Pharmaceutical compositions containing blood brain barrier penetrant AAV capsids

Abstract

The present invention provides compositions comprising a blood brain penetrant recombinant adeno-associated virus and one or more pharmaceutically acceptable excipients. The compositions have improved stability and shelf life as compared to other AAV compositions.

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING BLOOD BRAIN BARRIER PENETRANT AAV CAPSIDSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 678,478 filed on August 1, 2024 and entitled “Pharmaceutical Compositions Containing Blood Brain Barrier Penetrant AAV Capsids” the entire contents of each of which are incorporated by reference herein.SEQUENCE LISTING

[0002] A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via Patent Center encoded as XML. The electronic document, created on July 25, 2025, is entitled “P-0284-WOl-91355-12516.xml”, and is 4 KB bytes in size.FIELD

[0003] This application relates to pharmaceutical compositions of blood brain barrier penetrant AAV capsids.BACKGROUND OF THE INVENTION

[0004] The clinical translation of genomic medicines to treat disorders of the central nervous system (CNS) has been limited by inefficient gene delivery. AAV capsids that cross the blood brain barrier (BBB) in rodents exhibit widespread CNS transduction and efficacy; however, capsids that cross the BBB in non-human primates have been challenging to engineer.

[0005] A blood brain penetrant AAV capsid, such as STAC -BBB capsid, is engineered to enhance the delivery of AAV9 to neurons by crossing the blood-brain barrier via intravenous injection. This enhancement is achieved through the insertion of a small peptide in the STAC -BBB capsid sequence, displayed 60 times on each viral protein monomer compared to AAV9. These inserted peptides can affect the structure and conformational stability of the capsid and its colloidal stability through intermolecular interactions due to a change in the isoelectric point (pl).

[0006] Previously, the AAV9 platform formulation used an artificial cerebrospinal fluid (aCSF)-based formulation due to specific clinical recommendations for intrathecal administration. The aCSF buffer, being phosphate-based, creates a pH gradient during freezethaw cycles resulting in low stability and shelf life.

[0007] Thus, there is a need for developing improved formulations for the STAC-BBB capsid with improved stability and shelf life so that their therapeutic potential in gene therapy, particularly for intravenous delivery, can be fully implemented.SUMMARY OF THE INVENTION

[0008] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a recombinant blood-brain penetrant recombinant adeno-associated virus (rAAV) vector, sodium chloride (NaCl), potassium chloride (KC1), histidine, polyol, magnesium chloride (MgC12), and a poloxamer, optionally wherein the composition comprises about 1% w / v to about 6% w / v sucrose, optionally wherein the composition has a pH of about 6.0 to about 7.5, optionally wherein the composition comprises about 0.001% w / v to about 1% w / v poloxamer.

[0009] In certain embodiments, the pharmaceutical composition of the present disclosure has a pH about 6.3 to about 7.0. In certain embodiments, the pharmaceutical composition of the present disclosure has a pH about 6.5.

[0010] In certain embodiments, the pharmaceutical composition of the present disclosure comprises a polyol which is sucrose. In certain embodiments, the pharmaceutical composition of the present disclosure comprises a poloxamer which is poloxamer 188.

[0011] In certain embodiments, the pharmaceutical composition of the present disclosure contains about 1.0 mM to about 10.0 mM magnesium chloride. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 1.0 mM to about 3.0 mM magnesium chloride. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 1.5 mM to about 2.5 mM magnesium chloride. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 2.2 mM magnesium chloride.

[0012] In certain embodiments, the pharmaceutical composition of the present disclosure contains about 100 mM to about 300 mM. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 125 mM to about 175 mM sodiumchloride. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 150 mM sodium chloride.

[0013] In certain embodiments, the pharmaceutical composition of the present disclosure contains about 2.5 to about 3.5 mM, optionally about 3.0 mM, potassium chloride.

[0014] In certain embodiments, the pharmaceutical composition of the present disclosure contains about 1.0 to about 20.0 mM. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 1.0 to about 5.0 mM. In certain embodiments, the pharmaceutical composition of the present disclosure contains 3.0 mM, potassium chloride.

[0015] In certain embodiments, the pharmaceutical composition of the present disclosure contains about 5 mM to about 25 mM histidine. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 5 mM to about 15 mM histidine. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 10 mM, histidine.

[0016] In certain embodiments, the pharmaceutical composition of the present disclosure contains about 2% to about 4% (w / v) sucrose. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 3% (w / v) sucrose.

[0017] In certain embodiments, the pharmaceutical composition of the present disclosure contains about 0.001% to about 1.0% (w / v) poloxamer 188. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 0.005% to about 0.5% (w / v) poloxamer 188. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 0.01% to about 0.1% (w / v) poloxamer 188. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 0.03% to about 0.07% (w / v) poloxamer 188. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 0.05% poloxamer 188.

[0018] In certain embodiments, the pharmaceutical composition of the present disclosure contains an rAAV which comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the pharmaceutical composition of the present disclosure contains an rAAV which comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0019] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a recombinant blood-brain penetrant recombinant adeno-associated virus (rAAV) vector, about 150 mM sodium chloride, about 3.0 mM potassium chloride, about 2.2 mM magnesium chloride, about 3% (w / v) sucrose, and about 0.05% (w / v)pol oxamer 188, optionally wherein the composition has a pH of about 6.0 to about 7.0, optionally wherein the composition has a pH of about 6.5.

[0020] In certain embodiments, the pharmaceutical composition of the present disclosure contains an rAAV comprising a genome comprising an expression cassette encoding a therapeutic protein. In some embodiments, the therapeutic protein is a nucleic acid binding regulatory protein that binds to and regulates a gene target. In some embodiments, the gene target is a human microtubule associated protein tau polypeptide. In some embodiments, the gene target is a human major prion protein polypeptide.

[0021] In certain embodiments, the pharmaceutical composition of the present disclosure contains the blood-brain penetrant rAAV at about 1.0E+13 to about 1.0E+14 vector genomes (vg) per mL, optionally about 1.0E+13 to about 5.0E+13 vg per mL. In certain embodiments, the pharmaceutical composition of the present disclosure contains about 2.0E+13 vg per mL.

[0022] In certain embodiments, the present disclosure provides a vial comprising 2-10 mL, optionally 10 mL, of the pharmaceutical composition of present disclosure. In certain embodiments, the vial is made of cyclo-olefin copolymer. In certain embodiments, the vial has an in-place thermoplastic elastomer stopper.

[0023] In certain embodiments, the present disclosure provides a method of treating a patient in need of a therapeutic protein, comprising administering to the patient the pharmaceutical composition of the present disclosure.

[0024] In certain embodiments, the present disclosure provides a use of the pharmaceutical composition of the present disclosure for the manufacture of a medicament for treating a human subject. In certain embodiments, the present disclosure provides a use of the pharmaceutical composition of the present disclosure for treating a human subject.

[0025] Other features, objects, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0026] FIG. 1 shows an exemplary study of the stability of various compositions of a drug product by ITR ddPCR.

[0027] FIG. 2 shows an exemplary study of the vg titer loss of various compositions of a drug product by ITR ddPCR.

[0028] FIG. 3 shows an exemplary study of %HMW of various compositions of a drug product by SEC-MALS.

[0029] FIG. 4 shows an exemplary study of monomer mass of various compositions of a drug product by SEC-MALS.

[0030] FIG. 5 shows an exemplary study of % monomer mass loss of various compositions of a drug product by SEC-MALS.

[0031] FIG. 6 shows an exemplary study of % full capsid of various compositions of a drug product by SEC-MALS.

[0032] FIG. 7 shows an exemplary study of free DNA release of various compositions of a drug product.

[0033] FIG. 8 shows an exemplary study of subvisible particles of various compositions of a drug product.

[0034] FIG. 9 shows an exemplary study of infectivity of various compositions of a drug product.

[0035] FIG. 10 shows an exemplary study of vg titer of various compositions of a drug product by ITR ddPCR.

[0036] FIG. 11 shows an exemplary study of % HMW of various compositions of a drug product by SEC-MALS.

[0037] FIG. 12 shows an exemplary study of monomer mass loss of various compositions of a drug product by SEC-MALS.

[0038] FIG. 13 shows an exemplary study of % full capsid of various compositions of a drug product.

[0039] FIG. 14 shows an exemplary study of free DNA release of various compositions of a drug product.DETAILED DESCRIPTION OF THE INVENTION

[0040] The present disclosure provides pharmaceutical compositions comprising bloodbrain penetrant AAV vectors and one or more pharmaceutically acceptable excipients. The present AAV vector compositions may comprise blood-brain penetrant rAAV whose genome carries an expression cassette for a protein of interest (e.g., a therapeutic protein). The present inventors have unexpectedly discovered that blood-brain penetrant rAAV vector formulationshave improved stability and shelf life as compared to prior compositions. The inventors have discovered that the present blood-brain penetrant AAV vector compositions have improved stability (as determined by product quality attributes under freeze / thaw cycles and accelerated stability conditions).I. Preparation of Recombinant AAV

[0041] The viral preparations described herein may be obtained by any known production systems, such as mammalian cell AAV production systems (e.g., those based on 293T or HEK293 cells) and insect cell AAV production systems (e.g., those based on sf9 insect cells and / or those using baculoviral helper vectors). The viral preparations may be purified from the cell cultures by using well known techniques such as discontinuous cesium chloride density gradients (see, e.g., Grieger, Mol Ther Methods Clin Dev. (2016) 3: 16002).

[0042] The present compositions may comprise blood brain penetrant AAV of any or a combination of a variety of AAV serotypes, such as AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV8.2, AAV9, AAVrhlO, AAV10, and AAV11, as well as variants, hybrids, chimera or pseudo-types thereof. By “pseudo-typed” or “cross-packaged” rAAV is meant a recombinant AAV whose capsid is replaced with the capsid of another AAV serotype, to, for example, alter transduction efficacy or tropism profiles of the virus (see, e.g., Balaji et al., J SurgRes. (2013) 184(1):691 -8). By “chimeric” or “hybrid” rAAV is meant a recombinant AAV whose capsid is assembled from capsid proteins derived from different serotypes and / or whose capsid proteins are chimeric proteins with sequences derived from different serotypes (e.g., serotypes 1 and 2; see, e.g., Hauck et al., Mol Ther. (2003) 7(3):419-25). For example, the present compositions may comprise recombinant AAV whose genome such as the ITRs is derived from one serotype such as AAV2 while the capsids are derived from another serotype; e.g., AAV2 / 8, AAV2 / 5, AAV2 / 6, AAV2 / 9, or AAV2 / 6 / 9. See, e.g., U.S. Pats. 7,198,951 and 9,585,971.

[0043] In certain embodiments, the blood brain penetrant AAV capsid protein comprises serotype AAV9. In certain embodiments, the blood brain penetrant AAV capsid protein comprising serotype AAV9 includes an amino acid sequence comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 1 (YVNIMDDMD). In certain embodiments, the blood-brain penetrant AAV capsid protein of serotype AAV9 comprises a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 2. In certain embodiments, the blood-brain penetrant AAV capsid protein of serotype AAV9 comprises a sequence set forth in SEQ ID NO: 2.

[0044] SEQ ID NO: 1 : YVNIMDDMD

[0045] SEQ ID NO: 2:MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVI<TI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAYVNIMDDMDQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAI<IPHTDGNFHPSPLMGGFGMI<HPPPQILII<NTPVPADPPTAFNI<D KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYS EPRPIGTRYLTRNLII. Formulation of Recombinant AAV

[0046] Once purified, the AAV preparations can be formulated as described herein, for example, by buffer exchange through tangential flow filtration, normal flow filtration using stir-cells, gel filtration, dialysis, column chromatography, and / or desalting columns, to arrive at a composition comprising the desired ingredients. By way of illustration, the purified viral preparation may be concentrated first by ultrafiltration (UF) and then diafiltrated (DF) with 10 times or more equivalent volumes of the desired aqueous formulation solution.

[0047] The formulation solution may comprise tonicity agents, stabilizing agents, surfactants, and buffering agents. Buffering agents may include, for example, acetate, succinate (e.g., disodium succinate hexahydrate), succinic acid, gluconate, citrate, histidine, acetic acid, phosphate, phosphoric acid, ascorbate, ascorbic acid, tartaric acid, malate, maleic acid, glycine, lactate, lactic acid, bicarbonate, carbonic acid, sodium benzoate, benzoic acid, edetate, imidazole, tris, and mixtures thereof. In some embodiments, the formulation solution contains sodium chloride at, e.g., about 125-200 mM (e.g., about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 160 mM, about 165 mM, about 170 mM , about 175 mM , about 180 mM , about 185 mM , about 190 mM , about 195 mM , or about 200 m) and / or potassium chloride about 2.5-3.5 mM (e.g.,about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM, about 3.1 mM, about 3.2 mM, about 3.3 mM, about 3.4 mM, or about 3.5 mM).

[0048] The formulation solution may be histidine-buffered. In some embodiments, the total phosphate ion concentration in the formulation solution is about 5.0-15.0 mM (e.g., about 5.0 about 5.5 mM, about 6.0 mM, about 6.5 mM, about 7.0 mM, about 7.5 mM, about 8.0 mM, about 8.5 mM, about 9.0 mM, about 10 mM, about 10.5 mM, about 11.0 mM, about 11.5 mM, about 12.0 mM, about 12.5 mM, about 13.0 mM, about 13.5 mM, about 14.0 mM, about 14.5 mM, or about 15.0 mM). The formulation may have a pH of about 6.0-7.0 (e.g., about 6.3-6.7, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0).

[0049] The formulation solution may contain magnesium (e.g., magnesium chloride (MgCh)). In some embodiments, the formulation solution contains about 1.5 to 2.5 mM of magnesium chloride (e.g., about 2.1-2.3 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, or about 2.5 mM).

[0050] The formulation solution may contain a polyol such as mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, ethylene glycol, propylene glycol, polyethylene glycol, inositol, fructose, glucose, mannose, sucrose, sorbose, xylose, lactose, maltose, dextran, pullulan, dextrin, cyclodextrins, soluble starch, hydroxyethyl starch, water-soluble glucans, or mixtures thereof. In some embodiments, the formulation solution contains about 1% to 6% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, or about 6%) (w / v) sucrose.

[0051] The formulation solution may contain a nonionic or ionic hydrophilic surfactant. Example of surfactants are a polysorbate, poloxamer, triton, sodium dodecyl sulfate, sodium laurel sulfate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl- sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl- taurate, disodium methyl oleyl- taurate, dihydroxypropyl PEG-5 linoleammonium chloride, polyethylene glycol, polypropylene glycol, sorbitan monoesters (e.g., Spans), acid esters of glycerol, and mixtures thereof. In some embodiments, the surfactant can be polysorbate (PS) 20, PS-21, PS-40, PS-60, PS-61, PS-65, PS-80, PS-81, PS-85, PEG-3350, poloxamer 188,and mixtures thereof. In some embodiments, the formulation solution contains about 0.01% to 0.1% (e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1%) (w / v) poloxamer 188.

[0052] In some embodiments, the blood-brain penetrant rAAV comprises a genome comprising an expression cassette for a therapeutic protein. In some embodiments, the therapeutic protein is a human microtubule associated protein tau polypeptide. In some embodiments, the therapeutic protein is a human major prion protein polypeptide human major prion protein polypeptide.

[0053] The pharmaceutical compositions may further comprise one or more preservatives such as ascorbic acid (vitamin C), sulfites, sorbates, benzoates, phenol, m-cresol, benzyl alcohol, benzalkonium chloride, phenoxyethanol, and / or parabens (e.g., methyl paraben). In some embodiments, the pharmaceutical compositions do not contain any added preservatives.

[0054] The pharmaceutical compositions may comprise also other reagents that enhance the effectiveness of the pharmaceutical composition. The pharmaceutical composition may contain delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, and vesicles.III. Exemplary Recombinant AAV

[0055] In exemplary embodiments, the present disclosure provides improved pharmaceutical compositions comprising blood-brain penetrant rAAV vector. In the some embodiments, the rAAV vector comprises an AAV9 serotype comprising the sequence set forth in SEQ ID NO: 1. In the some embodiments, the rAAV vector comprises an AAV9 serotype comprising the sequence set forth in SEQ ID NO: 2.

[0056] In the exemplary Study 1 described below, the blood-brain penetrant rAAV vector comprises a genome comprising an expression cassette for a therapeutic protein, where the therapeutic protein is a nucleic acid binding regulatory protein that binds to and regulates a gene target encoding a human microtubule associated protein tau polypeptide. In the exemplary Study 2 described below, the blood-brain penetrant rAAV vector comprises a genome comprising an expression cassette for a therapeutic protein, where the therapeutic protein is a nucleic acid binding regulatory protein that binds to and regulates the gene target encoding a human major prion protein polypeptide.

[0057] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.EXAMPLES

[0058] The following Examples describe a study in which the inventors discovered STAC- BBB formulations that improved upon the intrathecal formulation originally developed for the wildtype AAV9.

[0059] A variety of assays were employed for the biophysical characterization of the capsid, including pH, osmolality, size exclusion chromatography with multi-angle static light scattering (SEC-MALS), dynamic light scattering (DLS), free DNA, % full capsids, and microflow imaging (MFI). The inverted terminal repeats (ITR) digital droplet polymerase chain reaction (ddPCR) assay was used to monitor the viral genome (vg) titer. A summary and description of these assays are provided in Table 1.Table 1: Analytical tools used for formulation screening

[0060] In this study, size-exclusion chromatography (SEC), coupled with multi-angle light scattering (MALS) and refractive index (RI) detectors alongside the conventional UV and fluorescence (FLD) detectors, was employed to collect supplementary insights. These include % full capsid, capsid titer, vector genome (vg) titer, and DNA integrity through molecular weight (Mw) measurements, as well as particle size.

[0061] This report presents %HMW as a common attribute to evaluate the purity and stability of the molecule. Additionally, we present monomer mass and % monomer mass loss. The monomer mass is calculated using UV and RI signals. Evaluating monomer mass and monomer mass loss is important because the SEC column, due to inherent design limitations, cannot pass larger aggregates, leading to an inaccurate representation of stability. Thecombination of %HMW, % monomer mass loss, and % full capsids of monomer provides a more holistic understanding of stability trends for an AAV.

[0062] In the studies described below, the drug product (DP) was stored in 6.3 mL fill in 10 mL CZ vials, with 20 mm West 4432-50 stopper and 20 mm flip top aluminum cap seal. The intended storage condition is frozen at < -65°C. While the intended presentation is 10 mL CZ vial with 20 mm 4432-50 stopper, due to material limitations in early screening 2 mL CZ vials and 13 mm 4432-50 stopper were used.

[0063] Materials for Study 1 were provided from small-scale production using a shake flask in an adherent system for upstream and CsCl gradient for downstream production. The gene-of-interest (GOI) / cargo of the capsid in Study 1 was the Tau zinc-finger, comparable in size to 2,700bp or 833kDa. Materials for Study 2 were provided from a 40L bioreactor in a suspension for upstream and column chromatography for downstream production. The gene- of-interest (GOI) / cargo of the capsid in Study 2 was the Tau zinc-finger, comparable in size to 2,700bp or 833kDa. The study designs are described in Table 2.Table 2: List of the material sources and descriptions used in formulation screeningDesign of Study 1

[0064] The design of Study 1 is described in Table 3. The main comparison was between histidine formulations and phosphate formulations. The initial material was received in DPBS with no sucrose, which was used as a control to understand the impact of sucrose on STAC- BBB stability. An arm of the study included histidine buffer at pH 7.0 with 6% sucrose to evaluate the impact of higher sucrose concentrations. An additional arm included 80mM arginine to test the hypothesis that charged amino acids could enhance colloidal stability by screening surface charges.Table 3: Study Design 1

[0065] In this study, different stability temperatures, i.e., real time storage (< -65°C), accelerated (5°C), and stress conditions (25°C and 40°C) were included for a comprehensive perspective on molecule stability (Table 4). The material for study 1 was received in Dulbecco’s phosphate-buffered saline (DPBS) buffer with no sucrose. Buffer exchange was performed using the Unagi system from Unchained Labs with 30kDa filters to the corresponding formulated buffer presented in Table 3. Samples were then filtered with 0.22 um syringe filter, filled into 2 mL CZ vials at 0.75 mL fill volume, and set under stability conditions.Table 4: Conditions tested for Study 1

[0066] Figure 1 shows ddPCR data for all conditions compared to -80°C samples as a control. Phosphate pH 8 and phosphate with no sucrose showed small decreases in vg / mL compared to control, while other formulations had no significant impact under the studied conditions. Histidine formulations were comparable to phosphate, with no measurable impact at 40°C for 1 week or after 5 freeze-thaw cycles. Storage at 5°C for up to 3 months and at 25°C for up to 1 month had no measurable impact on vg titer for all formulations.Additionally, the reference standard tested across all conditions for STAC-BBB in this reportis separately presented in Supplementary Figure 18 which visually presents the expected assay variability.

[0067] For comparative purposes, the drop of vg titer compared to the control is plotted in Figure 2. The no-sucrose phosphate formulation had much higher vg loss compared to all other formulations. This indicates the importance of presence of at least 1% w / v Sucrose in the formulation. Phosphate pH 8.0 had the highest vg loss at 40C for 1 week among the rest of the formulations.

[0068] SEC-MALS Method: Parameters such as %HMW, monomer mass, and % monomer mass loss were evaluated using SEC-MALS. Figure 3 shows the comparison between %HMW for each condition relative to -80°C controls. At 40°C for 1 week, all formulations had similar %HMW. Only phosphate pH 8 showed increased %HMW after 5 freeze-thaw cycles. Storage at 5°C for 3 months and 25°C for 1 month showed no meaningful increase of %HMW and comparable stability to AAV9 data.

[0069] Figure 4 shows the calculated monomer mass for all formulations exposed to 40°C for 1 week and 5 freeze-thaw cycles compared to the -80°C control samples. To enable a more accurate comparison between formulations and conditions, the %Monomer mass loss was calculated and plotted separately in Figure 5. Histidine formulations showed lower monomer mass loss compared to phosphate, with phosphate pH 8.0 having the highest mass loss at 40°C for 1 week. Storage at 5°C up to 3 months had no significant impact on monomer loss, while 25°C storage resulted in < 4% monomer loss. Phosphate with no sucrose showed the worst stability, indicating the importance of at least 1% w / v sucrose in the formulation.

[0070] % Full Capsids: Figure 6 shows the % full capsids in the monomer peak. All formulations showed a drop in % full capsids after 40°C for 1 week storage, expected due to heat impact on DNA ejection. Histidine formulations were comparable to phosphate. No impact on % full capsids was observed after 5 freeze-thaw cycles, 5°C for 3 months, or 25°C for 1 month storage, comparable to AAV9 data.

[0071] Free DNA Assay: Figure 7 shows free DNA release after exposure to tested conditions. Storage at 40°C-l week had the highest impact on DNA release, with phosphate formulations showing higher free DNA release compared to histidine. The higher free DNA levels at 40°C-l week is expected due to impact of heat on DNA ejection from the capsid. Addition of arginine to histidine pH 7.0 had an adverse impact on stability and DNA ejection. Phosphate with no sucrose had the highest free DNA release after 5 freeze-thaw cycles,suggesting at least 1% w / v sucrose is essential for protection against freeze-thaw stress. Similar trends were observed for storage at 5°C-3 months and 25°C-1 month.

[0072] Subvisible Particles: Subvisible particles (SVP) were evaluated for a limited sample panel (Figure 8). The main pathway of particle formation for a frozen drug product is freezethaw stress and exposure to solid-liquid interfaces during freezing and thawing. Therefore, the goal was mainly to evaluate the SVP post 5 times freeze-thaw, no-sucrose control had much higher subvisible particles compared to all other formulations. All formulations with 1% w / v sucrose showed the same level of SVP, indicating the importance of adding at least 1% w / v sucrose to the final formulation.

[0073] These exemplary studies showed that histidine formulations at pH 6.0 to 7.0 exhibited similar or better stability compared to phosphate buffers. Phosphate buffer at pH 8.0 showed the poorest stability performance with the CQAs analyzed in this study.

[0074] These exemplary studies also showed that addition of a cryoprotectant, sucrose, from 0% to 1% w / v in phosphate buffer significantly increased stability, particularly after freeze-thaw cycles. Similarly, increasing sucrose concentration from 1% to 6% w / v in histidine buffer also enhanced stability, as indicated by reduced monomer mass loss.Design of Study 2

[0075] Materials for Study 2 were allocated from a 40L bioreactor produced using the suspension HEK293 platform and purified with column chromatography. Study design and formulations are described in Table 5. The conditions that have been evaluated are presented in Table 6.Table 5: Study 2 design and formulationsTable 6: Conditions tested for Study 2

[0076] Similar to study 1, samples were buffer exchanged using Unagi system from Unchained Labs with 30kDa filters to the corresponding formulated buffer presented in Table 5. Post-buffer exchange, samples were tested for pH and osmolality. Samples were then filtered with 0.22 um syringe filter, filled into 2mL CZ vials at 0.5mL fill volume, and set under stability conditions.

[0077] TCID50 Assay: Figure 9 presents tissue culture infectious dose (TCID50) data for two formulations: Histidine pH 6.0 with 6% w / v Sucrose and 0.05% w / v P188, and Histidine pH 6.8 with 6% w / v Sucrose and 0.05% w / v P188. The data indicate that storage up to 3 months at 5°C does not impact the infectivity of the STAC-BBB, similar to AAV9. Furthermore, freeze-thaw cycles up to 5 times did not affect the infectivity of STAC-BBB. The two histidine pH conditions yielded similar results, with pH 6.8 showing higher infectivity at 25°C for 1 month but worse infectivity at 40°C for 1 week, while pH 6.0 exhibited higher infectivity at 40°C for 1 week but worse at 25°C for 1 month.

[0078] Vector Genome (ITR ddPCR Assay): Figure 10 presents the ITR ddPCR readout of all conditions compared to the -80°C control sample. No impact on vg titer was observed, except for the formulation with 1% w / v sucrose after five freeze-thaw cycles. Therefore, a higher level of sucrose i.e. 3% to 6% w / v sucrose is recommended for protecting the STAC- BBB against freeze-thaw conditions.

[0079] %HMW: Figure 11 represents the %HMW for all conditions relative to the -80C controls. Data shows no significant changes in %HMW across all formulations and conditions tested. Lower %HMW levels in this study compared to study 1 indicate improved process and purification of the source material.

[0080] Monomer Mass Loss: Figure 12 represents the monomer mass loss % relative to the -80C-control samples. Data shows the lowest monomer mass loss for 6% sucrose-0.05% P188 formulations at both pH 6.0 and pH 6.8. Storage at 5°C-3 months had no significantimpact, while 25°C storage showed the highest mass loss for 1% w / v sucrose at pH 6.0 formulation.

[0081] % Full Capsids: Figure 13 represents the %Full capsid in monomer peak theoretically calculated using SEC-MALS. Data shows a drop in % full capsids after 40°C for 1 week storage, expected due to heat impact on DNA ejection. Lower impact was observed for 6% sucrose-0.05% Pl 88 formulations. No impact was observed after 5 freeze-thaw cycles, 5°C for 3 months, or 25°C for 1 month storage, comparable to AAV9.

[0082] Free DNA Release: Figure 14 shows the Free DNA release after exposure to the tested conditions compared to the -80°C control sample. A trend was observed for both pH 6.0 and pH 6.8, where increasing the sucrose concentration reduced Free DNA release. The most optimal formulation, with the least Free DNA release, was the one containing 6% w / v sucrose and 0.05% w / v P188. Formulations with only 1% sucrose had higher Free DNA release . This data indicates that increasing the concentrations of sucrose and Pl 88 enhances the stability of the STAC-BBB capsid. Additionally, the overall values, including those of the -80°C control, were lower than previously measured for research-grade material. This suggests much lower impurities in the 40L bioreactor purified with column chromatography compared to the shake flask purified with a density gradient.

[0083] These exemplary studies also showed overall evaluation of infectivity by TCID50, vg titer by ddPCR, %HMW, %Full capsids, %Monomer mass loss by SEC-MALS and Free DNA release showed that formulations with 3% to 6% w / v sucrose had enhanced stability compared to those with 1% sucrose.

[0084] These exemplary studies also showed that increasing the Pl 88 concentration to 0.05% w / v further improved stability, particularly against freeze-thaw stress.

[0085] The exemplary studies also showed that histidine formulations at pH 6.0 and 6.8 containing 3% to 6% w / v sucrose were comparable in performance. These exemplary studies show showed that a pH of 6.5 can enable maximal buffering capacity with histidine buffer.

Claims

CLAIMS1. A pharmaceutical composition comprising a recombinant blood-brain penetrant recombinant adeno-associated virus (rAAV) vector; sodium chloride (NaCl); potassium chloride (KC1); histidine; a polyol; magnesium chloride (MgCh); and a poloxamer, optionally wherein the composition comprises about 1% w / v to about 6% w / v sucrose, optionally wherein the composition has a pH of about 6.0 to about 7.5, optionally wherein the composition comprises about 0.001% w / v to about 1% w / v poloxamer.

2. The composition of claim 1, wherein the pH is about 6.3 to about 7.0, optionally wherein the pH is about 6.5.

3. The composition of claims 1 or 2, wherein the polyol is sucrose.

4. The composition of claims 1-3, wherein the poloxamer is poloxamer 188.

5. The composition of any one of claims 1-4, wherein the composition contains about 1.0 mM to about 10.0 mM magnesium chloride, optionally wherein the composition contains about 1.0 mM to about 3.0 mM magnesium chloride, optionally wherein the composition contains about 1.5 mM to about 2.5 mM magnesium chloride.

6. The composition of claims 1-5, wherein the composition contains about 2.2 mM magnesium chloride.

7. The composition of any one of claims 1-6, wherein the composition contains about 100 mM to about 300 mM sodium chloride, optionally wherein the composition contains about 125 mM to about 175 mM sodium chloride, optionally about 150 mM sodium chloride.

8. The composition of any one of claims 1-7, wherein the composition contains about 1.0 to about 20.0 mM potassium chloride, optionally wherein the composition contains about 1.0 to about 5.0 mM potassium chloride, optionally wherein the composition contains about 3.0 mM potassium chloride.

9. The composition of any one of claims 1-8, wherein the composition contains about 5 mM to about 25 mM histidine, optionally wherein the composition contains about 5 mM to about 15 mM histidine.

10. The composition of any one of claims 1-9, wherein the composition contains about 10 mM histidine.

11. The composition of any one of claims 1-10, wherein the composition contains about 2% to about 4% (w / v) sucrose.

12. The composition of any one of claims 1-11, wherein the composition contains about 3% (w / v) sucrose.

13. The composition of any one of claims 1-12, wherein the composition contains about 0.005% to about 0.5% (w / v) poloxamer 188, optionally about 0.01% to about 0.1% (w / v) poloxamer 188.

14. The composition of any one of claims 1-13, wherein the composition contains about 0.05% poloxamer 188.

15. A pharmaceutical composition comprising a recombinant blood-brain penetrant recombinant adeno-associated virus (rAAV) vector, about 150 mM sodium chloride, about 3.0 mM potassium chloride, about 2.2 mM magnesium chloride, about 3% (w / v) sucrose, and about 0.05% (w / v) poloxamer 188, optionally wherein the composition has a pH of about 6.0 to about 7.0, optionally wherein the composition has a pH of about 6.5.

16. The composition of any one of the claims 1-15, wherein the rAAV comprises the amino acid sequence set forth in SEQ ID NO: 1.

17. The composition of any one of the claims 1-16, wherein the rAAV comprises the amino acid sequence set forth in SEQ ID NO: 2.

18. The composition of any one of claims 1-17, wherein the rAAV comprises a genome comprising an expression cassette encoding a therapeutic protein.

19. The composition of claim 18, wherein the therapeutic protein is a nucleic acid binding regulatory protein that binds to and regulates a gene target.

20. The composition of claim 19, wherein the gene target is a human microtubule associated protein tau polypeptide.

21. The composition of claim 19, wherein the gene target is a human major prion protein polypeptide.

22. The composition of any one of claims 1-21, the composition contains the blood-brain penetrant rAAV at about 1.0E+13 to about 1.0E+14 vector genomes (vg) per mL, optionally about 1.0E+13 to about 5.0E+13 vg per mL.

23. The composition of claim 22, wherein the composition contains about 2.0E+13 vg per mL.

24. A vial comprising 2-10 mL, optionally 10 mL, of the composition of any one of claims 1-23.

25. The vial of claim 24, wherein the vial is made of cyclo-olefin copolymer.

26. The vial of claim 24 or 25, wherein the vial has an in-place thermoplastic elastomer stopper.

27. A method of treating a patient in need of a therapeutic protein, comprising administering to the patient the composition of any one of claims 1-23.

28. Use of the pharmaceutical composition of any one of claims 1-23 for the manufacture of a medicament for treating a human subject in the method of claim 27.

29. The pharmaceutical composition of any one of claims 1-23 for use in treating a human subject in the method of claim 27.

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