulting values ​​should be proportional to the amount of circulating GLA-specific monkey antibody. Test samples with a signal >250 underwent confirmatory assays, in which the test samples were first pre-incubated at room temperature for 1 hour with or without excess (100 µg / mL) GLA protein before being added to the immunoassay plate.

[0172] Liver transduction and GLA activity At 3 and 6 months after administration, until 5.0 x 10 14 At a dose of gc / kg, liver vector DNA levels showed a dose-dependent increase. Figure 7 A). Administer 2.0 x 10 14 Up to 7.3 x 10 14 In animals with gc / kg liver transgenic GLAAm RNA levels, the level of transgenic GLAAm RNA was proportional to the level of vector DNA. Figure 7 B). At 4.0 x 10 13 At a dose of gc / kg, relatively higher transgenic GLA mRNA levels were observed compared to those expected based on vector DNA levels measured in this dose group. At 3 and 6 months post-administration, levels were observed in samples taken from 2.0 x 10 g / kg of gc / kg. 14 and 7.3 x 10 14 Comparable levels of vector DNA and drug-derived GLA mRNA (data not shown) were found in the livers of animals treated with the drug product at gc / kg. As expected, no vector DNA or transgenic GLA mRNA was found in the livers of animals treated with the drug product. GLA activity analysis in liver tissue showed increased hepatic GLA activity in all drug product treatment groups compared to animals treated with the drug product. Figure 7 C). Although animals treated with the carrier showed a mean hepatic GLA activity of 98 ± 9 nmol / h / mg (their endogenous GLA activity), an increase in hepatic GLA activity (corresponding to carrier-derived GLA) was observed in animals treated with the drug product. Individual variations in hepatic GLA activity were observed within each dose group.

[0173] Plasma GLA activity During the first two months after administration, when using 4.0 x 10 13 Up to 5.0 x 10 14 When the drug product was administered at gc / kg, NHP showed a peak in plasma GLA activity followed by a decline. Figure 8 A). Plasma GLA activity stabilized between weeks 10 and 13 and remained at that level until the study ended at week 26 post-dose. Figure 8 B). As the dose increases, individual variations within each dose group become more pronounced because each group has one or more “low” responders, with plasma GLA activity 1.5 to 2 times higher than the medium, showing a dose-dependent increase in plasma GLA activity in animals, from 4.0 × 10⁻⁶. 13 The gc / kg group was 4.5 times the medium level up to 5.0 × 10⁻⁶.14 The group treated with gc / kg of the drug product was 40 times higher than the mediator level. In the group treated with 7.3 × 10 14 The changes observed in the gc / kg dosing group were primarily caused by a high-response animal that showed a 98-fold increase in plasma GLA activity compared to the animal treated with the vehicle, while other animals in the group showed increases in plasma GLA activity ranging from 6 to 26-fold.

[0174] GLA activity in extrahepatic tissues Compared with animals treated with the drug, measurements of GLA activity in extrahepatic tissues did not show an increase in GLA activity. This is most likely due to the significant background levels of this method and the presence of endogenous GLA activity in healthy NHPs.

[0175] Safety pharmacology of prepared drug products Electrocardiogram, blood pressure measurement, and respiratory rate measurement were included as routine examinations in the general toxicity study of NHP and showed no effect on the drug product.

[0176] result The formulated drug product was based on an AAV5 recombinant vector designed to deliver liver-specific expression of the human GLA transgene via a single (one-time) IV infusion. Pharmacodynamic and efficacy studies of the drug product demonstrated a strong correlation between vector dose and GLA expression levels, and confirmed the biological activity of the expressed and fully functional GLA protein. Up to 5.0 × 10⁻⁶ 14 At a dose of gc / kg, treatment of NHP with the drug product resulted in a dose-dependent increase in liver vector DNA and mRNA levels. This was observed after receiving 7.3 × 10 gc / kg of the drug. 14No further increase was observed in animals receiving gc / kg. Compared to animals treated with the drug product, both hepatic and plasma GLA activity showed a dose-dependent increase in all drug product treatment groups. In the first two months after administration, NHP showed a peak followed by a decline (primarily at lower dose levels), and stable plasma GLA activity, which had been observed in previous gene therapy procedures involving liver-directed human transgene overexpression (Spronck EA et al., Molecular Therapy Methods & Clinical Development, 2019, Vol. 15, 221-231). Between 2 and 3 months after drug product treatment, some animals showed an increase in plasma GLA activity. This may reflect the induction of humoral tolerance to drug product-derived GLA proteins in NHP receiving IV AAV8-GLA, as described by Nietupski et al. (Nietupski JB et al., Molecular Therapy, 2011, Vol. 19(11):1999-2011). The response was durable in NHP because liver vector DNA levels, vector-derived GLA mRNA levels, and plasma GLA activity levels persisted for at least 6 months after drug product administration (the longest time point tested), indicating vector persistence and expression persistence. In summary, durable expression of vector-derived GLA can be achieved in NHP with IV drug product administration.

[0177] Pharmacokinetics Biodistribution of drug-derived vector DNA in NHP was assessed using validated qPCR in plasma and blood samples collected before administration and during the study period. Tissue levels of vector DNA were measured in liver, kidney, heart, brain, gonads, lung, spleen, dorsal root ganglion, injection site, and 14 other tissues harvested at 3 and 6 months post-drug administration. Furthermore, transduction in hepatocytes was analyzed using vector DNA-specific probes via fluorescence in situ hybridization (FISH). At these time points, transgene expression in the liver and 11 extrahepatic tissues was assessed by validated RT-qPCR.

[0178] Blood and tissue sampling procedures for pharmacokinetic endpoints Blood samples for pharmacokinetic assessment were collected periodically before and during the observation period after administration. In NHP studies, blood was collected from the cephalic vein or saphenous vein in the forearm. For plasma separation, venous blood was collected into tubes containing K2EDTA to prevent clotting and held on wet ice until centrifugation to harvest plasma. Plasma samples were stored frozen (<-65°C) until use. For serum separation, venous blood was collected into serum separation tubes and allowed to clot on ice at room temperature for at least 30 minutes until centrifugation to harvest serum. Serum samples were stored frozen (<-65°C) until use.

[0179] Pre-existing anti-AAV5 antibodies Since NHP is the natural host of AAV5, pre-existing (neutralizing) antibodies may be present due to natural infection with the wild-type virus. Evaluation of anti-AAV5 neutralizing antibodies showed that serum from 31 of the 36 NHP patients already possessed AAV5 neutralizing activity prior to administration, with titers ranging from 50 to 689. Similar to our experience with other AAV5-based gene therapy procedures in NHP patients with pre-administration anti-AAV5 neutralizing antibody titers up to 1,000 (Majowicz, Nijmeijer et al. 2019), no correlation was observed between neutralizing antibody titers and liver transduction levels (described below).

[0180] Liver vector DNA level Liver vector DNA levels are presented in Figure 9 In the middle. Four liver lobes of each animal were analyzed, and treated animals showed uniform diffusion of vector DNA between liver lobes. From low dose (4.0 × 10⁻⁶) 13 gc / kg) to 5.0 × 10 14 A dose-response relationship was observed in liver vector DNA copies at a drug product dose of gc / kg. At 7.3 × 10 14 No further increase in liver vector DNA copy number was observed at the highest dose of gc / kg. Figure 9 A). Similar levels of vector DNA were found in the livers of animals treated with the drug product at two time points, 3 and 6 months after administration. Figure 9 B).

[0181] Tissue vector DNA distribution Up to 5.0 × 10 14 At a dose of gc / kg, the biodistribution of the vector DNA in all tissues showed a clear dose-dependent effect. At a dose of 5.0 × 10⁻⁶ gc / kg, the biodistribution of the vector DNA in all tissues was significantly dose-dependent. 14 gc / kg and 7.3 × 10 14 Similar vector DNA levels were observed among animals treated with gc / kg ( Figure 10 Three months after administration, at 5.0 × 10⁻⁶ 14 and 7.3 × 10 14 At the two highest doses (gc / kg), the highest levels of vector DNA were found in the liver and adrenal glands (range 4 to 6 × 10⁻⁶ gc / kg). 6 (1 copy / ug DNA), followed by the aorta. Other tissues showed quantifiable, but less than 1 / 10 the level of vector DNA found in the liver.

[0182] level of carrier DNA in blood and plasma Measure the level of vector DNA in blood and plasma after drug product administration. Figure 11 A and Figure 11 B). Overall, blood and plasma carrier DNA clearance rates were comparable at each test dose. A sharp decline in blood and plasma levels was observed during the first 2 weeks after administration, followed by a slow decline until the final time point of 3 months post-administration.

[0183] GLA mRNA derived from drug products in tissues Based on the use of liver-specific promoters, RT-qPCR revealed the highest level of GLA transgene mRNA in the liver. Figure 12 Up to 5.0 × 10 14 At a dose of gc / kg, transgenic GLA mRNA levels were correlated with vector dose. Similar to vector DNA levels, the two highest doses (5.0 and 7.3 × 10⁻⁶) showed a correlation. 14 (gc / kg) showed comparable levels of GLA transgenic mRNA. At 3 and 6 months post-dose, the levels were observed in mRNA derived from 2.0 × 10⁻⁶ gc / kg. 14 and 7.3 × 10 14 Comparable levels of GLA transgenic mRNA were found in the livers of animals treated with the drug product at gc / kg. Approximately 1 / 10th the level of GLA transgenic mRNA was found in the adrenal glands and dorsal root ganglia (DRGs) compared to the liver, followed by the heart and spleen, showing lower but quantifiable levels. In the testes, at a concentration of 5.0 × 10⁻⁶ g / kg... 14 In some animals treated with gc / kg, mRNA levels were found to be just above LLOQ. In the kidney and sciatic nerve, no dose-dependent effect was observed in GLA transgenic mRNA levels, as most groups showed results near the measured LLOQ, except with 2 × 10⁻⁶ gc / kg. 14 Except for the kidney tissue of animals administered gc / kg, the expression of GLA transgenic mRNA showed high variability within this group. No quantifiable expression of GLA transgenic mRNA was observed in any other tissue (<5 × 10⁻⁶). 3 LLOQ of copy / µg RNA Figure 12).

[0184] In summary, a single-dose intravenous infusion of the prepared drug product resulted in uniform distribution of the vector DNA throughout the liver and widespread distribution in multiple tissues. Up to 5.0 × 10⁻⁶ 14 At a dose of gc / kg, tissue vector DNA levels were proportional to the administered dose. High levels of GLA transgenic mRNA were present in the liver. Comparable levels of vector DNA and vector-derived GLA mRNA were found in the liver at 3 and 6 months post-drug administration.

[0185] result In NHP, high levels of dose-related drug-derived vector DNA were found in the liver and adrenal glands. (5.0 × 10⁻⁶) 14 and 7.3 × 10 14 At the two highest doses (gc / kg), the vector genome copy number in the liver and adrenal glands of NHP ranged from 4 to 6 × 10⁻⁶. 6Vector DNA copies / ug DNA. Analysis of vector DNA from tissues other than the liver and adrenal glands was less than or 1 / 10 of that from the liver (target organ). An assessment of the biodistribution of vector DNA in reproductive tissues to evaluate potential reproductive toxicity showed dose-related levels in the ovaries, testes, epididymis, and seminal vesicles, and were approximately less than 1 / 400 of those in the liver. Blood and plasma vector DNA clearance was generally comparable at each tested dose, with vector DNA levels decreasing sharply in the first 2 weeks after administration followed by a slower decline. Depending on the use of a liver-selective promoter, vector DNA transcription resulted in dose-related, quantifiable GLA transgenic mRNA levels primarily in the liver, but mRNA expression was also observed in the adrenal glands, dorsal root ganglia, heart, and spleen. Similar vector DNA and GLA transgenic mRNA levels were found in the livers of NHPs treated with the drug product at 3 and 6 months post-administration, indicating persistence of vector and transgenic expression. Up to the highest titer of 689 measured in any pre-dose sample, no correlation was observed between pre-dose Nab and hepatic vector DNA and mRNA levels, indicating that pre-existing neutralizing antibodies do not affect transduction efficiency, as observed with similar AAV5 vectors for hemophilia B (where pre-dose neutralization titers in NHP are as high as 1,000) (Majowicz A et al., Molecular Therapy Methods & Clinical Development, 2019, Vol. 14, 27-36). In summary, the pharmacokinetic parameters included in the study indicate dose-dependent and persistent levels of vector DNA and GLA transgenic mRNA in the liver of Fabry disease mice and NHP. In NHP, the biodistribution of drug-derived vector DNA to extrahepatic tissues was less than or 1 / 10 of that to the liver, and even more so in reproductive organs (less than 1 / 400). The use of liver-specific promoters primarily restricts GLA transgenic mRNA expression to the liver.

[0186] Evaluate From arrival at the test site, monitor NHP mortality and any clinical signs or symptoms at least twice daily. Assess food consumption daily and measure animal weight weekly. Measure body temperature once before administration, and once at 4, 8, and 24 hours after administration, at week 4, and at necropsy at weeks 13 and 26. At weeks 4 and 26, perform electrocardiogram, blood pressure, and respiratory rate measurements and compare them with measurements taken before administration. Perform a full clinicopathological evaluation, including cardiac troponin levels and hematological and coagulation parameters, at relevant time points before administration and during the study. To closely monitor any effects on liver and cardiac function, perform additional measurements of the selected set (cardiac troponin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, and glutamate dehydrogenase) more frequently during the first two weeks after administration. To evaluate whether drug administration triggered adverse immune responses, a panel of cytokines was analyzed in blood samples collected before administration, 4 and 24 hours after administration, on days 4, 8, and 15 after administration, and at 4 weeks after administration. To assess the immunogenicity of the drug, anti-AAV5 IgM levels were measured in plasma samples collected before administration and on days 8 and 15 after administration. Anti-AAV5 IgG and neutralizing antibodies, as well as anti-GLA protein IgG antibodies, were measured in serum samples collected before administration and at 3 and 6 months after drug administration. At autopsy, organ weight measurements and histopathological analyses were performed on 48 relevant organs and tissues, including the liver, adrenal glands, kidneys, heart, brain, gonads, lungs, spleen, dorsal root ganglia, and injection sites.

[0187] General safety parameters During the study period, there were no unplanned deaths, and no clinical signs related to treatment with the drug product were observed. Food consumption, body temperature, weight, weight gain, electrocardiogram, blood pressure, and respiratory rate were not significantly affected (up to 7.3 × 10⁻⁶). 14 The therapeutic effect of gc / kg of drug products.

[0188] toxicology Clinical pathology and hematology Throughout the study, no effects related to the drug product on hematological or coagulation test results were observed. For administration of 4 × 10 13 Two males were administered gc / kg and 2 × 10 gc / kg of glucose. 14In one male at gc / kg, transient minimal to mild increases in aspartate transaminase and / or alanine transaminase activity and mild to significant increases in creatine kinase activity were observed on days 1 and / or 4; glutamate dehydrogenase (GLDH) activity did not increase significantly. Since the increases in enzyme activity were observed only in a small number of animals in the two lowest-dose groups, lasted only for a short duration, and were likely related to the surgical (treatment) of skeletal muscle perturbation, these were considered not adverse.

[0189] Immunology Blood cytokine analysis revealed no drug-product-related effects on inflammatory markers. Assessment of the humoral immunogenicity of the drug product showed that one female animal had a higher level of anti-AAV5 IgM prior to administration compared to other animals. Analysis of total anti-AAV5 IgG showed a signal-to-noise ratio ≥ 3 in samples from 14 out of 36 animals prior to administration, indicating the presence of pre-existing AAV5-specific IgG. Pre-administration serum samples from 17 out of 18 males and 14 out of 18 females showed AAV5 neutralizing activity, with titers ranging from 50 to 689. All samples from animals treated with the drug product, collected on days 8 and 15 (for IgM analysis) and at months 3 and 6 (for IgG and neutralizing antibody analysis), showed high levels of anti-AAV5 antibodies, as expected. In IgM analysis, the OD450nm level after administration was >10-fold higher than the pre-administration level, and the post-administration AAV5 neutralizing titer ranged from 33,828 to >729,000. A high signal-to-noise ratio was observed in the IgG analysis, indicating an increase in anti-AAV5 antibodies after administration. Analysis of anti-GLA antibodies in serum samples collected before administration showed that no animals were positive for anti-GLA antibodies prior to administration. Samples from two animals were reported positive 3 months after administration. Both animals were male, and one animal was treated with 4.0 × 10⁻⁶ mmol / L. 13 One animal was given gc / kg, and another animal was given 5.0 × 10 gc / kg. 14 Dosage was administered at gc / kg. In animals treated with the vector, no difference in anti-AAV5 or anti-GLA antibody levels was observed between pre- and post-drug administration.

[0190] Organ weight and histopathology No changes related to the drug product were observed in organ weight or in macroscopic or microscopic observations at 3 and 6 months post-administration.

[0191] In summary, when administered as a single intravenous dose, the concentration is as high as 7.3 × 10⁻⁶. 14 The drug product at gc / kg was well tolerated. For administrations up to 7.3 × 10 g / kg... 14No systemic or local toxicity related to the drug product was observed in animals at gc / kg; therefore, under study conditions, this dose is considered the No Observed Effect Level (NOAEL).

[0192] Integration site analysis in the livers of cynomolgus monkeys treated with drug products To investigate the integration of drug-derived vector DNA into the genomic DNA of liver tissue from NHP animals, analysis of vector DNA integration into the host genome was performed using liver tissue samples from GLP toxicity studies of the drug product in NHP animals. A combination of splice-extension primer tag selection ligation-mediated PCR (S-EPTS / LM-PCR) and high-throughput sequencing confirmed the integration pattern of the vector DNA in the NHP liver samples. All liver samples from animals treated with the drug product showed polyclonal integration site (IS) signatures, and no dominant IS (>30%) was observed. There were no signs of clonal amplification. This indicates that no safety issues have arisen.

[0193] Local tolerance Local tolerability at the injection site has been evaluated as part of a general toxicity study of GLP. No significant findings were observed upon microscopic examination of the injection site.

[0194] result The safety of the drug product was evaluated through a combination of GLP toxicity studies in NHP and subsequent assessments of vector DNA integration into host liver DNA. The observation periods for NHP were 3 and 6 months post-administration. In the GLP toxicity studies of NHP, the vector dose levels ranged from doses supporting low pharmacological effects (4.0 × 10⁻⁶). 13 From gc / kg) to 2.4 times the expected high human dose (7.3 × 10 g / kg) 14 The dosage was (gc / kg). Hepatotoxicity and / or thrombotic microangiopathy (TMA) have been reported in individuals receiving AAV-based gene therapy (CTCTAC_FDA 2021). In this GLP NHP study, no evidence of drug-related hepatotoxicity was observed. Transient, mild increases in liver enzymes were observed in three animals during the first few days after administration, two of which were males given a low dose (4 x 10 gc / kg). 13 gc / kg), and one male was given a medium dose (2 x 10 gc / kg). 14 (gc / kg), but not observed in animals receiving higher doses of the drug product.

[0195] The increase in anti-AAV5 IgM levels observed on day 8 post-drug administration was inconsistent with changes in cytokine levels that counteract the product-induced inflammatory response. Complement activation following AAV administration in NHP has been described in the literature (Hinderer C et al., Human Gene Therapy, 2018, Vol. 29, No. 3; Katz N et al., Human Gene Therapy Methods, 2018, 29(5):212-219; Hordeaux J et al., Molecular Therapy, 2018, 26(3):664-668), but was accompanied by severe hepatotoxicity or significant transaminase elevation, which was not observed in our study. The combination of liver function data and the absence of a cytokine response made product-induced complement activation highly unlikely. This is further supported by our clinical experience with AMT-061 for hemophilia B using an AAV5 capsid similar to that of the current product. No TMA-related adverse events were observed in patients in clinical studies using AMT-061.

[0196] In summary, toxicity studies conducted with the drug product did not reveal any safety issues related to vector administration or high expression of the GLA transgenic product. NOAEL was set at the highest dose tested in NHP (7.3 x 10⁻⁶). 14 This dose (gc / kg) is 2.4 times the expected high human dose of the drug product and 12 times the expected starting (low) dose in the first clinical trial. Integration studies of vector DNA into liver genomic DNA using the drug product indicated a low risk of tumorigenicity. In conclusion, the beneficial toxicity profile of the drug product was achieved even at very high doses.

Claims

1. A type of equal-tension compound, comprising: Buffer solution; Recombinant adeno-associated virus vector containing a transgene encoding a therapeutic protein; and Cyclodextrin or its derivatives.

2. The isotropic formulation of claim 1, wherein the formulation has a weight osmolality of 250 to 330 mOsm / kg.

3. The isotropic formulation as described in claim 1 or 2, wherein the formulation is substantially free of visible particles.

4. The isotonic formulation as described in any of the preceding claims, wherein the recombinant adeno-associated virus vector encoding a transgenic protein encoding an enzyme, preferably an enzyme associated with lysosomal storage impairment.

5. The isotonic formulation as described in any of the preceding claims, wherein the recombinant adeno-associated virus vector comprises AAV2 serotype, AAV5 serotype, or a combination thereof, preferably a hybrid AAV2 / 5 serotype.

6. The isotonic formulation as described in any of the preceding claims, wherein the concentration of the recombinant adeno-associated virus vector is up to 5E15 gc / ml.

7. The isotonic formulation as described in any of the preceding claims, wherein the cyclodextrin is an unsubstituted or substituted β-cyclodextrin.

8. The isotonic formulation as described in any of the preceding claims, wherein the concentration of the cyclodextrin is from about 0.05% w / v to about 4% w / v.

9. The isotensive formulation as claimed in any of the preceding claims, wherein the isotensive formulation has a pH value of 6.5 to 8.

10. The isotonic formulation as described in any of the preceding claims, further comprising a pharmaceutically acceptable salt at a concentration of at least 50 mM, wherein the salt is preferably NaCl, KCl, CaCl2, MgCl2, or a combination thereof.

11. The isotonic formulation as described in any of the preceding claims, wherein the buffer solution is a Tris buffer solution.

12. The equal-tension article as described in any of the preceding claims, wherein the equal-tension article comprises Approximately 15-25 mM Tris; Recombinant adeno-associated virus vector containing the AAV5 serotype; Approximately 110-140 mM NaCl; Approximately 1.5%-2.5% (w / v) hydroxypropyl-β-cyclodextrin; The formulation has a pH of approximately 7 to 8.

13. The isotonic formulation as described in any of the preceding claims, wherein the recombinant adeno-associated virus vector comprises a transgene, the transgene comprising or consisting of SEQ ID NO: 01 or a variant thereof, and / or wherein the transgene encodes an amino acid sequence comprising or consisting of SEQ ID NO: 02 or a variant thereof.

14. The isotonic formulation as described in any of the preceding claims, the isotonic formulation being used in a pharmaceutical preparation.

15. The isotonic formulation for use as claimed in claim 14, the isotonic formulation being used to treat a disorder related to a defective or deficient protein, preferably an enzyme, more preferably a lysosomal storage disorder, and even more preferably Fabry disease.

16. The isotonic formulation for use as described in claim 14 or claim 15, wherein the formulation is used to administer a viral vector transducible to the liver.

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