Formulations for viral drug products

Abstract

The invention relates to the field of gene therapy. In addition, the invention relates to the field of diseases caused by defective or absent proteins. In particular, the invention relates to gene therapy for enzyme replacement and more in particular to pharmaceutical formulations with improved stability for the treatment of diseases that can benefit from enzyme replacement, including lysosomal storage disorders.

Description

[0001] Formulations for viral drug products

[0002] Field of the invention

[0003] The invention relates to the field of gene therapy. In addition, the invention relates to the field of diseases caused by gene defects resulting in proteins that are partly or completely defective (non-functional) or absent or both. In particular, the invention relates to gene therapy for protein replacement and more in particular to pharmaceutical formulations of those gene therapies with improved stability for the treatment of diseases that can benefit from such gene therapy for protein replacement, especially enzyme replacement including lysosomal storage disorders.

[0004] Background

[0005] The elucidation of DNA as the carrier of genetic information, and therefore also as the source of inherited diseases, has led to envisaged therapies in which mutant, damaged genes could be replaced or silenced. Many genes or other nucleic acids have now been identified to play a role in (genetic) disease. If the mutant gene(s) could be replaced by a healthy one, or if the genes expressing aberrant (sometimes toxic) products could be silenced, the disease could be treated at the molecular level and potentially cured. Gene therapy provides a promising concept, in particular for diseases caused by mutations in a single gene, wherein such mutation results in reduced expression or reduced function or both (i.e. a partly or completely absence of a protein and / or a partly or completely non-functional protein). However, the delivery of the desired nucleic acid to cells is not an easy task. Numerous (viral) delivery systems have been investigated, all of them having advantages and drawbacks. One of the viral delivery vehicles that are used for gene therapy is recombinant Adeno-Associated Virus (rAAV).

[0006] Wildtype AAV has a single-stranded DNA genome of approximately 4.8 kilobases (kb). AAV belongs to the parvovirus family and is dependent for replication on co-infection with other viruses, in particular adenoviruses. The genome comprises Rep (Replication) and Cap (Capsid) genes. These coding sequences are flanked by inverted terminal repeats (ITRs) that assist genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), replicates the viral genome, and facilitates packaging, while Cap gene expression gives rise to the viral capsid proteins (VP, namely VP1 , VP2, and VP3), which form the outer capsid shell. For gene therapy, the viral DNA of the wildtype AAV is almost completely removed. Recombinant AAV (rAAV) for gene therapy is formed by a protein capsid containing a desired nucleic acid, the transgene, that is to be delivered to target cells. The desired nucleic acid is flanked by the ITRs of AAV.

[0007] Protein replacement therapy refers to medical treatment that aim to substitute or replenish specific protein deficiencies that result either from the protein being absent or non-functional due to mutations in affected patients. For example, Enzyme replacement therapy (ERT) replaces an enzyme that is deficient or absent in the body. This can be done by giving the patient an intravenous infusion of a composition comprising the enzyme. Such therapy is available for some lysosomal storage disorders: Gaucher disease, Fabry disease, MPS I, MPS II (Hunter syndrome), MPS VI, and Pompe disease. Enzyme replacement therapy does not correct the underlying genetic defect. Therefore, treatment requires lifelong intravenous infusions of the therapeutic enzyme. This procedure is expensive; in the United States, it may cost over $200,000 annually per patient. In addition, the biodistribution of the enzyme after these infusions is not uniform; the enzyme is less available to certain areas in the body, like the bones, lungs, brain. For this reason, many symptoms of diseases requiring ERT remain untreated, especially neurological symptoms. Additionally, the efficacy of ERT is often reduced due to an unwanted immune response against the enzyme, which prevents metabolic function. Furthermore, when using ERT, the enzyme levels typically do not remain at the same level but decrease after administration. This results in peaks and troughs in the enzyme activity. During the trough phases, any detrimental symptoms or damage caused by the disease may still take place. Use of an rAAV gene therapy vector can enable cells to produce the curative enzyme by themselves, allowing a more longterm solution with improved and stable biodistribution. Furthermore, costs of treatment are reduced because gene therapy is anticipated to be only given once in a lifetime, which is not only cost-effective but also increases patient quality of life.

[0008] Formulations intended for the storage of rAAVs, and later administration of rAAVs to a patient, present special challenges regarding stability, such as the need to avoid undesirable formation of aggregated or agglomerated particles in the formulation that may be detrimental to the therapeutic compound and / or a patient. In particular, such formulations comprising rAAV may be stored frozen and then thawed before use. Aggregates or agglomerates could problematically form during or after thawing the formulation comprising rAAV, and also during the time following thawing but before administration of the formulation comprising rAAV, as well as during administration of the formulation comprising rAAV. In some instances, where administration is over several hours, a sustained, aggregate-free formulation is required. It would therefore be highly desirable to provide an innovative formulation for an rAAV vector, which reduces or eliminates aggregate formation for extended in-use shelf life.

[0009] Summary of the invention

[0010] In one aspect, there is provided an isotonic formulation comprising: a buffer; a recombinant adeno-associated viral vector comprising a transgene encoding a therapeutic protein; and a cyclodextrin or a derivative thereof.

[0011] In a further aspect, there is provided the isotonic formulation as disclosed herein for use in a medicament. Detailed description of the invention

[0012] The present invention seeks to provide a new pharmaceutical formulation comprising rAAVs that has reduced aggregate or agglomerate formation during processing and is essentially free of visible particles for an extended time at room temperature after thawing of the formulated drug product. Such aggregates can include, but are not limited to, aggregates comprising rAAV particles, salt crystal aggregates and combinations thereof. The present invention also seeks to provide a method of treating diseases associated with defective enzymes, such as lysosomal storage disorders, by administering the formulation of the invention comprising rAAVs to a patient. Accordingly the invention provides an isotonic formulation comprising: a buffer; a recombinant adeno-associated viral vector comprising a transgene encoding a therapeutic protein; and a cyclodextrin or a derivative thereof.

[0013] It has been surprisingly found that the presence of a cyclodextrin or a derivative thereof in the isotonic formulation of the invention helps to improve the stability of the drug product. The term “drug product” as used herein refers to an rAAV drug product. A drug product comprised within the isotonic formulation as described herein may remain stable without substantial formation of aggregates or agglomerates, for instance for at least about 6 months, preferably at least about 12 months, more preferably at least about 24 months, for example at least 36 months, at a storage temperature of for example -65 ° C. A drug product comprised within the isotonic formulation as described herein may also remain stable at room temperature (15°C-25 °C) without substantial formation of aggregates or agglomerates for an extended time, preferably at least about 12 hours, more preferably at least about 24 hours, for instance after thawing from a frozen state. Thus, in certain embodiments, the drug product comprised within the isotonic formulation as described herein remains stable at room temperature (15°C-25°C) without substantial formation of aggregates or agglomerates for at least 12 hours after thawing from a frozen state. As presented in the example contained herein, other formulations comprising an rAAV drug product do form aggregates or agglomerates within 12 hours after thawing from a frozen state.

[0014] Preferably, the formulation of the invention is substantially isotonic to human blood. Tonicity is a measure for the effective osmotic pressure that a liquid formulation can exert, and depends primarily on the number of dissolved particles in solution. Osmotic pressure is an important factor affecting biological cells. Hypertonicity is the presence of a solution that causes cells to shrink. Hypotonicity is the presence of a solution that causes cells to swell. Isotonicity is the presence of a solution that produces no change in cell volume. When a biological cell is in a hypotonic environment, the cell interior accumulates water, water flows across the cell membrane into the cell, causing it to expand. For mammalian cells this can lead to cytolysis, and tonicity is therefore important when fragile cells are to be exposed to a composition. Tonicity agents can therefore be added to preparations such as injectable preparations to prevent osmotic shock at the site of injection upon administration, and thereby reduce local irritation or even damage to tissues or blood cells. Preferably, the isotonic formulation as described herein is substantially isotonic to human blood, which has a tonicity or osmotic concentration of about 290 mOsm / kg. In particular, the formulation of the invention may have an osmolality, also referred to as tonicity or osmotic concentration, of from 250 to 330 mOsm / kg, such as 260 to 310 mOsm / kg. In some preferred embodiments, the isotonic formulation as described herein has an osmolality of from 260 to 320 mOsm / kg, more preferably from 270 to 315 mOsm / kg, most preferably from 274 to 310 mOsm / kg. For example, the isotonic formulation as described herein can have an osmolality of 290 mOsm / kg.

[0015] Typical tonicity agents are excipients used for tonicity adjustment, and are known in the art. Tonicity agents can include dextrose, glycerin, mannitol, and metal salts. Metal salts are preferred, and preferred metal salts are pharmaceutically acceptable metal salts. Thus in preferred embodiments the isotonic formulation further comprises a pharmaceutically acceptable salt at a concentration of at least 50 mM, wherein the salt is preferably NaCI, KCI, CaCh, MgCh, or combinations thereof.

[0016] Pharmaceutically acceptable metal salts may comprise a periodic group 1 or group 2 metal salt, preferably a periodic group 1 or group 2 metal chloride salt, preferably selected from the group consisting of NaCI, KCI, CaCh, MgCh, and combinations thereof. NaCI is particularly preferred.

[0017] The isotonic formulation as described herein preferably comprises the pharmaceutically acceptable metal salt at a concentration greater than about 55, 60, or 65 mM, particularly 75 mM, which has been found beneficial for the stability of the drug product, as measured through absorbance and visual inspection per the Examples disclosed herein. Preferably, the concentration of pharmaceutically acceptable salt may be about 75 mM to about 200 mM, preferably about 80 mM to about 175 mM, more preferably about 85 mM to about 160 mM, more preferably about 90 to about 155 mM, more preferably about 95 to about 150 mM, more preferably about 100 to about 145 mM, more preferably about 105 to about 140 mM, more preferably about 115 to about 135 mM, more preferably about 120 to about 130 mM, such as most preferably about 125 mM. Thus, in some embodiments, the isotonic formulation of the invention comprises a pharmaceutically acceptable salt selected from the group consisting of NaCI, KCI, CaCh, MgCh, and combinations thereof, at a concentration of about 100 mM to about 150 mM, preferably of about 115 mM to about 135 mM, more preferably about 120 to about 130 mM, such as most preferably about 125 mM.

[0018] In some specific embodiments, the pharmaceutically acceptable salt comprises NaCI, present in the formulation 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 of about 115 mM to about 135 mM, most preferably of about 125 mM.

[0019] The isotonic formulation of the invention may have a pH value compatible with human blood. For example, the isotonic formulation may have a pH of about 6.5 or higher, preferably about 7 or higher. In some embodiments, the formulation may have a pH value of from 6.5 to 8.5, preferably from 7 to 8, more preferably from 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 from 6.5 to 8.5, preferably of 7.5 to 8, most preferably of 7.5. In a preferred embodiment, the isotonic formulation has a pH value of 7.2 to 7.8, most preferably of 7.3 to 7.5. In one embodiment, the isotonic formulation as described herein comprises a buffer. Buffering agents are known in the art, and help maintain the pH of the composition stable within a given range. A buffering agent is often a buffer salt. Thus, the isotonic formulation of the invention may comprise a buffer selected from acetate, citrate, phosphate, Tris (tris(hydroxymethyl)aminomethane or tromethamine), and derivatives (e.g. Tris hydrochloride) and combinations thereof, including tromethamine in combination with Tris hydrochloride. In a preferred embodiment, Tris is tromethamine. The isotonic buffer may be a Tris buffer at a pH of about 7.5 to about 8.0, a citrate buffer at a pH of about 5.5 to about 6.5, or a phosphate buffer at a pH of about 7.0 to about 7.5. Preferably, the buffer is a Tris buffer. In some embodiments, the isotonic formulation comprises a Tris buffer at a pH of about 7.5 to about 8.0. In some specific embodiments, the buffer is a Tris buffer at a pH of 7.5. The buffering agent is preferably present at 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, most preferably about 15 to about 25 mM. The buffering agent can also be present at about 16 to about 24 mM, more preferably about 17 to about 23 mM, still more preferably about 18 to about 22 mM, most preferably at about 19 to 21 mM, such as at 20 mM. In some preferred embodiments, the buffer is a 20 mM T ris buffer at a pH of 7.5.

[0020] The isotonic formulation of the invention comprises a cyclodextrin or a derivative thereof. The inventors have found that, surprisingly, cyclodextrins can contribute to an improvement in the preservation of the stability of recombinant adeno-associated viruses, and / or are particularly advantageous in providing a stable drug product that substantially reduces the formation of aggregates or agglomerates. Cyclodextrins are a family of cyclic oligosaccharides, consisting of a macrocyclic ring of glucose subunits joined by a-1 ,4 glycosidic bonds. Cyclodextrins can be a (alpha)-cyclodextrin having 6 glucose subunits, p (beta)-cyclodextrin having 7 glucose subunits, or y (gamma)-cyclodextrin having 8 glucose subunits. Preferred cyclodextrins for the invention are p-cyclodextrins. Combinations of cyclodextrins can also be used.

[0021] Cyclodextrins can be substituted or unsubstituted. Preferably, the cyclodextrin is an unsubstituted or substituted p-cyclodextrin. Substituted cyclodextrins are generally modified at their hydroxyl moieties, preferably at all of them, preferably having the same modification at all of them. Examples of substitutions are methylation, acetylation, and hydroxypropylation such as 2- hydroxypropylation (having for instance derivatized the hydroxyl moieties using propylene oxide). A preferred cyclodextrin is a substituted cyclodextrin, particularly a substituted p-cyclodextrin. A preferred substituted cyclodextrin is hydroxypropyl-cyclodextrin, and 2-hydroxypropyl-p-cyclodextrin is particularly preferred (CAS number 128446-35-5).

[0022] In some embodiments, the cyclodextrin is present in the isotonic formulation in an amount less than about 4% w / v (weight per volume percent), 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, most preferably about 1 .9% w / v to about 2.2% w / v such as about 2% w / v. In some specific embodiments of the isotonic formulation, the cyclodextrin is a substituted p-cyclodextrin at a concentration of from 1 .5% w / v to 2.5% w / v.

[0023] In some embodiments, the isotonic 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 and combinations thereof. For example, the sugar or sugar alcohol is mannitol. It has been found that, sugar or sugar alcohols may act as cryoprotectants and may be particularly advantageous in providing a stable pharmaceutical formulation. In preferred embodiments, the formulation does not further comprise a sugar or sugar alcohol.

[0024] 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, preferably about 0.1 % w / v to about 1 % w / v. Thus, in some embodiments, the isotonic formulation of the invention comprises a sugar or sugar alcohol at a concentration of from about 0.05% w / v to about 2% w / v. In some specific embodiments, the isotonic formulation comprises a sugar or sugar alcohol selected from the group consisting of trehalose, sucrose, maltose, mannitol, and derivatives and combinations thereof, at a concentration of from about 0.05% w / v to about 2% w / v.

[0025] Where the sugar or sugar alcohol is mannitol, it may be present in the isotonic formulation in an amount 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.

[0026] In some embodiments, the isotonic formulation comprises the combination of 2-hydroxypropyl- p-cyclodextrin and mannitol, preferably in the amount of about 0.1 w / v% 2-hydroxypropyl-p-cyclodextrin and about 0.1 w / v% mannitol.

[0027] In some embodiments, the isotonic formulation further comprises an amino acid, which were found to be beneficial for the stability of the drug product. Amino acids are natural osmolytes that can stabilize proteins when in solution. For example, the amino acid may be selected from cysteine, arginine, histidine, glycine, and derivatives and combinations thereof. Preferably, the amino acid may comprise histidine and / or glycine. Where 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 the isotonic formulation does not further comprise an amino acid.

[0028] When present, in some embodiments, the isotonic formulation comprises an amino acid selected from the group consisting of cysteine, arginine, histidine, glycine, and derivatives and combinations thereof, at a concentration of from about 2 mM to about 3 mM. In some specific embodiments, the isotonic formulation comprises histidine, or a derivative thereof, at a concentration of about 2.5 mM. Preferably, the isotonic formulation does not comprise a surfactant. Surfactants and their characteristics are well known; surfactants generally comprise at least one polar head group and at least one apolar or hydrophobic tail and are preferably charge neutral, i.e., they do not have a net charge at the conditions for their use. For example, the isotonic formulation does not comprise Polysorbate 20, Polysorbate 80, or Poloxamer 188. The inventors have found that the usual effect of surfactants can be achieved by using the formulation as disclosed, and that therefore there is no need to expose subjects to surfactants.

[0029] In one embodiment, the transgene comprised in the recombinant adeno-associated viral vector comprised in the isotonic formulation described herein is a transgene that encodes a therapeutic protein. Examples of suitable therapeutic proteins include proteins for use in the therapy of a x-linked recessive disorder, preferably haemophilia A, haemophilia B or glucose-6-phosphate dehydrogenase deficiency. X-linked recessive inheritance is a mode of inheritance in which a mutation in a gene on the X chromosome causes the phenotype to be always expressed in males (who are necessarily homozygous for the gene mutation because they have one X and one Y chromosome) and in females who are homozygous for the gene mutation. Females with one copy of the mutated gene are carriers. In some embodiments, the transgene that encodes a therapeutic protein, encodes an enzyme. Examples of suitable enzymes are aspartylglucosaminidase, alfa-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine transporter, acid ceramidase, acid alfa-L-fucosidase, protective protein / cathepsin A, acid beta-glucosidase or glucocerebrosidase, acid beta-galactosidase, iduronate-2-sulfatase, alfa-L-iduronidase, galactocerebrosidase, acid alfa-mannosidase, acid beta-mannosidase, arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid beta-galactosidase, N-acetylglucosamine-1- phosphotransferase, acid sphingomyelinase, NPC intracellular cholesterol transporter 1 , acid alfa- glucosidase, beta-hexosaminidase B, heparan N-sulfatase, alfa-N-acetylglucosaminidase, acetyl- CoA:alfa-glucosaminide N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase, alfa-N- acetylgalactosaminidase, alfa-neuramidase, beta-glucuronidase, beta-hexosaminidase A, and acid lipase. A preferred enzyme is a galactosidase, particularly alfa-galactosidase such as alfa- galactosidase A. In preferred embodiments, the transgene does not encode a microRNA. Additionally, in preferred embodiments, the therapeutic protein is not a biomarker or encoded by a suicide gene.

[0030] In a particularly preferred embodiment, the transgene comprised in the recombinant adeno- associated viral vector comprised in the isotonic formulation described herein encodes alfa- galactosidase A.

[0031] In a preferred embodiment, the transgene comprised in the recombinant adeno-associated viral vector comprised in isotonic formulation described herein comprises a nucleic acid sequence comprising or consisting of SEQ ID NO: 01 , or a variant thereof.

[0032] In a further embodiment, the transgene comprised in the recombinant adeno-associated viral vector comprised in the isotonic formulation described herein encodes an amino acid sequence comprising or consisting of SEQ ID NO: 02, or a variant thereof. In some embodiments, the recombinant adeno-associated viral vector with a transgene encoding an enzyme may encode an enzyme associated with a lysosomal storage disorder. A group of metabolic disorders known as lysosomal storage disorders or diseases (LSD) includes over forty genetic disorders, many of which involve genetic defects in various lysosomal enzymes such as hydrolases. Representative lysosomal storage disorders and the associated defective enzymes are listed in the table below. In preferred embodiments, the formulation is for treating the disorder in the left column, and the transgene encoding an enzyme encodes the associated enzyme in the right column. In preferred embodiments, the isotonic formulation described herein is for treating the disorder in the left column, and the transgene encoding an enzyme encodes the associated enzyme in the right column.

[0033] In one embodiment, the isotonic formulation described herein comprising a recombinant adeno- associated viral vector is for use in the treatment of Fabry’s disease.

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

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

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

[0037] In some embodiments, the recombinant adeno-associated viral vector comprises a capsid protein of an AAV2 serotype, an AAV5 serotype, or a combination thereof. Said serotypes have been found particularly preferred for the treatment of diseases or conditions associated with defective enzymes, such as for instance lysosomal storage disorders. In some embodiments, the recombinant adeno-associated viral vector comprises an AAV5 serotype. In one example, the recombinant adeno- associated viral vector comprises an AAV5 variant. In some embodiments, the recombinant adeno- associated viral vector comprises a combination of capsid proteins from different serotypes, forming a hybrid AAV serotype. By way of example, the hybrid AAV serotype may be a hybrid AAV2 / AAV5 serotype. Preferably, only a single serotype is comprised in the viral vector. Most preferably, the viral vector is of the AAV5 serotype and of the AAV2 serotype, which can also be referred to as an AAV2 / 5 serotype. Herein the first 136 residues of the AAV5 VP1 protein are replaced with the first 137 residues of the AAV2 VP1 protein.

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

[0039] Some recombinant adeno-associated viral (rAAV) vectors, such as of the AAV5 serotype (rAAV5), have been shown to direct stable gene transfer and expression in hepatocytes i.e. have increased liver transduction, when compared to other rAAVs. Therefore, in some embodiments, the isotonic formulation as described herein is for administration of a viral vector as described herein that is preferably transducible to the liver. The isotonic formulation as disclosed herein, has particular application as a formulation suitable for use in the treatment of diseases that can be treated by rAAV- based therapy having increased transduction to the liver, such as the above-mentioned LSDs or coagulation disorders such as haemophilia A or B. It may also be envisioned that, in another embodiment, the isotonic formulation as disclosed herein is suitable for the storage of drug products for use in the treatment of liver associated diseases. Liver diseases and liver associated diseases include, but are not limited to: diseases caused by viruses, such as hepatitis A, hepatitis B, and hepatitis C; diseases caused by drugs, poisons, such as alcohol, which lead to fatty liver disease and cirrhosis; autoimmune hepatitis; liver cancer; inherited diseases, such as hemochromatosis, Alpha-1 antitrypsin deficiency (AATD) and Wilson disease; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis (NASH); and biliary atresia. In such cases, the transgene encodes a relevant therapeutic protein. Therefore, in some embodiments, the recombinant adeno-associated viral vector with a transgene encoding an enzyme may encode an enzyme associated with a storage disease of the liver or metabolic liver disease.

[0040] In some embodiments, the concentration of the recombinant adeno-associated viral vector in the isotonic formulation as described herein is up to 5E15 gc / ml (genome copies per milliliter), preferably 1 E15 gc / ml, more preferably 5E14 gc / ml (genome copies per milliliter), still more preferably up to 1 E14 gc / ml. Preferably the concentration of the recombinant adeno-associated viral vector is 1 E10 to 5E14 gc / ml, preferably 5E10 to 2E14 gc / ml, more preferably 1 E11 to 1 E14 gc / ml, more preferably 5E11 to 8E13 gc / ml, more preferably 1 E12 to 6E13 gc / ml, more preferably 5E12 to 5E13 gc / ml. In other embodiments the concentration of the recombinant adeno-associated viral vector is from 1 E13 to 1 E15 gc / ml, preferably from 5E13 to 5E14 gc / ml, more preferably from 6E13 to 2E14 gc / ml. In one example, the isotonic formulation comprises an rAAV5 with a transgene encoding a therapeutic protein, preferably an enzyme, for example a-galactosidase A, and the concentration of rAAV5 is 5E13 gc / ml or 6E13 gc / ml, preferably 5E13. In another example, the isotonic formulation comprises an rAAV5 with a transgene encoding a therapeutic protein, preferably an enzyme, for example a-galactosidase A, and the concentration of rAAV5 is 1 E14 gc / ml or 2E14 gc / ml, preferably 2E14 gc / ml. In a further embodiment, the isotonic formulation as described herein comprises an rAAV5 vector comprising 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 from 5E13 to 6.5E13. In a preferred embodiment, the therapeutic protein is an enzyme. In a further preferred embodiment, the enzyme is a-galactosidase A. The current invention has particular application in instances wherein high doses of rAAV are required, and longer administration time may be required, for example, where administration is over 1 or more hours, wherein a sustained, aggregate-free formulation is required for the duration of the administration.

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

[0042] In a preferred embodiment, there is provided the use of the isotonic formulation as described herein in the manufacture of a medicament for the treatment of Fabry disease, wherein the formulation comprises a recombinant adeno-associated viral vector comprising a transgene comprising 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, preferably wherein the concentration of the recombinant adeno-associated viral 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 viral vector with a transgene encoding a therapeutic protein refers to a vector comprising one or more polynucleotide sequences of interest, genes of interest or "transgenes" that are flanked by AAV inverted terminal repeat sequences (ITRs). An rAAV preferably comprises a nucleic acid construct comprising a gene of interest encoding a therapeutic protein, the transgene, that is flanked by at least one ITR. In a further preferred embodiment, the rAAV vector comprises an expression cassette for the transgene, wherein i) the transgene is operably linked to a promoter, preferably a liver-specific promoter such as the LP1 promoter, more preferably a promoter selected from the liver-specific promoters described in W02020 / 104424, which is incorporated by reference herein, and / or ii) the transgene is operably 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 a further 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 in between the two AAV ITR nucleotide sequences. In another embodiment, the expression cassette is flanked by one ITR engineered with two D regions, wherein the expression cassette is located on either side of the engineered ITR. In a preferred embodiment the 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, the isotonic formulation as disclosed herein, has particular application as a formulation suitable for the storage of drug products for use in the treatment of diseases treated using liver-tropic AAV based therapy. In some embodiments, the isotonic formulation as disclosed herein, has particular application as a formulation suitable for the storage of drug products for use in the treatment of diseases treated via the liver. Therefore, the isotonic formulation as described herein is for use as a medicament. The medicament is preferably for treating a disorder associated with a defective or absent protein, preferably an enzyme, wherein more preferably the disorder is a lysosomal storage disorder. This is because the isotonic formulation comprises a recombinant adeno-associated viral vector with a transgene encoding a protein, which can be conveniently used for replacing or complementing defective or impaired enzymes. Examples of suitable encoded enzymes and combinations with conditions to be treated have been provided elsewhere herein. Suitable subjects are subjects in need of treatment. The isotonic formulation as disclosed herein comprising a recombinant adeno-associated viral vector comprising a transgene encoding a therapeutic protein, i.e. the product or products, may be conveniently used in methods of treatment. The products per se can also be used in the manufacture of a medicament, preferably wherein the medicament is for treating the condition as indicated. It was found that the isotonic formulations according to the invention are suitable for treating lysosomal storage disorders, and can therefore be beneficial for administering treatment to a subject, for example a treatment intended for the liver of the subject administered via intravenous injection.. In all embodiments the isotonic formulation is for intravenous administration. In preferred embodiments, the isotonic formulation is not for administration to the central nervous system. In preferred embodiments, the isotonic formulation is not for administration to the brain.

[0045] The invention provides a method for preparing a drug product for administration to the liver, the method comprising the steps of: i) providing an isotonic formulation according to the invention; and ii) aliquoting the isotonic formulation into a suitable dosage form.

[0046] The prepared drug product is suitable for administration to the liver. Accordingly, the 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 regime from 1 E12 to 1 E15 genome copies per kilogram (gc / kg), for example from 6E12 to 6E14 genome copies per kilogram. For example, when the isotonic formulation is administered intravenously, the injection volume may be 100 - 800 mL at a concentration (of the recombinant adeno-associated viral vector) of 2E12 or 2E13 gc / ml.

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

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

[0050] The range of 1 E13 to 1 E15 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. It was found that the isotonic formulations according to the invention have exceptional stability, and that aggregates or precipitates do not readily form. In preferred embodiments, the formulation is essentially free of visible particles. In other words, the isotonic formulation is preferably substantially free of particles visible to the naked human eye, as determined and characterized according to U.S. Pharmacopeial Convention (USP) Chapter 790 “VISIBLE PARTICULATES IN INJECTIONS” guidance of 2014 regarding parenteral medical products being “essentially free” of visible particulate matter (USP 790). Visible particles may be a sign of aggregation, agglomeration, and / or degradation of rAAV particles, such that a lack of visible particles may be advantageous to indicate a particularly stable formulation free from substantial aggregation or agglomeration. Preferably, the isotonic formulation also comprises limited subvisible particles, as determined and characterized according to U.S. Pharmacopeial Convention (USP) Chapter 787 “ SUBVISIBLE PARTICULATE MATTER IN THERAPEUTIC PROTEIN INJECTIONS” (USP 787), which is incorporated herein by reference. For example, the number of particles having a diameter of 25 pm is no more than 600 per vial, and the number of particles having a diameter of / a 10 pm is no more than 6000 per vial, in the formulation. Optionally, the isotonic formulation has a Dv90 of less than 50 pm, preferably less than 25 pm, more preferably less than 10 pm. The Dv90 values represent the percent (90% in this case) of the formulation that has a size no larger than the specified value. The term Dv90 as used herein defines the point in the size distribution at which 90% of the total volume of the formulation has a certain size or is smaller than that size.

[0051] The following are some preferred embodiments of the invention:

[0052] • The isotonic formulation having an osmolality of from 250 to 330 mOsm / kg and a pH value of from 6.5 to 8.

[0053] • The isotonic formulation wherein it is substantially isotonic to human blood. For example, the formulation has an osmolality of 260 to 310 mOsm / kg and a pH value of 7.1 to 7.7.

[0054] • The isotonic formulation comprising about 15-25 mM buffer; a recombinant adeno-associated viral vector with a transgene encoding a therapeutic protein; about 110-140 mM pharmaceutically acceptable salt; about 1 .5-2.5% (w / v) cyclodextrin or a derivative thereof; wherein the formulation has a pH of about 7 to 8.

[0055] • The isotonic formulation comprising about 15-25 mM Tris; a recombinant adeno-associated viral vector comprising an AAV5 serotype; about 110-140 mM NaCI; about 1.5-2.5% (w / v) hydroxypropyl-beta-cyclodextrin; wherein the formulation has a pH of about 7 to 8. • The isotonic formulation comprising about 18-22 mM buffer; a recombinant adeno-associated viral vector with a transgene encoding a therapeutic protein; about 120-130 mM pharmaceutically acceptable salt; about 1 .8-2.2% (w / v) cyclodextrin or a derivative thereof; wherein the formulation has a pH of about 7.2 to 8.

[0056] • The isotonic formulation comprising about 20 mM buffer; a recombinant adeno-associated viral vector with a transgene encoding a therapeutic protein; about 125 mM pharmaceutically acceptable salt; about 2% (w / v) cyclodextrin or a derivative thereof; wherein the formulation has a pH of about 7 to 8 such as 7.5 to 8 such as 7.5.

[0057] • The isotonic formulation comprising about 18-22 mM Tris; a recombinant adeno-associated viral vector comprising an AAV5 serotype; about 120-130 mM NaCI; about 1.8-2.2% (w / v) hydroxypropyl-beta-cyclodextrin; wherein the formulation has a pH of about 7.2 to 8.

[0058] • The isotonic formulation comprising about 20 mM Tris; a recombinant adeno-associated viral vector comprising an AAV5 serotype; about 125 mM NaCI; about 2% (w / v) hydroxypropyl-beta-cyclodextrin; wherein the formulation has a pH of about 7 to 8 such as 7.5 to 8 such as 7.5.

[0059] Methods for manufacturing a recombinant adeno-associated viral (rAAV) vector suitable for incorporation into the formulation according to the invention are known in the art. In general, suitable methods for producing an rAAV vector as described herein in mammalian or insect host cells, and means therefore (such as expression constructs for expression of AAV rep proteins), are described, for mammalian cells in: Clark et al. (Hum. Gene Ther. 1995, 6, 1329-134), Gao et al. (Hum. Gene Ther. 1998, 9, 2353-2362), Inoue and Russell (J. Virol. 1998, 72, 7024-7031), Grimm et al. (Hum. Gene Ther. 1998, 9, 2745-2760), Xiao et al. (J. Virol. 1998, 72, 2224-2232) and Judd et al. (Mol Ther Nucleic Acids. 2012; 1 : e54), and for insect cells in: Urabe et al. (Hum. Gene Ther. 2002, 13:1935-1943), WG2007 / 046703, WG2007 / 148971 , WG2009 / 014445, WG2009 / 104964, WO2011 / 122950, WO2013 / 036118, WO2015 / 137802, WO2019 / 016349, WO2021 / 198508, WG2021 / 198510 and WO2022 / 253955, all of which are incorporated herein in their entirety. AAV Rep and ITR sequences are particularly conserved among most serotypes. The Rep78 proteins of various AAV serotypes are e.g. more than 89% identical and the total nucleotide sequence identity at the genome level between AAV2, AAV3A, AAV3B, and AAV6 is around 82% (Bantel-Schaal et al., J. Virol., 1999, 73(2):939-947). Moreover, the Rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (i.e., functionally substitute) corresponding sequences from other serotypes in production of AAV particles in mammalian cells. US2003148506 reports that AAV Rep and ITR sequences also efficiently cross-complement other AAV Rep and ITR sequences in insect cells. Modified "AAV" sequences also can be used in this context, e.g. forthe production of rAAV vectors in insect cells. Such modified sequences e.g. include 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 more nucleotide and / or amino acid sequence identity to an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , AAV12 or AAV13 ITR or Rep can be instead of place of wild-type AAV ITR or Rep sequences.

[0060] In one embodiment, a mammalian cell for producing an rAAV vector as described herein is selected from cell lines among any mammalian species, including, but not limited to: A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1 , COS 7, BSC 1 , BSC 40, BMT 10, VERO, WI38, HeLa, a HEK 293 cell, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster. The selection of the mammalian species providing the cells is not a limitation of this disclosure; nor is the type of mammalian cell, i.e., fibroblast, hepatocyte, tumour cell. Mammalian cell lines for producing rAAV vectors in particular include a broad range of HEK293 cell lines, of which the HEK293T cell line is preferred.

[0061] Insect cell lines for producing an rAAV vector as described herein can be any cell line that is suitable for the production of heterologous proteins. Preferably the insect cell allows for replication of baculoviral vectors and can be maintained in culture, more preferably in suspended culture. In a preferred embodiment, the insect cell allows for replication of recombinant parvoviral vectors, including rAAV vectors. For example, the cell line used can be from Spodoptera frugiperda, Drosophila, or mosquito, including Aedes albopictus-demed cell lines. In a preferred embodiment, the insect cell or cell line is derived from insect species which are susceptible to baculovirus infection, including but not limited to: S2 (CRL-1963, ATCC), Se301 , SelZD2109, SeUCRI , Sf9, Sf900+, Sf21 , BTI-TN-5B1-4, MG- 1 , Tn368, HzAml , Ha2302, Hz2E5, High Five (Invitrogen, CA, USA) and expresSF+® (US 6,103,526; Protein Sciences Corp., CT, USA).

[0062] The rAAV vectors that are released into the supernatant of the mammalian or insect cell culture can be recovered and / or purified using suitable techniques which are known to those of skill in the art. In one embodiment, monolith columns (e.g., in ion exchange, affinity or IMAC mode), chromatography (e.g., capture chromatography, fixed method chromatography, and expanded bed chromatography), centrifugation, filtration and / or precipitation, are used for purification and concentration. These methods may be used alone or in combination. In one embodiment, capture chromatography methods, including column-based or membrane-based systems, are utilized in combination with filtration and precipitation. Suitable precipitation methods, including, but not limited to, chromatography methods utilizing polyethylene glycol (PEG) 8000 and NH3SO4, can be readily selected by one of skill in the art. Thereafter, the precipitate can be treated with enzymes, including but not limited to: benzonase, and purified using suitable techniques. In addition, recovery preferably comprises the step of affinitypurification of the rAAV vector using an anti-AAV antibody, preferably an immobilised antibody. The anti-AAV antibody preferably is a monoclonal antibody. A particularly suitable antibody is a single chain camelid antibody or a fragment thereof as e.g. obtainable from camels or llamas (see e.g. Muyldermans, Biotechnol. 2001 , 74: 277-302). The antibody for affinity-purification of rAAV vectors preferably is an antibody that specifically binds an epitope on an rAAV capsid protein, whereby preferably the epitope is an epitope that is present on capsid protein of more than one AAV serotype. E.g. the antibody may be raised or selected on the basis of specific binding to AAV6 capsid but at the same time also it may also specifically bind to rAAV5 capsids.

[0063] In a preferred embodiment, the recombinant adeno-associated viral (rAAV) vector as described herein is obtained using a method comprising the steps of: I) culturing a host cell comprising: i) an expression vector for expression of a rAAV vector, and; ii) an expression vector encoding an expression cassette flanked by at least one AAV inverted terminal repeat (ITR), comprising a nucleic acid molecule encoding at least one gene product; said host cell preferably further comprising a nucleotide sequence encoding a parvoviral replication (Rep) protein under conditions such that the rAAV vector is produced; and, II) recovery of the rAAV vector, preferably wherein recovery of the rAAV vector comprises at least one of: 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 having a nominal pore size of 30-70 nm.

[0064] General definitions

[0065] In this document and in its claims, the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a combination or a composition as defined herein may comprise additional components) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0066] Whenever a parameter of a substance is discussed in the context of this invention, it is assumed that unless otherwise specified, the parameter is determined, measured, or manifested under physiological conditions. Physiological conditions are known to a person skilled in the art, and comprise aqueous solvent systems, atmospheric pressure, pH-values between 6 and 8, a temperature ranging from room temperature to about 37° C (from about 20° C to about 40° C), and a suitable concentration of buffer salts or other components. The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10% of the value, optionally more or less 5%. In the context of this invention, a decrease or increase of a parameter to be assessed preferably means a change of at least 5% of the value corresponding to that parameter. More preferably, a decrease or increase of the value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it can be the case that there is no longer a detectable value associated with the parameter, “substantially” - the term “substantially” as used herein is a broad term, and is to be given its ordinary and customary meaning to a person of ordinary skill in the art (and is not to be limited to a special or customized meaning), and refers without limitation to being largely but not necessarily wholly that which is specified.

[0067] Each embodiment as identified herein may be combined together unless otherwise indicated. The invention has been described above with reference to a number of embodiments. A skilled person could envision trivial variations for some elements of the embodiments. These are included in the scope of protection as defined in the appended claims. All patent and literature references cited are hereby incorporated by reference in their entirety

[0068] A “nucleic acid construct” is defined as a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids which are combined or juxtaposed in a manner which would not otherwise exist in nature. A nucleic acid molecule is represented by a nucleotide sequence. Optionally, a nucleotide sequence present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production or expression of said peptide or polypeptide in a cell or in a subject.

[0069] A “variant” of a nucleic acid sequence as referred to herein has 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% up to 99,999% nucleic acid sequence identity to a recited nucleic acid sequence.

[0070] A “variant” of an amino acid sequence as referred to herein has 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% up to 99,999% amino acid sequence identity to a recited amino acid sequence.

[0071] The term “homologous” when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species. The term “heterologous” may be used to indicate that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of a different species.

[0072] "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 the expression control sequence controls and regulates the transcription and / or the translation of the nucleotide sequence. Thus, an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a protein-encoding gene, splicing signals for introns, and stop codons. The term "expression control sequence" is intended to include, at a minimum, a sequence whose presence is designed to influence expression, and can also include additional advantageous components. For example, leader sequences and fusion partner sequences are expression control sequences. The term can also include the design of the nucleic acid sequence such that undesirable potential initiation codons in and out of frame are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It includes sequences or polyadenylation sequences (pA) which direct the addition of a polyA tail, i.e., a string of adenine residues at the 3'-end of a mRNA, which may be referred to as polyA sequences. It also can be designed to enhance mRNA stability. Expression control sequences which affect the transcription and translation stability, e.g., promoters, as well as sequences which effect the translation, e.g., Kozak sequences, suitable for use in insect cells are well known to those skilled in the art. Expression control sequences can be of such nature as to modulate the nucleotide sequence to which it is operably linked such that lower expression levels or higher expression levels are achieved.

[0073] As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter, including e.g. attenuators or enhancers, but also 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 only active in specific types of tissues or cells.

[0074] A “3’ UTR” or “3’ non-translated sequence” (also often referred to as 3’ untranslated region, or 3’end) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal (such as e.g. AAUAAA or variants thereof). After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the cytoplasm (where translation takes place).

[0075] A "vector" is a nucleic acid molecule (typically DNA or RNA) that serves to transfer a passenger nucleic acid sequence (i.e., DNA or RNA) into a host cell. Three common types of vectors include plasmids, phages and viruses. Preferably, the vector is a virus. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and / or in vitro transcription, it may be necessary to remove, add or alter 5' and / or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription ortranslation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.

[0076] A "viral vector" refers to a vector comprising some or all of the following: viral genes encoding a gene product, control sequences and viral packaging sequences. A "parvoviral vector" is defined as a recombinantly produced parvovirus or parvoviral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of parvoviral vectors include e.g., adeno-associated virus vectors. Herein, a parvoviral vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. A “recombinant adeno-associated viral vector” as used herein is defined as a recombinantly produced adeno-associated viral vector or recombinantly produced adeno-associated viral particle that comprises a polynucleotide or expression cassette to be delivered into a host cell, either in vivo, ex vivo or in vitro.

[0077] As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to 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 only active in specific types of tissues or cells.

[0078] The promoter may be any appropriate promoter sequence, which shows transcriptional activity in the cell, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extra-cellular or intracellular polypeptides either homologous (native) or heterologous (foreign) to the cell.

[0079] The term “flanked” with respect to a sequence that is flanked by another elements) herein indicates the presence of one or more of the flanking elements upstream and / or downstream, i.e., 5’ and / or 3’, relative to the sequence. The term “flanked” is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and a flanking element. A sequence that is “flanked” by two other elements (e.g. ITRs), indicates that one element is located 5’ to the sequence and the other is located 3’ to the sequence; however, there may be intervening sequences therebetween. In a preferred embodiment a nucleotide sequence of (i) is flanked on either side by parvoviral inverted terminal repeat nucleotide sequences.

[0080] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, referto a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or isochemically modified, non-natural, or derivatized nucleotide bases. "Oligonucleotide" generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- 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 known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art.

[0081] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes:

[0082] (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it;

[0083] (b) inhibiting the disease, i.e., arresting its development; and

[0084] (c) relieving the disease, i.e., causing regression of the disease.

[0085] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman Wunsch) which aligns the sequences optimally overthe entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) 1 8 (proteins) and gap extension penalty = 3 (nucleotides) 12 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred. Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc.

[0086] Where herein reference is made to a dose of e.g. 1 E14 gc / kg, this can also be referred to as a dose of 1x1014gc / kg, or 1 ,0x1014gc / kg.

[0087] For the avoidance of doubt, the terms “formulation as described herein”, “formulation of the invention”, “isotonic formulation”, “isotonic formulation of the invention”, “isotonic formulation as disclosed herein” and “isotonic formulation as described herein” may be used interchangeably.

[0088] Description of the Figures

[0089] Fig. 1 - 280 nm absorbance and 335 nm fluorescence measurements as a function of the accelerated stress conditions and duration for the different salt concentrations. The different pHs were pooled.

[0090] Fig. 2 - 280 nm absorbance and 335 nm fluorescence measurements as a function of the accelerated stress conditions and duration. The different pHs are shown as a gradient from dark (pH 8.0) to light (pH 5.0). The different salt concentrations were pooled.

[0091] Fig. 3A - genome copies for various formulations after an accelerated stability study at 25°C.

[0092] Fig. 3B - as 3A but showing the ratio of genome copies over infective particles.

[0093] Fig. 3C - as 3A but showing sub-visible particles >10pm.

[0094] Fig. 3D - as 3A but showing visible particles.

[0095] Fig. 4A - the ratio of genome copies over infective particles for various formulations after degradation studies performed at 40°C for 4 weeks.

[0096] Fig. 4B - as 4A but showing sub-visible particles >10pm.

[0097] Fig. 4C - as 4A but showing polydispersity.

[0098] Fig. 5A - genome copies for various formulations after long-term stability studies at -80°C.

[0099] Fig. 5B - as 5A but showing sub-visible particles >10pm.

[0100] Fig. 5C - as 5A but showing the ratio of total particles to genome copies.

[0101] Fig. 6A - genome copies for various formulations after long-term stability studies at 2-8°C.

[0102] Fig. 6B - as 6A but showing visible particles.

[0103] Fig. 6C - as 6A but showing sub-visible particles >10pm.

[0104] Fig. 6D - as 6A but showing sub-visible particles >0.3pm.

[0105] Fig. 7 Liver transduction and GLA activity - Liver samples were collected from non-human primates (NHPs) at 3 months and 6 months after dosing and analysed for vector DNA (A), transgene alpha galactosidase A (GLA) mRNA (B), and GLA activity (C). Closed symbols represent samples taken 3 months post-dose, open symbols samples collected after 6 months follow up. Squares depict the male animals, circles the female animals. The horizontal lines represent the mean + / - SD for each group. Group mean and SD for vector DNA and transgene GLA mRNA were calculated using the mean value of the different liver lobes for each animal. For vector DNA, the lower limit of quantification (LLOQ) is 50 vector DNA copies per pg DNA and for transgene GLA mRNA the LLOQ is 5,000 copies per pg RNA. Total GLA activity in liver returned values within the reportable range of the assay for all analysed samples.

[0106] Fig. 8 Plasma GLA activity - Plasma samples were collected at different timepoints before and after dosing and analysed for GLA activity. Values are depicted as mean + SEM for each group. (A) plasma GLA activity in all animals up to 3 months post-dose. (B) plasma GLA activity in the animals which were followed up until 6 months post-dose (n=4 per group). GLA activity in plasma was in the dynamic range of the assay for all analysed samples.

[0107] Fig. 9 Vector DNA levels in liver from NHPs at 3 months and 6 months after IV infusion with drug product - (A) Vector DNA in liver, 3 months post-dose, from NHPs treated with different doses of drug product are shown (mean ± SD). (B) Vector DNA in liver 3 and 6 months post-dose from NHPs treated with 2.0x1014and 7.3 xi o14gc / kg (mean ± SD). Each symbol represents one animal, the bars depict the group mean and the whiskers the SD. Group mean and SD were calculated using the mean value of the different liver lobes for each animal. The LLOQ is 50 vector DNA copies per pg DNA.

[0108] Fig. 10 Biodistribution of vector DNA in NHPs treated with drug product at 3 months post-dose

[0109] - Vector DNA in tissues, 3 months post-dose, from NHPs treated with different doses of drug product are shown (mean ± SD). The LLOQ is 50 vector DNA copies per pg DNA (dotted line). The limit of detection (LOD) is 12.5 vector DNA copies per pg DNA. For some samples of epididymis, heart, muscle, sciatic nerve, dorsal root ganglia, thyroid, dosing site, spinal cord, aorta, vena cava, skin of abdomen, and skin of fingertip less than 1 pg DNA could be extracted and, depending on the amount of DNA input per reaction, the LOD was maximally 117 copies / pg DNA and the LLOQ was maximally 467 copies / pg DNA. To calculate a group mean and standard deviation, samples with a result between the LLOQ and LOD were given a value of 18.75 (=(LLOQ-LOD) / 2) and samples with a result below LOD were given a value of 6.25 (=LOD / 2).

[0110] Fig. 11 Vector DNA levels in blood and plasma from NHPs treated with drug product - Vector DNA in blood (A) and plasma (B) up to 3 months post-dose, from NHPs treated with different doses of drug product are shown (mean ± SD). The LLOQ is 50 vector DNA copies per pg DNA in blood. For some timepoints, less than 1 pg DNA could be extracted from blood samples and, depending on the amount of DNA input per reaction, the LLOQ was maximally 6.485 x103copies / pg DNA. No LLOQ was determined for plasma, as a carrier nucleic acid was used to extract DNA from these samples which precludes assessment of the sample DNA concentration.

[0111] Fig. 12 Drug product derived GLA mRNA expression in selected tissues of drug product treated NHPs at 3 months post-dose - Drug product derived GLA mRNA in tissues, 3 months post-dose, from NHPs treated with different doses of drug product are shown (mean ± SD). The LLOQ is 50 mRNA copies per reaction, corresponding to 5000 copies per pg RNA (dotted line). The LOD is 3.125 mRNA copies per reaction (312.5 copies per pg RNA). To calculate a group mean and standard deviation, samples with a result between the LLOQ and LOD were given a value of 23.44 x103(=(LLOQ-LOD) / 2) and samples with a result below LOD were given a value of 1 .5625 (=LOD / 2). Examples

[0112] Materials

[0113] The following materials were used in the examples and are available as specified below in Table 1 . Table 1 : Materials used in the examples and their suppliers

[0114] Data Points

[0115] The stress conditions and time points for data collection for each Example are as specified below in Table 2. Table 2: Stress Conditions and Data Collection Time Points for the Examples

[0116] *RT: room temperature

[0117] **N.A.: not available

[0118] Measurements

[0119] 1 . The read-outs obtained were as follows:

[0120] Absorbance at 260 nm, which correlates with DNA concentration.

[0121] Absorbance at 280 nm, which correlates with protein concentration. • Absorbance at 350 nm (turbidity), which correlates with protein aggregation, or agglomeration and / or particle formation.

[0122] • Absorbance at 900 nm, which correlates with the background signal from the plastic of the wells and seal.

[0123] • Absorbance at 975 nm measures the water absorbance, thus, the path length.

[0124] • Tryptophan fluorescence at 280 / 335 nm, measured from the top and the bottom of the micro-well plate. This measurement correlates with protein and / or capsid unfolding and denaturation. The bottom read-out was used in the figures.

[0125] 2. Visual inspection

[0126] Visual inspection was carried out as generally accepted.

[0127] 3. Data processing

[0128] The protein and DNA concentrations were obtained as follows: According to the Lambert-Beer law, the absorbance is proportional to the concentration and the light path length. A constant named the extinction coefficient turns this into an equation A= cc.L.e, where A is the absorbance, cc is the concentration, L is the light path length and £ is the extinction coefficient. The light path length is measured by the water absorbance measured at 975 nm (A975) minus the plastic background measured at 900 nm (A900) thus: L=A975-A900

[0129] The protein concentration is measured by measuring the absorbance at 280nm (A280), thus: A280=cc(protein).L.£(Protein). The background from the precipitated and aggregated proteins or turbidity is measured by the absorbance at 350 nm (A350). It is assumed to be similarto the contribution of turbidity at 280nm, and thus, deducted from the absorbance at 280 nm. Then:

[0130] CC(protein).£(protein)=(A28O—A350) / (A975-A900)

[0131] Since £(protein) is a constant, we can conclude that cc(protein).£(protein), is proportional to the protein concentration, which is computed from the different absorbance values measured (A280, A350, A975 & A900). For the DNA concentration at 260 nm, a similar reasoning was applied.

[0132] Example 1 : Pre-formulation

[0133] The initial solution used in Example 1 was recombinant adeno-associated viral vector serotype 5 (rAAV5) with a transgene at a concentration of 4E13 gc / mL in PBS - / - (NaCI 137 mM; KCI 2.7 mM; Na2HPO410 mM and KH2PO4 1.8 mM), 5% w / v sucrose, rAAV5-protein, where the protein is for the treatment of Fabry disease, i.e. a-Galactosidase A.

[0134] The initial solution containing rAAV5 was buffer exchanged in a multi-well system into thirty different formulations comprising different pHs, buffer types, and salt contents using 96 well filter plates (100 kDa cut-off). After the buffer was exchanged and filtered through a 0.2 pm filter, the formulations were aliquoted into the plates according to the plate layout and buffer formulation compositions as specified in Table 3 below. Each formulation was prepared in duplicate. The outer rows and columns were filled with WFI to rule out plate location effects. The buffers had overlapping pHs, to differentiate between a buffer and a pH effect.

[0135] Table 3: Buffer Plate Layout and Buffer Formulation Compositions for Example 1

[0136] Salt Buffer WFI Acetate Citrate Phosphate Tris WFI

[0137] WFI is water for injection The different preparations were monitored for absorbance (at 280nm), fluorescence (at 335nm), and turbidity (at 350nm). Corrections in the absorbance for background due to turbidity were carried out, as well as corrections for the path length. Three plates were prepared per each stress condition as specified above in Table 2 above, and each condition (or each well) was measured in duplicate and the duplicate values were averaged. A tabular representation of the results obtained at T=0 is shown in Table 4. Table 4 shows that the three multi-well plates have similar values for the corrected absorbance at 280 nm (protein concentration) as expected. In addition, for the three plates at T=0, the absorbances are higher in the presence of salt, particularly at 150 mM NaCI, therefore showing a higher protein content and a lower degree of aggregation or agglomeration. This can be concluded since the values measured include the correction for the background as explained in the previous section. A positive effect for the presence of NaCI was shown. Buffers with a pH of 6.5 to 8 (in particular with the addition of salt) are preferable as higher absorbance (protein concentration) is observed. Table 4: T=0 Protein Content as followed by Absorbance at 280 nm (corrected for turbidity and path length in three multi-well plates; arbitrary units). A: F / T at -80°C; B: 28°C and C: 40°C.

[0138] The results obtained from the fluorescence measurements (protein and / or capsid unfolding and denaturation) are shown in Table 5. The lower the fluorescence, the more unfolded the proteins are.

[0139] These results suggest that the buffers which best stabilize the virus capsids 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 NaCI, with the 150 mM NaCI concentration seeming to be improved over the 75 mM NaCI concentration.

[0140] Table 5: T=0 Fluorescence as followed by Fluorescence at 335 nm (bottom measurement from three multi-well plates; arbitrary units). A: F / T at -80°C; B: 28°C and C: 40°C. Accelerated studies were carried out for a various conditions. The various assays were followed after two and seven days for the samples at 28°C and 40°C; and after 1 , 3, and 10 cycles for the samples under F / T stress.

[0141] Fig. 1 shows the 280 nm absorbance and the 335 nm fluorescence (Trp) measured in the accelerated stress studies for the different formulations. It is seen that the higher the salt concentration, the higher the 280 nm absorbance and the 335 nm fluorescence. This observation, which is the same as observed for T=0, shows that higher salt concentrations are beneficial for the formulation in terms of protein concentration.

[0142] In Fig. 2, the 280 nm absorbance and the 335 nm fluorescence are shown as a function of the different stresses applied. In this graph, there are three panels for the different salt concentrations: 0 mM left panel, 75 mM middle panel, and 150 mM right panel. As in the previous figure, the observed effect of the salt on the absorbance and fluorescence is clear: the higher the salt concentration, the higher the absorbance and fluorescence, showing the favourable effect of salt. The effect of the pH is also seen: the higher the pH, the higher the absorbance and the fluorescence. This effect is especially observed for the fluorescence (Trp). Its magnitude is lower than the one observed for the salt effect.

[0143] It is therefore concluded from Example 1 that the salt concentration (above 75 mM) and the pH (above 7.0) have a beneficial effect on enhancing the stability of the capsids. Tris pH 7.5 and 8.0, and phosphate pH7.0 buffers were chosen for further tests and optimizations.

[0144] Example 2: Excipient Screening

[0145] Accelerated studies were carried out for a various conditions. The various assays were followed after one and two days for the samples at 28°C and 40°C; after 1 and 3 cycles for the samples under F / T stress and after 1 hour of shaking at RT. Different excipients cysteine, arginine and MgCh do not appear to have an obvious or measurable effect on the stability of the product when compared to the control conditions (buffer alone) at any of the tested pHs: 7.0; 7.5 or 8.0.

[0146] A second excipients screening experiment was performed to assess the effect of two carbohydrates on the stability of the drug product: mannitol and a cyclodextrin. The main variables of this Example are the concentrations of mannitol and cyclodextrin on their own or in combinations. The concentration of mannitol and cyclodextrin ranged from low (0.05% w / v) to high (4% w / v).

[0147] While testing the effect of addition of mannitol and / or cyclodextrin (here hydroxypropyl-beta-cyclodextrin was used), various buffer formulations were subject to further tests and optimizations. Several multiwell plates were prepared and assigned to different stress conditions as specified in Table 2 above. The basic buffer, referred to as TH+, is a Tris 20 mM buffer pH 7.5. The buffer further included tonicity agents to set the buffer at a desired osmolality (290 mOsm / kg) to mimic physiological conditions (here: 5 mM histidine, 2.5 mM MgCh, 2.5 mM KCI, 2.5 mM CaCh, and NaCI). The drug product used in Example 2 was the same rAAV5 as used in Example 1 . Distinct percentages of mannitol or cyclodextrin were added to the TH+ buffer (see Table 6). As controls, a PBS buffer at pH 7 supplemented with 5% sucrose (Ctrl 1) and TH+ buffer alone (Ctrl 2) were used.

[0148] The formulation matrix tested for Example 2 is shown in Table 6. Each formulation is prepared in triplicates. The outer rows and columns are filled with WFI to rule out plate location effects. Table 6: Buffer Plate Layout and Buffer Formulation Compositions for Example 3

[0149] 1 2 3 4 5 6 7 8 9 10 11 12

[0150] Table 7 shows the results of the formulation confirmation experiment. It shows the 280 nm absorbance of the plates as measured at T=0. In general, the formulations that contain cyclodextrin seem to always have a better formulation profile.

[0151] Table 7: T=0 protein content as followed by Absorbance at 280 nm (corrected for turbidity and path length in four multi-well plates; arbitrary units). A: F / T at -80°C; B: 28°C; C: 40°C and D: Shaking.

[0152] The protein content was measured under different stresses for different durations. The stress conditions included incubation at 28°C and 40°C for 0, 3, or 7 days; F / T for 2 and 5 cycles, and 2 hours of shaking (SHK). The different mannitol or cyclodextrin concentrations do not show significant differences as stability-enhancing excipients beyond T=0 under the different stress conditions or durations (data not shown). This may point out that the stabilizing effects of the different formulations achieved at T=0 are still valid under the stresses applied.

[0153] Conclusions based on Examples 1 and 2:

[0154] The conclusions of this study are as follows:

[0155] • Cyclodextrin shows a beneficial stabilizing effect.

[0156] • A salt, such as NaCI, also assists in stabilizing the drug product. Salt addition at concentration levels of 75 mM or higher is beneficial.

[0157] • Neutral or slightly basic formulations pH (7.0-8.0) improve stability of the drug product.

[0158] Example 3: Testing the stability of rAAV5 in the formulation.

[0159] Based on the above two example the following formulation of the invention is therefore specifically exemplified:

[0160] • rAAV5 with a transgene encoding a therapeutic protein,

[0161] • 20 mM Tris at pH 7.5,

[0162] • 125 mM sodium chloride and,

[0163] • 2% w / v HP-p-cyclodextrin

[0164] This buffer is referred to herein as FB. The rAAV5 used in this example has a transgene encoding a therapeutic protein.

[0165] Table 8: Materials

[0166] Visual inspection rAAV5-protein in FB has no visual particles under the stress conditions of 1 F / T +24h at RT (15 °C -25 °C), 1 D agitation at RT and 3F / T (Table 9). The results showed small particles at 37°C (1 W and 2 W) and at 25°C (2 W and 4 W). In 1-2 vials out of the 3 vials, particles were observed in FB at 2-8°C for 1 M. However, no visual particles were observed in FB at 2-8°C for 3 M and 6 M time points. Therefore, the particles observed at 2-8°C (1 M) can be disregarded. There were also no visual particles observed at 2-8°C 1 M in one 500 L batch where the concentration is 5E13 gc / ml instead of 1 E14 gc / ml as in this study. Table 9: Overview of visual inspection results for platform formulation fit study for rAAV5- protein in FB with target gc at 1E14 gc / mL

[0167] Sub-visible particles Two replicates from each time-point I condition were analysed for sub-visible particle content. For each particle size range, data of the two replicates were averaged. Table 10 summarizes the cumulative subvisible particles count / per vial with sizes >10 pm and >25 pm. Under all stress conditions the sub- visible particles for rAAV5-enzyme in FB remain within acceptance criteria (USP). Table 10. Cumulative subvisible particles count / per vial for rAAV5-protein in FB with target gc at 1E14 gc / ml (production batch 1). Genome copies, total particles and infectivity

[0168] For platform formulation fit study, the gc concentration of rAAV5-protein in FB remains at around 1 E14 gc / ml within assay variation under different conditions (% CV < 25%, Determination of Genome Copy Content of rAAV5-enzyme Test Samples by Quantitative PCR) . Accordingly, it can be concluded that there is no effect of F / T-cycles, temperature stresses and 1 D agitation on the genome copy content of rAAV5-protein in FB under the tested conditions.

[0169] The total particle content of rAAV5-protein in FB is stable between 1.1 E+14 total particles (tp) / mL and 1 .3E+14 tp / mL except under stress conditions at 37°C (1 W and 2 W) and at 25°C (2 W) where the total particle content decreases from about 1 .2E14 tp / ml to 7E13 tp / ml which might indicate the presence of aggregates of rAAV5, which correlates with the conclusions from visual inspection results. The tp / gc ratio for rAAV5-protein in FB under various conditions remains below 2.

[0170] The infectivity expressed as infectious particles (ip) / mL shows a variable data set, mainly due to the variability of the method (CV% is < 80%, Replication-based infectious vector titer assay). In general, the infectivity of rAAV5-protein in FB decreases at 37°C and at 25°C. Infectivity of rAAV5- protein in FB showed no significant different results under 3 F / T cycles and 1 D agitation compared to control at To.

[0171] Dynamic light scattering (DLS)

[0172] DLS measurements yield the polydispersity index (PDI) and the z-average. For rAAV5-enzyme in FB, the PDI value under various conditions is lowerthan 0.1 which means monodisperse with size remaining around 25 nm.

[0173] Example 4: Assessing the stability in a 10-fold larger batch size

[0174] The obtained stability results on rAAV5-protein in FB were obtained from a 50 L production batch. A comparability study was performed between a batch scale from 50 L to 500 L to compare the stability of rAAV5-protein produced from the differentially sized batches with a comparable titer of 1 E14 gc / mL. This was done under four different stress conditions: 1 F / T +24h holding at RT (15 °C -25 °C), 3 F / T cycles, 1 D agitation and 1 W 37°C.

[0175] The 50 L batch data (production batch 1) was compared to rAAV5-protein from a 500 L batch under the 4 different stress conditions mentioned above. The results are summarized in Table 11 . The attributes as monitored-visual inspection, subvisible particles, genome copy concentration, total particles, infectious vector titer, monomeric particle purity and particle size distribution, gc / ip ratio and tp / gc ratio all remain within the rAAV5-protein drug product specifications (MS-07014) and there is no significant difference for rAAV5-protein from a 50 L (production batch 1 , at 1 E14 gc / mL) and rAAV5-protein from a 500 L batch (production batch 2, at 1 E14 gc / mL). Therefore, based on overall collected data rAAV5- protein from 50 L (production batch 1 , at 1 E14gc / mL) and 500 L (production batch 2, at 1 E14gc / mL) batches are comparable. Table 11 : Overview data on rAAV5-protein in FB from the 50 L production batch 1 vs rAAV5- enzyme from the 500 L production batch 2 under 4 different stress conditions (at IE14gc / mL). Example 5: further comparison of FB with other buffers

[0176] Using materials and methods as described for previous examples, with rAAV5-protein where the therapeutic protein is factor VIII, the following buffers were compared with formulations comprising around 4E13-5E13 genome copies:

[0177] 1 . PBS + 5% Sucrose + 0,02% PS20;

[0178] 2. FB (20mM Tris pH: 7,5 + 125mM NaCI + 2% HP-p-Cyclodextrin);

[0179] 3. 20mM Citrate / phosphate pH: 7,5 + 125mM NaCI + 0,1 % PS80; and

[0180] 4. 20mM Tris pH: 7,5 + 125mM NaCI + 0,1 % PS80.

[0181] The formulations were put on an accelerated development stability study at 25°C for a maximum time of three months. The four formulations behaved similarly (Fig. 3A) with the exception of formulation 3 where the GC / IP was above the acceptance criteria at 3 months (Fig. 3B). For formulation 3 and 4, an increase in the number of sub-visible particles was observed (Fig. 3C). There were no visible particles for any of the formulations even after 3 months (Fig. 3D).

[0182] Degradation studies were also performed at 40°C for the period of 4 weeks. The GC / IP for all formulations increased, with formulations 2 and 4 showing the best performance (Fig. 4A). There were also increases in sub-visible particles for all the formulations except for formulation 2 (Fig. 4B). Formulations 1 and 3 showed an increased polydispersity after 2 weeks at 40°C (Fig. 4C).

[0183] The four formulations were placed in long-term development stability studies at -80°C. Almost parallel curves for genome copies showed that there are no large differences between the four formulations for this parameter (Fig. 5A). Formulation 1 showed a visible particle after 6 months. Formulation 3 showed an increase in the sub-visible particles, and all formulations except formulation 2 showed increased >10pm particles (Fig. 5B). Furthermore, formulations 3 & 4 showed a ratio of total particles to genome copies (tp / gc) that was higher than the other samples, implying more impurities after 1 month (Fig. 5C). In addition, formulations 3 & 4 showed an increase in polydispersity index at the first months tested.

[0184] The four formulations were also placed in long-term development stability studies at 2-8°C. The almost parallel curves for genome copies again show that there were no large differences between the four formulations for this parameter (Fig. 6A). Again formulations 3 and 4 were less stable, showing visible particles after 6 and 12 months respectively (Fig. 6B), and showing an increase in sub-visible particles after 6 months (Fig. 6C). All formulations except formulation 2 showed an increase in their sub-visible particles >0.3pm (Fig. 6D).

[0185] Example 6: Determination of formulated drug product stability

[0186] 2-8°C up to 12 months

[0187] < -65°C up to 24 months For 5x1013gc / mL

[0188] 500L scale, non-GMP (Good Manufacturing Practice) certified rAAV5 vector comprising a transgene encoding a-Galactosidase A. The drug product (DP) was formulated at 5 X 1013(gc) / mL in a formulation buffer containing 20 mM T ris, 125 mM NaCI, 2% HP- 6 -CD (w / v), pH 7.5 with a fill volume of 1 .2 mL DP in 2 mL glass vials. The DP vials were stored upright at the storage conditions (sS-65 °C) up to 24 months and in accelerated conditions (2-8°C) up to 12 months. At six predefined time-points (0, 1 , 3, 6, 12, and 24 months), the stored DP was sampled, and quality attributes (QA) were determined. aValue based on 1 replicate, gc = Genome copies, ip = Infectious particles, tp=Total particles aValue based on 1 replicate, gc = Genome copies, ip = Infectious particles, tp=Total particles

[0189] Subvisible particles The number of subvisible particles was determined by light obscuration (Accusizer). Both >10 pm and >25 pm particles are shown in Tables 12 and 13 (particles per 10.7 mL DP). The acceptance criteria complies with the small volume parenteral specification of USP<787>: i.e. <6000 particles per vial (>10 pm) and <600 particles per vial (>25 pm). Subvisible particle counts were within compendial specification, far below the acceptance criteria for all tested timepoints and corresponding storage conditions. Shown results (Tables 12 and 13) are extrapolated to 10.7 mL as this is the DP volume per clinical vial.

[0190] Genome copies, total particles & infectivity

[0191] The gc concentration remained within the DP specification for GMP material (3.1 xi o13- 6.8x1013) at both storage conditions up to 24M (Tables 12 and 13). Variation in-between TO and the different timepoints was observed, but was within assay variation for both storage conditions (2-8°C and <- 65°C) at all tested time-points. At both storage conditions, size exclusion chromatography (SEC)-purity was a 100% in DP 5x1013gc / mL at all time-points. The total particle (TP) content of the DP, 5x1013 gc / mL, varied slightly more than assay variation (% relative standard deviation (RSD) <8%) throughout storage at 2-8°C and <-65 °C up to 24M. However, no clear trend in-between the timepoints was observed and no variation was observed between 12M and 24M at <-65 °C. The tp / gc ratio remained within DP product specification (<2.0 tp / gc) throughout storage at both 2-8°C and <-65°C up to 24M.

[0192] The infectious titer of the DP varied within assay variation (Coefficient of Variation% <67). Therefore, it was concluded that no difference in infectious titer was observed. The similar observation was made with the DP at 1 xi o14gc / mL. The genome copies over infectious particles (gc / ip) ratio remains equal to or below 48 gc / ip for DP stored at 2-8°C and <-65°C up to 12 or 24M (Tables 12 and 13). The DP gc / ip specification is <70 and thus the studied DP (5x1013gc / mL) complied with the specifications set for the GMP batches.

[0193] Dynamic light scattering (DLS)

[0194] DLS was performed according to standard procedures. Both Z-average and polydispersity index (Pdl) of the DP only varied slightly in-between the timepoints for both storage conditions from TO onwards (Z-average range: 26.8-27.1 nm and Pdl range: 0.036-0.060). At TO Z-average and Pdl were higher, this is probably due to sample or assay artifacts (e.g. dust, air-bubble) as all other time points have comparable lower values. Both the Z-average and Pdl were in line with the observations with the DP at 1 xi o14gc / mL in long-term stability studies (data not shown).

[0195] Results

[0196] Based on the 12M and 24M results of the DP long-term stability study (500 L) it was concluded that: At 2-8 °C, all quality attributes (QA) of the DP at 5x1013gc / mL (500L) remained comparable to TO with exception of visible particles. Visible particles were observed at the 6- & 12-month time-points. At <- 65°C, QA of the DP at 5x1013gc / mL (500L) remained comparable to TO with exception of visible particles. Visible particles were observed only at the 12-month time-point.

[0197] However, the DP at 1 xi o14gc / mL (50L) (data not shown) was free of visible particles up to 24M when stored at <-65°C. No differences compared to TO in all other QA were observed in the DP at 1 xi o14gc / mL (50L) up to 24M at both storage conditions. In conclusion, the DP QA prove that the formulation is capable of accommodating high concentrations of rAAV vectors while maintaining favourable characteristics which allow for patient dosing, and maintain those characteristics for at least 24 months when stored at <-65°C, and for at least 12 months when stored at 2-8 °C. Example 7: Determination of GMP-grade formulated drug product stability

[0198] Stability data at the long-term storage condition of <-65°C for a clinical, GMP-grade drug product (DP) batch is provided in Table 14. Values are obtained as described above. Osmolality was determined using standard procedures.

[0199] Similar to the conclusions drawn from the non-GMP-grade DP, the above-depicted data show that the formulated drug product can be stored for extended periods of time at temperatures <-65°C.

[0200] Example 8: dose evaluation of a 26-week single intravenous administration biodistribution and toxicity study (GLP) in the cynomolgus monkey

[0201] The objective of the study was to evaluate biodistribution, alpha-galactosidase A (GLA) pharmacodynamic markers and toxicity of drug product formulated in 20 mM Tris pH 7.5, 125 mM NaCI, 2% HP-beta-cyclodextrin, following a single IV administration in male and female NHPs followed by a 3-month and 6-month post-dosing observation period (see Table 15). The GLA pharmacodynamic markers are described below. Detailed evaluation of pharmacokinetic parameters, vector DNA biodistribution and transgene mRNA expression and detailed description of all safety evaluations are also provided below.

[0202] Table 15: Design of single-dose toxicity study in NHPs with formulated drug product aVehicle: Formulation buffer: 20 mM Tris pH 7.5, 125 mM NaCI, 2% HP-beta-cyclodextrin Pharmacology

[0203] To determine whether durable and functional hepatic drug product-derived GLA protein expression and transport to the plasma could be obtained in a large animal model, GLA activity was measured in liver, at 3 and 6 months post-dose, and in plasma during the course of the study. Values obtained in liver and plasma of drug product-treated animals are compared to the endogenous NHP GLA activity found in vehicle treated NHPs. In addition, GLA activity was measured in selected extrahepatic tissues; adrenals, brain, dorsal root ganglia, heart, kidney, and spleen.

[0204] Test methods for pharmacodynamic endpoints

[0205] GLA activity was measured using the same methodology as is usually employed in the clinic for diagnosis and follow-up of Fabry patients. Suitability of the assay for the analysis of tissue and plasma samples from NHPs was confirmed and the assay was qualified for use. In addition to the GLA activity measurements, transduction of the liver by assessing vector DNA and vector-derived GLA mRNA expression was taken along as an additional pharmacokinetic / pharmacodynamic parameter. More information on the development and validation of the qPCR methods to detect vector DNA and vector- derived GLA mRNA is presented below.

[0206] Blood and tissue sampling procedures for pharmacodynamic endpoints

[0207] Blood samples for pharmacodynamic assessments were taken pre-dose and at regular intervals during the post-dose observation period. Blood was collected from the Vena cephalica antebrachia or Vena saphena. For the isolation of plasma, venous blood was collected into tubes containing K2EDTA to prevent clotting and kept on wet ice until centrifugation to harvest the plasma. For serum, venous blood was collected into serum tubes and allowed to clot for >30 min at room temperature, before centrifugation to isolate the serum. Plasma and serum samples were stored frozen (<-65°C) until use.

[0208] Measurement of antibodies in NHPs against drug product derived GLA

[0209] Antibodies against the GLA-protein in NHP serum samples were determined using a semi-quantitative MesoScale Discovery (MSD) assay. As no calibrators are available, results were expressed as relative electroluminescence units. Although this approach does not produce a titer, the generated value should nonetheless be proportional to the amount of circulating GLA-specific monkey antibodies. Test-samples with signals >250 were subjected to a confirmatory assay in which test-samples were first pre-incubated for 1 hour at room temperature with or without an excess (100 pg / mL) of GLA-protein prior to be added to the immunoassay plates.

[0210] Liver transduction and GLA activity

[0211] At 3 and 6 months post dosing, liver vector DNA levels show a dose-dependent increase up to a dose of 5.0x1014gc / kg (Fig. 7A). Liver transgene GLA mRNA levels were proportionate to the vector DNA levels in animals dosed 2.0x1014to 7.3x1014gc / kg (Fig. 7B). At a dose of 4.0x1013gc / kg relatively higher transgene GLA mRNA levels were observed than expected based on the vector DNA levels measured in this dose group. At 3 months and 6 months post-dose, comparable levels of vector DNA and drug product derived GLA mRNA were found in livers from animals treated with 2.0x1014and 7.3x1014gc / kg drug product (data not shown). As expected, no vector DNA or transgene GLA mRNA was found in livers of vehicle-treated animals. GLA activity analysis in liver tissue demonstrated an increase in liver GLA activity in all drug product treated groups, compared to vehicle treated animals (Fig. 7C). While vehicle treated animals showed an average liver GLA activity of 98 ± 9 nmol / h / mg, their endogenous GLA activity, an increase in liver GLA activity, corresponding to vector-derived GLA, was seen in drug product treated animals. Within each dose group, individual variation in liver GLA activity was observed.

[0212] Plasma GLA activity

[0213] In the first two months after dosing, NHPs show a peak and subsequent decline in plasma GLA activity when dosed with 4.0x1013up to 5.0x1014gc / kg drug product (Fig. 8A). The plasma GLA activity stabilizes between week 10 and week 13 and then remains at that level until end of the study at 26 weeks post-dose (Fig. 8B). With higher doses, individual variation within each dose group become more apparent, as each group has one or more “low” responders, with a plasma GLA activity of 1 .5 to 2-fold over vehicle, amidst animals which show a dose-dependent increase in plasma GLA activity, from 4.5-fold over vehicle level in the 4.0x1013gc / kg group up to 40-fold over vehicle level in the group treated with 5.0x1014gc / kg drug product. The variation seen in the group dosed with 7.3x1014gc / kg is mostly caused by one high responding animal, which had a 98-fold increase in plasma GLA activity compared to vehicle treated animals, while the other animals in this group showed an increase in plasma GLA activity between 6- to 26-fold.

[0214] GLA activity in extrahepatic tissues

[0215] Measurement of GLA activity in extrahepatic tissues did not show an increase in GLA activity compared to vehicle treated animals. Most likely, this is due to the substantial background level of the method and the endogenous GLA activity present in healthy NHPs.

[0216] Safety pharmacology of the formulated drug product

[0217] Electrocardiograms, blood pressure measurements and measurements of the respiratory rate were included as a routine examination in the general toxicity study in NHPs and showed no effect of the drug product.

[0218] Results

[0219] The formulated drug product is a recombinant AAV5-based vector designed to deliver liver-specific expression of the human GLA transgene by a single (one-time) IV infusion. Studies on the pharmacodynamics and efficacy of the drug product demonstrate a strong correlation between vector dose and GLA expression levels, and confirmed the biological activity of the expressed and fully functional GLA protein. Treatment of NHPs with the drug product resulted in a dose-dependent increase in liver vector DNA and mRNA levels up to a dose of 5.0x1014gc / kg. No further increase was observed in the animals receiving 7.3x1014gc / kg. GLA activity in both liver and plasma demonstrated a dosedependent increase in all drug product treated groups, compared to vehicle treated animals. In the first two months after dosing, NHPs show a peak followed by a subsequent decline, mainly in at the lower dose levels, and stabilization in plasma GLA activity, which has been observed in previous liver-directed human transgene overexpressing gene therapy programs (Spronck EA et al., Molecular Therapy Methods & Clinical Development 2019, Vol. 15, 221-231). Between 2 to 3 months after drug product treatment, some animals demonstrate an increase in plasma GLA activity. This might reflect induction of humoral tolerance against the drug product derived GLA protein, as described by Nietupski et al (Nietupski JB et al., Molecular Therapy, 2011 , Vol. 19(11):1999-2011) in NHPs receiving IV AAV8-GLA. Durability of the response was demonstrated in NHPs, as the liver vector DNA levels, vector-derived GLA mRNA levels and plasma GLA activity levels are sustained for at least 6-months post drug product administration, the longest time point tested, indicating vector persistence and durability of expression. In summary, durable expression of vector-derived GLA can be achieved with IV drug product administration in NHPs.

[0220] Pharmacokinetics

[0221] Biodistribution of drug product derived vector DNA in NHP was assessed using a validated qPCR in plasma and blood samples taken pre-dose and during the course of the study and tissue levels of vector DNA were measured in liver, kidney, heart, brain, gonads, lung, spleen, dorsal root ganglia, the injection site, and 14 othertissues, harvested 3 and 6 months after administration of the drug product. In addition, transduction of liver cells was analysed by fluorescence-in-situ-hybridization (FISH), using a vector DNA specific probe. Expression of the transgene was assessed by a validated RT-qPCR in liver and in 11 extrahepatic tissues at these timepoints.

[0222] Blood and tissue sampling procedures for pharmacokinetic endpoints

[0223] Blood samples for pharmacokinetic assessments were taken pre-dose and at regular intervals during the post-dose observation period. Blood was collected from the Vena cephalica antebrachia or Vena saphena in the NHP studies. For the isolation of plasma, venous blood was collected into tubes containing K2EDTA to prevent clotting and kept on wet ice until centrifugation to harvest the plasma. Plasma samples were stored frozen (<-65°C) until use. For the isolation of serum, venous blood was collected into serum separator tubes and allowed to clot for at least 30 minutes at room temperature ice until centrifugation to harvest the serum. Serum samples were stored frozen (<-65°C) until use.

[0224] Pre-existing anti-AAV5 antibodies

[0225] As NHP are natural hosts of AAV5, pre-existing (neutralizing) antibodies can be present due to a natural infection with the wild type virus. Assessment of anti-AAV5 neutralizing antibodies showed that predose the serum of 31 out of 36 NHPs already had AAV5-neutralizing activity with titers ranging from 50 to 689. Similar to our experience with other AAV5- based gene therapy programs in NHPs with pre-dose anti-AAV5 neutralizing antibody titers up to 1 ,000 (Majowicz, Nijmeijer et al. 2019), no correlation between neutralizing antibody titers and liver transduction levels (described below) were observed.

[0226] Liver vector DNA levels

[0227] Liver vector DNA levels are presented in Figure 9. Per animal, 4 liver lobes were analyzed and the treated animals demonstrated homogenous spread of vector DNA among the liver lobes. A doseresponse relationship in liver vector DNA copies was observed from the low dose, 4.0 xi o13gc / kg, to a dose of 5.0 xi o14gc / kg of drug product. No further increase in liver vector DNA copies was seen at the highest dose of 7.3 xi o14gc / kg (Fig. 9A). Similar vector DNA levels were found in livers of drug product treated animals at both the 3-month and 6-month post-dose timepoint (Fig. 9B).

[0228] Tissue vector DNA distribution

[0229] The biodistribution of vector DNA showed a clear dose-dependency in all tissues up to a dose of 5.0x1014gc / kg. Similar vector DNA levels were observed between animals treated with 5.0x1014gc / kg and 7.3x1014gc / kg (Fig. 10). At 3 months post-dose, the highest vector DNA levels were found in liver and adrenals (ranging from 4 to 6x106vector DNA copies / ug DNA) at the two highest doses of 5.0 xi o14and 7.3 xi o14gc / kg, followed by aorta. Other tissues showed quantifiable, but >10-fold lower vector DNA levels compared to liver.

[0230] Vector DNA levels in blood and plasma

[0231] Vector DNA levels were measured in blood and plasma after drug product administration (Fig. 11A and 11 B). Blood and plasma vector DNA clearance was generally comparable at each dose tested. A steep decline in blood and plasma levels was observed within the first 2 weeks after dosing, followed by a slow decline until the last timepoint at 3 months post-dose.

[0232] Drug product derived GLA mRNA in tissue

[0233] RT-qPCR revealed the highest GLA transgene mRNA levels in the liver, in accordance with the use of a liver specific promotor (Fig. 12). Up to a dose of 5.0X1014gc / kg, transgene GLA mRNA levels correlated with the vector dose. Similar to the vector DNA levels, the two highest doses (5.0 and 7.3x 1014gc / kg) showed comparable levels of GLA transgene mRNA. At 3 months and 6 months post-dose, comparable levels of GLA transgene mRNA were found in livers from animals treated with 2.0x1014and 7.3 xi o14gc / kg drug product. GLA transgene mRNA levels approximately 10-fold lower, compared to liver, were found in adrenals and dorsal root ganglia (DRG), followed by heart and spleen, showing lower but quantifiable levels. In testis, mRNA levels just above the LLOQ were found in some animals dosed with 5.0x1014gc / kg. In kidney and sciatic nerve, no dose-dependence is seen in the GLA transgene mRNA levels, as most groups show results around the assay’s LLOQ except for kidney tissue of animals dosed with 2x1014gc / kg which shows high variability within that group. All other tissues examined did not show quantifiable GLA transgene mRNA expression (< LLOQ of 5x103copies / pg RNA) (Fig. 12).

[0234] In conclusion, single dose intravenous infusion of formulated drug product resulted in homogenous vector DNA distribution throughout the liver with widespread distribution into multiple tissues. Tissue vector DNA levels were proportional to the dose administered up to a dose of 5.0 1014gc / kg. High GLA transgene mRNA levels were present in liver. Comparable levels of vector DNA and vector-derived GLA mRNA were found in liver at 3 months and 6 months after drug product administration. Results

[0235] In NHPs high, dose-related drug product derived vector DNA levels are present in liver and adrenal gland. Vector genome copy numbers in the liver and adrenals of the NHPs ranged from 4 to 6X106vector DNA copies / ug DNA at the two highest doses of 5.0x1014and 7.3x1014gc / kg. Vector DNA analysis from tissues other than liver and adrenals were at least 10-fold lower compared to the liver (target organ). Assessment of vector DNA biodistribution to reproductive tissues, for evaluation of possible reproductive toxicity, showed that vector DNA levels in the ovaries, testis, epididymis and seminal vesicles correlated with the dose and were around >400-fold lower as compared to the liver. Blood and plasma vector DNA clearance was generally comparable at each dose tested, with a sharp decrease in vector DNA levels in the first 2 weeks post-dose and subsequent slower decline. Vector DNA transcription resulted in dose-related quantifiable GLA transgene mRNA levels mainly in the liver, in accordance with the use of a liver-selective promoter, but mRNA expression was also observed in adrenals, dorsal root ganglia, heart and spleen. Similar vector DNA levels and GLA transgene mRNA levels were found in livers of drug product treated NHPs at the 3-month and 6-month post-dose timepoint, indicating persistence of the vector and transgene expression. No correlation was observed between pre-dose Nabs and liver vector DNA and mRNA levels up to the highest titer measured in any pre-dose sample, 689, indicating that pre-existing neutralizing antibodies do not impact transduction efficiency, as was observed for a similar AAV5 vector for hemophilia B, with neutralizing pre-dose titers in NHPs up to 1 ,000 (Majowicz A et al., Molecular Therapy Methods & Clinical Development 2019, Vol. 14, 27-36). Taken together, the pharmacokinetic parameters included in the studies indicate dosedependent and persistent levels of vector DNA and GLA transgene mRNA in the liver of Fabry mice and NHPs. Biodistribution of drug product-derived vector DNA to extra-hepatic tissues was at least 10- fold lower than liver in NHPs and even more reduced in reproductive organs (>400-fold). The use of a liver-specific promoter restricted GLA transgene mRNA expression mainly to the liver.

[0236] Assessments

[0237] Starting from arrival at the test site, NHPs were monitored at least twice daily for mortality and any clinical signs or symptoms. Food consumption was assessed daily, and body weight of the animals was determined weekly. Body temperature was measured once pre-dose, 4, 8, and 24 hours after dosing, in week 4 post-dose, and at necropsy at week 13 and week 26. In week 4 and week 26, electrocardiogram, blood pressure, and respiratory rate measurements were performed and compared to measurements taken pre-dose. Full clinical pathology evaluations, including cardiac troponin levels, and measurements of hematology and coagulation parameters were performed pre-dose and at relevant time points during the study. To be able to closely monitor any effects on liver and cardiac function, additional measurements of a selected panel (cardiac troponin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyltransferase and glutamate dehydrogenase) were performed more frequently in the first two weeks after dosing. To evaluate whether drug product administration triggers an adverse immune response, a panel of cytokines was analyzed in blood samples taken pre-dose, 4 and 24 hours after dosing, on days 4, 8, and 15 after dosing, and 4 weeks post-dose. To assess the immunogenicity of the drug product, anti-AAV5 IgM levels were determined in plasma samples taken pre-dose and at day 8 and day 15 post-dose. Anti- AAV5 IgG and neutralizing antibodies and anti-GLA protein IgG antibodies were determined in serum samples taken pre-dose and at 3 and 6 months after drug product administration. At necropsy, organ weight measurement and histopathological analysis of 48 relevant organs and tissues, including liver, adrenals, kidney, heart, brain, gonads, lung, spleen, dorsal root ganglia, and the injection site was performed.

[0238] General safety parameters

[0239] During the study, there were no unscheduled deaths and no clinical signs related to treatment with drug product were observed. Food consumption, body temperature, body weight, body weight gain, electrocardiography, blood pressure, and respiratory rate were unaffected by treatment up to 7.3x1014gc / kg drug product.

[0240] Toxicology

[0241] Clinical pathology and Hematology

[0242] No drug product-related effects on hematology or coagulation test results were noted throughout the study. Transient, minimally to mildly increased aspartate aminotransferase and / or alanine aminotransferase activities and mildly to markedly increased creatine kinase activity were observed on Days 1 and / or 4 for two males administered 4x1013gc / kg and one male administered 2x1014gc / kg; glutamate dehydrogenase (GLDH) activity was not notably increased. As the elevations in enzyme activity were only observed in a small number of animals of the two lowest dose groups, only for a short duration, and likely partly associated with procedural (handling) skeletal muscle perturbation, these were considered not to be adverse.

[0243] Immunology

[0244] Blood cytokine analysis revealed no drug product related effects on inflammatory markers. Assessment of the humoral immunogenicity of the drug product showed that at pre-dose 1 female animal had a higher anti-AAV5 IgM level compared to the other animals. Analysis of total anti-AAV5 IgG showed that 14 out of 36 animals had a signal-to-noise ratio of > 3 in the pre-dose sample, indicating the presence of pre-existing AAV5-specific IgG. Pre-dose serum of 17 out of 18 males and 14 out of 18 females showed AAV5-neutralizing activity with titers ranging from 50 to 689. Post-dose all samples from drug product treated animals taken on day 8 and 15 (for IgM analysis) and taken at 3 and 6 months (for IgG and neutralizing antibody analysis) showed high anti-AAV5 antibody levels, as expected. In the IgM analysis, post-dose the OD450nm levels were >10-fold compared to pre-dose levels, post-dose AAV5- neutralizing titers ranged from 33,828 to >729,000 and high signal-to-noise ratios were observed in the IgG analysis, indicating a post-dose increase in anti-AAV5 antibodies. Analysis of anti-GLA antibodies in serum samples taken pre-dose showed that none of the animals were positive for anti-GLA antibodies before dosing. At 3 months after dosing, samples from two animals reported positive. Both animals were male, one animal was dosed with 4.0x1013gc / kg and the other animal with 5.0x1014gc / kg. In the vehicle treated animals, no difference in anti-AAV5 or anti-GLA antibody levels pre- versus post-dose was observed.

[0245] Organ weight and histopathology

[0246] No drug product-related changes were found in organ weights and in macroscopic or microscopic observations at both 3 months and 6 months post-dose.

[0247] In conclusion, up to 7.3x1014gc / kg drug product was well tolerated when administered as a single intravenous dose. No drug product-related systemic or local toxicity findings were noted for animals administered up to 7.3x1014gc / kg; as such, this dose is considered the no observed adverse effect level (NOAEL) under the condition of the study.

[0248] Integration site analysis on liver of cynomolgus macaques treated with drug product

[0249] To investigate integration of drug product derived vector DNA into genomic DNA of liver tissue of NHPs, liver tissue samples from the GLP toxicity study with drug product in NHPs were used for integration analysis of vector DNA into the host genome. Shearing extension primer tag selection ligation-mediated PCR (S-EPTS / LM-PCR) combined with high throughput sequencing demonstrated integrated forms of the vector DNA in NHP liver samples. All liver samples from drug product treated animals showed a polyclonal integration site (IS) profile and no dominant IS (>30 %) have been observed. There is no indication of clonal expansion. This indicates that no safety concerns are raised.

[0250] Local Tolerance

[0251] Local tolerance at the injection site has been assessed as part of the GLP general toxicity studies. No notable findings were observed at microscopical examination of the injection site.

[0252] Results

[0253] Safety of the drug product was evaluated by a GLP toxicity study in NHPs combined with subsequent evaluation of vector DNA integration into host liver DNA. The observation periods in the the NHPs were 3 and 6 months post administration. In the GLP toxicity study in NHPs the vector dose levels ranged from a dose supporting a low pharmacological effect (4.0x1013gc / kg), to a dose corresponding to 2.4- fold (7.3x1014gc / kg) the intended human high dose. In humans receiving AAV-based gene therapy hepatotoxicity and / or thrombotic microangiopathy (TMA) has been reported (CTCTAC_FDA 2021). In this GLP NHP study, no evidence for drug product related hepatotoxicity was observed. Within the first days after dosing, some mild, transient, increases in liver enzymes were observed in 3 animals, two males administered low dose, 4x1013gc / kg, and one male administered mid dose, 2x1014gc / kg, but not in animals which received higher doses of drug product.

[0254] The increase in anti-AAV5 IgM levels, observed on day 8 post-dose, did not coincide with changes in cytokine levels negating a drug product induced inflammatory response. Complement activation following AAV administration in NHPs has been described in 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 liver toxicity or overt transaminitis, which was not observed in our study. The liver function data in combination with the absence of a cytokine response, make drug product induced complement activation highly unlikely. This is further supported by clinical experience with AMT-061 for hemophilia

[0255] B, using an AAV5 capsid similar to the current drug product. Patients in the clinical studies with AMT- 061 did not show any TMA-associated adverse events.

[0256] In conclusion, the toxicity studies conducted with the drug product did not reveal any safety concerns associated with administration of the vector or related to high expression of the GLA transgene product.

[0257] The NOAEL was set at the highest dose tested in the NHPs, 7.3x1014gc / kg, which is 2.4-fold above the intended human high dose of the drug product and 12-fold above the intended starting (low) dose in a first clinical trial. Integration studies of vector DNA in liver genomic DNA conducted with the drug product indicated a low risk for tumorigenicity. In conclusion, a beneficial toxicity profile was obtained for the drug product, even at very high doses.

Claims

Claims1 . An isotonic formulation comprising: a buffer; a recombinant adeno-associated viral vector comprising a transgene encoding a therapeutic protein; and a cyclodextrin or a derivative thereof.

2. The isotonic formulation of claim 1 , wherein the formulation has an osmolality of from 250 to 330 mOsm / kg.

3. The isotonic formulation of claim 1 or 2, wherein the formulation is essentially free of visible particles.

4. The isotonic formulation of any one of the preceding claims, wherein the recombinant adeno- associated viral vector with a transgene encoding a therapeutic protein encodes an enzyme, preferably an enzyme associated with a lysosomal storage disorder.

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

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

7. The isotonic formulation of any one of the preceding claims, wherein the cyclodextrin is an unsubstituted or substituted p-cyclodextrin.

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

9. The isotonic formulation of any one of the preceding claims, wherein it has a pH value of from 6.5 to 8.

10. The isotonic formulation of any one of the preceding claims, further comprising a pharmaceutically acceptable salt at a concentration of at least 50 mM, wherein the salt is preferably NaCI, KCI, CaCh, MgCh, or combinations thereof.

11. The isotonic formulation of any one of the preceding claims, wherein the buffer is a Tris buffer.

12. The isotonic formulation of any one of the preceding claims, comprising about 15-25 mM Tris; a recombinant adeno-associated viral vector comprising an AAV5 serotype; about 110-140 mM NaCI; about 1.5-2.5% (w / v) hydroxypropyl-beta-cyclodextrin; wherein the formulation has a pH of about 7 to 8.

13. The isotonic formulation of any one of the preceding claims, wherein the recombinant adeno- associated viral vector comprises a 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 of any one of the preceding claims for use in a medicament.

15. The isotonic formulation for use according to claim 14, for treating a disorder associated with a defective or absent protein, preferably an enzyme, more preferably wherein the disorder is a lysosomal storage disorder, more preferably wherein the disorder is Fabry disease.

16. The isotonic formulation for use according to claim 14 or claim 15, wherein the formulation is for administration of a viral vector transducible to the liver.

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