AAV preparations

Abstract

The present disclosure provides a composition comprising a recombinant adeno-associated virus (AAV) vector containing a heterologous nucleic acid, sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, and a non-ionic surfactant at a pH of 7.2 to 7.4. The present disclosure further provides a method of treating a neurological disorder in a subject, the method comprising administering the composition directly to the central nervous system of a subject in need thereof.

Description

[Technical Field]

[0001] The present disclosure relates to compositions comprising recombinant adeno-associated vectors.

[0002] Incorporation by Reference of Electronically Submitted Materials The computer-readable nucleotide / amino acid sequence listing identified by the file name "55330A_SeqListing.XML", which was filed concurrently herewith and created on December 27, 2023, and is 175,534 bytes, is incorporated by reference in its entirety. [Background technology]

[0003] Adeno-associated virus (AAV) is a small, replication-deficient, non-enveloped animal virus belonging to the Parvoviridae family. The AAV genome consists of a linear, single-stranded DNA approximately 4.7 kb in length. The genome consists of two open reading frames (ORFs) flanked by inverted terminal repeat (ITR) sequences approximately 145 bp in length. The ITRs consist of a nucleotide sequence at the 5' end (5' ITR) and a nucleotide sequence located at the 3' end (3' ITR) that contains a palindromic sequence. The ITRs function in cis by folding to form a T-shaped hairpin structure through complementary base pairing, which serves as a primer during the initiation of DNA replication for second-strand synthesis. The two open reading frames encode the rep and cap genes, which are involved in virion replication and packaging.

[0004] AAV infects both dividing and quiescent cells. Due to the specificity, efficiency, and safety associated with AAV, AAV vectors have emerged as the expression vector of choice for gene therapy applications. AAV vector production conditions and formulations must be carefully selected to ensure vector stability, purity, and potency while maintaining patient safety. Delivery to the central nervous system presents unique challenges. The brain and spinal cord are compartmentalized organs, and compositions must possess appropriate physicochemical properties adapted for administration to these regions, in addition to avoiding inflammatory and adverse immunogenic responses. There is a need in the art for optimized, stable compositions containing AAV vectors that are suitable for administration to the central nervous system. Summary of the Invention

[0005] The present disclosure provides a composition comprising a recombinant adeno-associated virus (AAV) vector containing a heterologous nucleic acid, sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, and a non-ionic surfactant at a pH of 7.2 to 7.4.

[0006] The present disclosure also provides a composition comprising a recombinant adeno-associated virus (AAV) vector comprising a heterologous nucleic acid, sodium chloride, potassium chloride, phosphate buffer, and a non-ionic surfactant at a pH of 7.2 to 7.4. The present disclosure further provides a composition comprising a recombinant adeno-associated virus (AAV) vector comprising a heterologous nucleic acid, sodium chloride, potassium chloride, magnesium chloride, Tris buffer, and a non-ionic surfactant at a pH of 7.2 to 7.4. The present disclosure further provides a composition comprising a recombinant adeno-associated virus (AAV) vector comprising a heterologous nucleic acid, sodium chloride, potassium chloride, Tris buffer, and a non-ionic surfactant at a pH of 7.2 to 7.4.

[0007] Optionally, the nonionic surfactant is present at a concentration in the range of about 0.001% to about 0.01% (w / v). In various embodiments, the nonionic surfactant is a poloxamer, such as Poloxamer 188. In this regard, various embodiments of the composition include Poloxamer 188 present at a concentration of about 0.005% (w / v). In various embodiments, the composition includes about 145 mM to about 150 mM sodium chloride and / or about 1.5 mM to about 4.5 mM potassium chloride and / or about 0.05 mM to about 1 mM magnesium chloride. For example, in some embodiments, the composition includes about 148 mM sodium chloride, about 3 mM potassium chloride, and about 0.8 mM magnesium chloride. In various embodiments, the phosphate buffer is present in an amount sufficient to provide about 0.5 mM to about 2 mM (e.g., about 1 mM) phosphate. Optionally, the phosphate buffer is sodium phosphate. In various embodiments, the composition comprises about 5×10 13 vg / mL ~ approx. 1×10 14 vg / mL (e.g., approximately 8 × 10 13 vg / mL). In some embodiments, the composition does not include calcium, such as calcium chloride. In some embodiments, the composition exhibits a conductivity of about 15.0 to about 17.0 mS / cm.

[0008] The present disclosure further provides a method for treating a neurological disorder in a subject, the method comprising administering a composition described herein directly to the central nervous system of a subject in need thereof. Optionally, the method comprises administering the composition to the subject via intraventricular injection. Examples of neurological conditions suitable for treatment include, but are not limited to, Dravet syndrome and epilepsy. The use of the compositions described herein for treating a subject in need thereof, and the use of the compositions described herein in preparing a medicament for treating a subject in need thereof, are contemplated. A kit is provided comprising the compositions described herein and instructions for use.

[0009] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Indeed, features of the present invention described herein, regardless of whether a combination of features is designated as an aspect or embodiment of the invention, can be recombined into additional embodiments that are also contemplated as aspects of the invention. The entire document is intended to be linked as a unified disclosure, and it should be understood that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of the specification. [Brief explanation of the drawings]

[0010] [Figure 1] Shown is the percent loss of rAAV genomic DNA (vg), as represented by loss of absorbance at 260 nm (y-axis), for samples from Table 1 at time 0, after six freeze-thaw cycles ("6FT"), after 7 or 17 days at room temperature ("RT d7" or "RT d17"), and after 3 or 7 days at 37°C ("37C d3" or "37C d7"). [Figure 2] Shown is the percent loss of rAAV capsid (cp), as represented by loss of absorbance at 280 nm (y-axis), for samples from Table 1 at time 0, after six freeze-thaw cycles ("6FT"), after 7 or 17 days at room temperature ("RT d7" or "RT d17"), and after 3 or 7 days at 37°C ("RT d7" or "RT d17"). [Figure 3] RP-HPLC results for a representative rAAV sample at t=0, after 17 days of incubation at room temperature (RT), and after 7 days of incubation at 37°C are shown. [Figure 4] Fold changes at P1 (according to Figure 3) are shown for samples from Table 1 at time point 0, after 6 freeze-thaw cycles ("6xFT"), after 7 or 17 days at room temperature ("RT d7" or "RT d17"), and after 3 or 7 days at 37°C ("37C d3" or "37C d7"). [Figure 5] Fold changes at P3 (per Figure 3) are shown for samples from Table 1 at time point 0, after 6 freeze-thaw cycles ("6xFT"), after 7 or 17 days at room temperature ("RT d7" or "RT d17"), and after 3 or 7 days at 37°C ("37C d3" or "37C d7"). [Figure 6] Fold changes at time 0, after 6 freeze-thaw cycles ("6xFT"), at 7 or 17 days at room temperature ("RT d7" or "RT d17"), and at P5 (according to Figure 3) after 3 or 7 days at 37°C ("37C d3" or "37C d7") are shown for samples from Table 1. [Figure 7] Particle size (Z average) is shown for the samples from Table 1. The bars in the graph correspond, from left to right, to Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, Sample 6, Sample 7, Sample 8, Sample 9, Sample 10, and Sample 11. [Figure 8] The polydispersity index (PDI) is shown for each sample from Table 1. The bars in the graph correspond, from left to right, to Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, Sample 6, Sample 7, Sample 8, Sample 9, Sample 10, and Sample 11. [Figure 9] Shown are vector genome titers measured by ddPCR for samples from Table 2 at time 0, after 1 month at below -70°C, after 1 month or 1 week at room temperature, after 2 weeks or 1 month at 37°C, or after 10 freeze-thaw cycles. [Figure 10] Aggregation measured by SE-HPLC for samples from Table 2 at time 0, after 1 month below -70°C, after 1 month or 1 week at room temperature, after 2 weeks or 1 month at 37°C, or after 10 freeze-thaw cycles is shown. Two bars are provided for each sample. The left bar for each sample corresponds to the A260 high molecular weight species (HMWS) (%), and the right bar for each sample corresponds to the A280 high molecular weight species (HMWS) (%). [Figure 11] Fold change in the P1 species by RP-HPLC of samples from Table 2 at time 0, after 1 month below -70°C, after 1 month at room temperature, after 2 weeks or 1 month at 37°C, or after 10 freeze-thaw cycles is shown. Two bars are provided for each condition. The left bar for each condition corresponds to Formulation 1, and the right bar for each condition corresponds to Formulation 2. [Figure 12] The fold change in the P3 species by RP-HPLC of samples from Table 2 is shown at time 0, after 1 month below -70°C, after 1 month at room temperature, after 2 weeks or 1 month at 37°C, or after 10 freeze-thaw cycles. Two bars are provided for each condition. The left bar for each condition corresponds to Formulation 1, and the right bar for each condition corresponds to Formulation 2. [Figure 13] Fold changes in the P5 species by RP-HPLC of samples from Table 2 are shown at time 0, after 1 month below -70°C, after 1 month at room temperature, after 2 weeks or 1 month at 37°C, or after 10 freeze-thaw cycles. Two bars are provided for each condition. The left bar for each condition corresponds to Formulation 1, and the right bar for each condition corresponds to Formulation 2. [Figure 14] Results of in vitro potency assays are shown for samples from Table 2 after 1 month at room temperature, 2 weeks or 1 month at 37°C, or after 10 freeze-thaw cycles, and are expressed as a percentage of potency at time 0. [Figure 15] Particle sizes are shown for samples from Table 2 at time 0, after 1 week or 1 month at 4°C, after 1 month at room temperature, after 2 weeks or 1 month at 37°C, after 1 month below -70°C, or after 10 freeze-thaw cycles. Two bars are provided for each sample. The left bar for each sample corresponds to the 15C Z-average diameter (nm), and the right bar for each sample corresponds to the 95C Z-average diameter (nm). [Figure 16]Normalized polydispersity indices are shown for samples from Table 2 at time 0, after 1 week or 1 month at 4°C, after 1 month at room temperature, after 2 weeks or 1 month at 37°C, after 1 month at below -70°C, or after 10 freeze-thaw cycles. Two bars are provided for each condition. The left bar for each condition corresponds to Formulation 1, and the right bar for each condition corresponds to Formulation 2. [Figure 17] External fluorescence at 15°C after 1 week or 1 month at 4°C, 1 month at room temperature, 2 weeks or 1 month at 37°C, 1 month below -70°C, or after 10 freeze-thaw cycles is shown for samples from Table 2. Two bars are provided for each condition. The left bar for each condition corresponds to Formulation 1, and the right bar for each condition corresponds to Formulation 2. [Figure 18] For samples from Table 2, Tonset / Tm1 (°C) is shown at time 0, after 1 week or 1 month at 4°C, after 1 month at room temperature, after 2 weeks or 1 month at 37°C, after 1 month at below -70°C, or after 10 freeze-thaw cycles. Two bars are provided for each condition. The left bar for each condition corresponds to Formulation 1, and the right bar for each condition corresponds to Formulation 2. [Figure 19] Tm2 (°C) is shown for samples from Table 2 at time 0, after 1 week or 1 month at 4°C, after 1 month at room temperature, after 2 weeks or 1 month at 37°C, after 1 month at below -70°C, or after 10 freeze-thaw cycles. Two bars are provided for each condition. The left bar for each condition corresponds to Formulation 1, and the right bar for each condition corresponds to Formulation 2. [Figure 20] Subvisible particle analysis is shown for formulation buffer 1 alone (1B), formulation buffer 1 with AAV (1S), formulation buffer 2 alone (2B), and formulation buffer 2 with AAV (2S) at time 0, after 1 month at below -70°C, after 1 week or 1 month at 4°C, after 1 month at room temperature, or after 2 weeks or 1 month at 37°C. Three bars are provided for each sample. The left bar for each sample corresponds to 2 microns, the middle bar corresponds to 10 microns, and the right bar for each sample corresponds to 25 microns. [Figure 21]Figure 1 shows subvisible particle analysis after 10 freeze-thaw cycles for formulation buffer 1 alone (1B), formulation buffer 1 with AAV (1S), formulation buffer 2 alone (2B), and formulation buffer 2 with AAV (2S). Three bars are provided for each sample. The left bar for each sample corresponds to 2 microns, the middle bar corresponds to 10 microns, and the right bar for each sample corresponds to 25 microns. [Figure 22] For the formulations in Table 4, vector genome concentrations (vg / mL) by ddPCR are shown at time 0, after 10 freeze-thaw cycles, or after 7 or 14 days at 37°C. [Figure 23] The statistical variation of Tonset (°C) under t=0 and stress conditions is shown for the formulations in Table 4. The bars in the graph correspond to, from left to right, Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, Sample 6, Sample 7, Sample 8, and Sample 9. [Figure 24] For the formulations in Table 4, external fluorescence (RFU (490-650 nm)) at 15°C is shown at time 0 (first bar for each sample), after 10 freeze-thaw cycles (second bar for each sample), or after 7 or 14 days at 37°C (third and fourth bars, respectively, for each sample). DETAILED DESCRIPTION OF THE INVENTION

[0011] The present disclosure provides a stable composition suitable for administering gene therapy. Gene therapy can be delivered via a range of different vectors, for example, via viral vectors (e.g., lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors) or non-viral vectors (e.g., naked DNA, particle-based, and chemical-based). In one example, the composition is suitable for administering AAV vectors. The compositions described herein can also be suitable for administration to the central nervous system of a subject in need thereof.

[0012] The composition may comprise a recombinant adeno-associated virus (AAV) vector comprising a heterologous nucleic acid and one or more of sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, and a non-ionic detergent at a pH of 7.2-7.4. In certain embodiments, the composition comprises a recombinant AAV vector comprising a heterologous nucleic acid, sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, and a non-ionic detergent at a pH of 7.2-7.4. In various embodiments, the composition comprises a recombinant AAV vector comprising a heterologous nucleic acid, sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, and a non-ionic detergent at a pH of 7.30. In various embodiments, the composition comprises a recombinant AAV vector comprising a heterologous nucleic acid, sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, trehalose, and a non-ionic detergent at a pH of 7.2-7.4. In various embodiments, the composition comprises a recombinant AAV vector comprising a heterologous nucleic acid, sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, trehalose, and a non-ionic surfactant at pH 7.30. Characteristics of the composition are further described below.

[0013] Surfactants improve the stability of compositions, for example, by minimizing surface-induced degradation. The hydrophobic portion of the surfactant molecule occupies an interfacial position (e.g., air / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. Pharmaceutically acceptable nonionic surfactants include, but are not limited to, polysorbate 80 (Tween 80, PS80), polysorbate 20 (Tween 20, PS20), digitonin, Triton X-100, Triton X-144, and poloxamers. Poloxamers, also known as Pluronics®, are amphiphilic block copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO). Bodratti et al., J Funct Biomater. 2018 Mar;9(1):11. BASF, the original manufacturer of Pluronics®, has introduced a specific nomenclature, where the first letter indicates the physical state (paste (P), liquid (L), or flake (F)) and a series of numbers, the first one or two of which relate to the molecular weight and the last number indicates the weight percent of the PEO block. Commercially available Pluronics® include, for example, L64, P65, P84, P85, F88, P103, P104, P105, F108, P123, and F127. In various embodiments, the nonionic surfactant in the composition is a poloxamer. In various embodiments, the nonionic surfactant is poloxamer 188.

[0014] Optionally, the compositions of the present disclosure comprise from about 0.001% (w / v) to about 0.02% nonionic surfactant, e.g., from about 0.001% (w / v) to about 0.01% (w / v), or from about 0.001% to about 0.005%, or from about 0.0025% (w / v) to about 0.0075% (w / v), or from about 0.003% (w / v) to about 0.007% (w / v), or from about 0.004% (w / v) to about 0.006% (w / v), or from about 0.003% to about 0.005%, or from about 0.003% to about 0.0046%. In exemplary embodiments, the composition may comprise about 0.001% (w / v), about 0.0015% (w / v), about 0.002% (w / v), about 0.0025% (w / v), about 0.003% (w / v), about 0.0035% (w / v), about 0.004% (w / v), about 0.0045% (w / v), about 0.005% (w / v), about 0.0055% (w / v), about 0.006% (w / v), about 0.0065% (w / v), or about 0.0075% (w / v). (w / v), about 0.007% (w / v), about 0.0075% (w / v), about 0.008% (w / v), about 0.0085% (w / v), about 0.009% (w / v), about 0.0095% (w / v), about 0.01% (w / v), about 0.015% (w / v), or about 0.02% (w / v) non-ionic surfactant (as well as ranges including any of these values ​​as endpoints). In certain embodiments, the compositions of the present disclosure comprise about 0.005% (w / v) non-ionic surfactant, such as a poloxamer (e.g., poloxamer 188).

[0015] The compositions of the present disclosure further comprise one or more pharmaceutically acceptable salts. Suitable "pharmaceutically acceptable salts" include, but are not limited to, metal salts (e.g., sodium, potassium, and cesium salts) and alkaline earth metal salts (e.g., magnesium salts). Non-limiting examples of pharmaceutically acceptable salts include, but are not limited to, sodium salts, magnesium salts, and potassium salts (e.g., sodium chloride, magnesium chloride, and potassium chloride; sodium acetate, magnesium acetate, and potassium acetate; sodium citrate, magnesium citrate, and potassium citrate; sodium phosphate, magnesium phosphate, and potassium phosphate; sodium fluoride, magnesium fluoride, and potassium fluoride; sodium bromide, magnesium bromide, and potassium bromide; and sodium iodide, magnesium iodide, and potassium iodide). In various embodiments, the formulation comprises one or more of sodium chloride, magnesium chloride, and potassium chloride, and optionally, the formulation comprises sodium chloride, magnesium chloride, and potassium chloride. In some embodiments, the formulation is substantially free of magnesium chloride.

[0016] In exemplary embodiments, compositions of the present disclosure contain about 0 mM to about 200 mM, about 1 mM to about 175 mM, about 3 mM to about 150 mM, or about 5 mM to about 100 mM of a pharmaceutically acceptable salt. In various embodiments, compositions contain about 0.1 mM to about 10 mM, about 0.5 mM to about 8 mM, about 0.5 mM to about 6.5 mM, about 0.5 mM to about 3 mM, about 0.5 mM to about 1 mM, or about 0.7 mM to about 0.9 mM of a pharmaceutically acceptable salt, such as any one of the pharmaceutically acceptable salts described above (e.g., magnesium chloride). In various embodiments, the composition contains about 1 mM to about 10 mM, about 1 mM to about 5 mM, 0.5 mM to about 6.5 mM, about 1.5 mM to about 4.5 mM, or about 2 mM to about 4 mM of a pharmaceutically acceptable salt, such as any one of the pharmaceutically acceptable salts described above (e.g., potassium chloride). In various embodiments, the composition contains about 100 mM to about 175 mM, about 120 mM to about 160 mM, about 130 mM to about 150 mM, about 140 mM to about 150 mM, or about 145 mM to about 150 mM of a pharmaceutically acceptable salt, such as any one of the pharmaceutically acceptable salts described above (e.g., sodium chloride).

[0017] In exemplary embodiments, the composition comprises about 0.1 mM, about 0.5 mM, about 0.8 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, or about 10 mM of one or more pharmaceutically acceptable salts. In exemplary embodiments, the composition comprises about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, about 155 mM, about 165 mM, or about 170 mM of one or more pharmaceutically acceptable salts. In certain embodiments, the composition comprises about 145 mM to about 150 mM (e.g., about 148 mM) sodium chloride, about 1.5 mM to about 4.5 mM (e.g., about 3 mM) potassium chloride, and about 0.05 mM to about 1 mM (e.g., about 0.8 mM) magnesium chloride.

[0018] The composition further comprises a buffering agent. Pharmaceutically acceptable buffering agents are well known in the art and include, but are not limited to, phosphate buffer (e.g., sodium phosphate), histidine, citrate buffer (e.g., sodium citrate), HEPES, Tris, glycine, acetate buffer (e.g., sodium acetate), sodium carbonate, lysine, arginine, and mixtures thereof. In some embodiments, the composition may comprise two or more buffering agents. In exemplary embodiments, the buffer is a phosphate buffer, such as a sodium phosphate buffer, optionally provided as sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In some cases, the phosphate buffer may comprise potassium phosphate. In this regard, the composition optionally comprises a phosphate buffer in an amount sufficient to provide about 0.01 mM to about 10 mM, about 0.05 mM to about 5 mM, about 0.1 mM to about 3 mM, about 3 mM to about 7 mM, or about 0.5 mM to about 2 mM phosphate. In some embodiments, the composition optionally contains about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, about 10 mM, about 10.5 mM, about 11 mM The composition comprises a phosphate buffer in an amount sufficient to provide about 1 mM, about 11.5 mM, about 12 mM, about 12.5 mM, about 13 mM, about 13.5 mM, about 14 mM, about 14.5 mM, about 15 mM, about 15.5 mM, about 16 mM, about 16.5 mM, about 17 mM, about 17.5 mM, about 18 mM, about 18.5 mM, about 19 mM, about 19.5 mM, or about 20 mM phosphate. In various embodiments, the composition comprises about 1 mM phosphate, optionally provided as sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate). In various embodiments, the composition comprises about 5 mM phosphate, optionally provided as sodium phosphate (e.g., monosodium phosphate and / or disodium phosphate).

[0019] In exemplary embodiments, the buffer is a Tris buffer, optionally provided as Tris hydrochloride or Tris acetate. In this regard, the composition optionally includes Tris buffer in an amount sufficient to provide about 0.01 mM to about 30 mM, about 0.01 mM to about 20 mM, about 0.01 mM to about 10 mM, about 0.05 mM to about 5 mM, about 0.1 mM to about 3 mM, about 3 mM to about 7 mM, or about 0.5 mM to about 2 mM Tris. In some embodiments, the composition optionally contains about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, about 10 mM, about 10.5 mM, about 11 mM The composition comprises Tris buffer in an amount sufficient to provide about 1 mM, about 11.5 mM, about 12 mM, about 12.5 mM, about 13 mM, about 13.5 mM, about 14 mM, about 14.5 mM, about 15 mM, about 15.5 mM, about 16 mM, about 16.5 mM, about 17 mM, about 17.5 mM, about 18 mM, about 18.5 mM, about 19 mM, about 19.5 mM, or about 20 mM Tris. In various embodiments, the composition comprises about 1 mM Tris. In various embodiments, the composition comprises about 5 mM Tris.

[0020] The composition has a physiologically compatible pH. For example, the pH of the composition is about 6.5 to about 9.0, about 6.5 to about 8.0, about 6.9 to about 7.7, about 6.9 to about 7.4, about 7.0 to about 7.5, about 7.0 to about 7.4, about 7.2 to about 7.4, about 7.0 to about 7.3, about 7.1 to about 7.4, or about 7.2 to about 7.5. In various embodiments, the pH of the formulation is about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, or about 7.8. In exemplary embodiments, the pH of the composition is about 7.2, about 7.3, or about 7.4. In certain embodiments, the pH of the composition is about 7.3.

[0021] In various aspects, the composition does not include a sugar (e.g., a monosaccharide, a disaccharide, a cyclic polysaccharide, a sugar alcohol, a linear branched dextrans, or a linear unbranched dextrans such as sucrose, trehalose, glucose, mannitol, or sorbitol). In various aspects, the composition does not include an amino acid such as glycine, glutamine, asparagine, arginine, or lysine. In various aspects, the formulation does not include calcium, such as calcium chloride.

[0022] The composition optionally includes a recombinant adeno-associated virus (AAV) vector, such as a recombinant AAV vector containing a heterologous nucleic acid. The abbreviation "rAAV" refers to a recombinant adeno-associated virus. The term "AAV" includes all serotypes of AAV, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hul4), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrhlO, AAV-DJ, and AAV-DJ8, as well as hybrids thereof (i.e., chimeric AAV vectors). The genomic sequences of various serotypes of AAV, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. "Heterologous nucleic acid" refers to a polynucleotide sequence not of AAV origin, typically a sequence of interest for delivery to a host cell. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeats (ITRs). As used herein, the term AAV vector encompasses both AAV particles (i.e., viral particles consisting of at least one AAV capsid protein and an encapsidated polynucleotide) and AAV vector plasmids (i.e., polynucleotides containing AAV components not encapsulated in AAV coat proteins). AAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV). See, e.g., Raj et al., Expert Rev Hematol. 2011 Oct;4(5):539-549. AAV can contain genomic components and capsids from multiple serotypes (e.g., pseudotyped vectors). For example, AAV can contain the serotype 2 genome (e.g., ITRs) packaged in a capsid from serotype 5 or serotype 9. Pseudotyped vectors may demonstrate improved transduction efficiency as well as altered tropism. In some cases, AAV serotypes that can cross the blood-brain barrier or infect cells of the CNS are preferred.In some aspects, the recombinant AAV vector is AAV1, AAV8, AAV9, AAVDJ, or a chimeric AAV comprising features of two or more of these serotypes. In various embodiments, the AAV vector is an AAV9 vector or an scAAV9 vector. In certain embodiments, the AAV vector is an AAV9 vector or an scAAV9 vector and comprises heterologous nucleic acid flanked by ITRs from an AAV serotype other than AAV9. In certain embodiments, the AAV vector is an AAV9 vector or an scAAV9 vector and comprises heterologous nucleic acid flanked by AAV serotype 2 ITRs (i.e., ITR2).

[0023] In exemplary embodiments, the composition comprises at least about 1×10 7 AAV viral genome (vg), at least about 1 × 10 8 vg, at least about 1 × 10 9 vg, at least about 1 × 10 10 vg, at least about 1 × 10 11 vg, at least about 1 × 10 12 vg, at least about 5 × 10 12 vg, at least about 6 × 10 12 vg, at least about 7 × 10 12 vg, at least about 8 × 10 12 vg, at least about 9 × 10 12 vg, at least about 9.5 × 10 12 vg, at least about 9.8 × 10 12 vg, at least about 1 × 10 13 vg, at least about 1.5 × 10 13 vg, at least about 1.6 × 10 13 vg, at least about 1.7 × 10 13 vg, at least about 1.8 × 10 13 vg, at least about 1.9 × 10 13 vg, at least about 2 × 10 13 vg, at least about 3 × 10 13 vg, at least about 4 × 10 13 vg, at least about 5 × 10 13 vg, at least about 6 × 10 13 vg, at least about 7 × 10 13vg, at least about 8 × 10 13 vg, at least about 9 × 10 13 vg, at least about 1 × 10 14 vg, at least about 2 × 10 14 vg, at least about 3 × 10 14 vg, at least about 4 × 10 14 vg, at least about 5 × 10 14 vg, at least about 6 × 10 14 vg, at least about 7 × 10 14 vg, at least about 8 × 10 14 vg, at least about 9 × 10 14 vg, or at least about 1 × 10 15 In various embodiments, the composition comprises about 1×10 12 vg / mL ~ approx. 5×10 14 vg / mL, approximately 1×10 13 vg / mL ~ approx. 5×10 14 vg / mL, approximately 5×10 12 vg / mL ~ approx. 1×10 14 vg / mL, approximately 5×10 13 vg / mL ~ approx. 1×10 14 vg / mL, approximately 7×10 13 vg / mL ~ approx. 9×10 13 In various embodiments, the composition comprises about 1 x 10 vg / mL of AAV. 13 vg / mL, approximately 2×10 13 vg / mL, approximately 3×10 13 vg / mL, approximately 4×10 13 vg / mL, approximately 5×10 13 vg / mL, approximately 6×10 13 vg / mL, approximately 7×10 13 vg / mL, approximately 8×10 13 vg / mL, approximately 9×10 13 vg / mL, approximately 1×10 14 vg / mL, approximately 2×10 14 vg / mL, approximately 3×10 14 vg / mL, approximately 4×10 14 vg / mL, or approximately 5 × 10 14 In various embodiments, the composition comprises about 5 x 10 vg / mL of AAV. 13 vg / mL ~ approx. 1×10 14vg / mL (e.g., approximately 8 × 10 13 In various embodiments, the composition comprises about 7 x 10 AAV. 12 vg / mL ~ approx. 1.3×10 13 vg / mL, approximately 1.4×10 13 vg / mL ~ approx. 2.6×10 13 vg / mL, or approximately 9.8 × 10 12 vg / mL ~ approx. 1.82×10 13 Contains vg / mL.

[0024] The present disclosure provides (a) a recombinant adeno-associated virus (AAV) vector comprising a heterologous nucleic acid, optionally comprising about 5×10 13 vg / mL ~ approx. 1×10 14 vg / mL (e.g., approximately 8 × 10 13 (vg / mL) of a recombinant AAV vector; (b) sodium chloride, optionally from about 145 mM to about 150 mM sodium chloride; (c) potassium chloride, optionally from about 1.5 mM to about 10 mM potassium chloride (e.g., from about 1.5 mM to about 4.5 mM); (d) magnesium chloride, optionally from about 0.05 mM to about 1 mM magnesium chloride; (e) a phosphate buffer (e.g., sodium phosphate), optionally present in an amount sufficient to provide from about 0.5 mM to about 2 mM (e.g., about 1 mM) phosphate; and (f) a non-ionic surfactant (e.g., a poloxamer such as Poloxamer 188), optionally present at a concentration ranging from about 0.001% to about 0.01% (w / V), at a pH of 7.2 to 7.4. For example, the surfactant can be poloxamer 188 present at a concentration of about 0.005% (w / V). In some embodiments, the composition is calcium-free, such as calcium chloride. In some embodiments, the composition exhibits a conductivity of about 15.0 to about 17.0 mS / cm.

[0025] The present disclosure provides (a) a recombinant adeno-associated virus (AAV) vector comprising a heterologous nucleic acid, optionally comprising about 5×10 13 vg / mL ~ approx. 1×10 14 vg / mL (e.g., approximately 8 × 10 13(vg / mL) recombinant AAV vector; (b) sodium chloride, optionally about 145 mM to about 150 mM sodium chloride; (c) potassium chloride, optionally about 1.5 mM to about 10 mM potassium chloride (e.g., about 1.5 mM to about 4.5 mM); (d) phosphate buffer (e.g., sodium phosphate), optionally present in an amount sufficient to provide about 0.5 mM to about 2 mM phosphate (e.g., about 1 mM); and (e) a non-ionic surfactant (e.g., a poloxamer, such as poloxamer 188), optionally present at a concentration ranging from about 0.001% to about 0.01% (w / v), at a pH of 7.2 to 7.4. For example, the surfactant can be poloxamer 188 present at a concentration of about 0.005% (w / v). In some embodiments, the composition is calcium-free, such as calcium chloride. In some embodiments, the composition exhibits a conductivity of about 15.0 to about 17.0 mS / cm.

[0026] The present disclosure provides (a) a recombinant adeno-associated virus (AAV) vector comprising a heterologous nucleic acid, optionally comprising about 5×10 13 vg / mL ~ approx. 1×10 14 vg / mL (e.g., approximately 8 × 10 13(vg / mL) recombinant AAV vector; (b) sodium chloride, optionally from about 145 mM to about 150 mM sodium chloride; (c) potassium chloride, optionally from about 1.5 mM to about 4.5 mM potassium chloride; (d) Tris buffer, optionally present in an amount sufficient to provide from about 0.5 mM to about 10 mM phosphate (e.g., about 1 mM, or about 5 mM); and (e) a non-ionic surfactant (e.g., a poloxamer, such as poloxamer 188), optionally present at a concentration ranging from about 0.001% to about 0.01% (w / v), at a pH of 7.2 to 7.4. For example, the surfactant can be poloxamer 188 present at a concentration of about 0.005% (w / v). In some embodiments, the composition is calcium-free, such as calcium chloride. In some embodiments, the composition exhibits a conductivity of about 15.0 to about 17.0 mS / cm. In some embodiments, the composition further comprises magnesium chloride, optionally from about 0.05 mM to about 1 mM magnesium chloride.

[0027] The present disclosure further contemplates compositions comprising the components described herein (e.g., sodium chloride, potassium chloride, optionally magnesium chloride, phosphate buffer, and non-ionic surfactant), but excluding rAAV. In this regard, the present disclosure provides a composition comprising: (a) sodium chloride, optionally from about 145 mM to about 150 mM sodium chloride; (b) potassium chloride, optionally from about 1.5 mM to about 4.5 mM potassium chloride; (c) magnesium chloride, optionally from about 0.05 mM to about 1 mM magnesium chloride; (d) a phosphate buffer (e.g., sodium phosphate), optionally present in an amount sufficient to provide from about 0.5 mM to about 10 mM (e.g., about 1 mM or about 5 mM) phosphate; and (e) a non-ionic surfactant (e.g., a poloxamer such as Poloxamer 188), optionally present in a concentration ranging from about 0.001% to about 0.01% (w / V), at a pH of 7.2 to 7.4.

[0028] In another aspect, the disclosure provides a composition comprising: (a) sodium chloride, optionally from about 145 mM to about 150 mM sodium chloride; (b) potassium chloride, optionally from about 1.5 mM to about 4.5 mM potassium chloride; (c) magnesium chloride, optionally from about 0.05 mM to about 1 mM magnesium chloride; (d) Tris buffer, optionally present in an amount sufficient to provide from about 0.5 mM to about 10 mM Tris (e.g., about 1 mM or about 5 mM); and (e) a non-ionic surfactant (e.g., a poloxamer such as Poloxamer 188), optionally present at a concentration ranging from about 0.001% to about 0.01% (w / V), at a pH of 7.2 to 7.4. In this regard, the present disclosure provides a composition comprising: (a) sodium chloride, optionally from about 145 mM to about 150 mM sodium chloride; (b) potassium chloride, optionally from about 1.5 mM to about 4.5 mM potassium chloride; (c) a phosphate buffer (e.g., sodium phosphate), optionally present in an amount sufficient to provide from about 0.5 mM to about 10 mM (e.g., about 1 mM or about 5 mM) phosphate; and (d) a non-ionic surfactant (e.g., a poloxamer such as Poloxamer 188), optionally present at a concentration ranging from about 0.001% to about 0.01% (w / V), at a pH of 7.2 to 7.4. In another aspect, the disclosure provides a composition comprising: (a) sodium chloride, optionally at about 145 mM to about 150 mM sodium chloride; (b) potassium chloride, optionally at about 1.5 mM to about 4.5 mM potassium chloride; (c) Tris buffer, optionally present in an amount sufficient to provide about 0.5 mM to about 10 mM Tris (e.g., about 1 mM or about 5 mM); and (e) a non-ionic surfactant (e.g., a poloxamer such as Poloxamer 188), optionally present at a concentration ranging from about 0.001% to about 0.01% (w / V), at a pH of 7.2 to 7.4.

[0029] In various examples, the surfactant is poloxamer 188 present at a concentration of about 0.005% (w / V). In some embodiments, the composition does not include calcium, such as calcium chloride.

[0030] In some embodiments, the composition can be such that the rAAV particles present in the composition are substantially stable therein. For example, the vector genome titer, as measured by ddPCR, can change by less than 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% after exposure to storage and / or handling conditions. Examples of storage or handling conditions include, but are not limited to, storage at about 4°C, storage at about room temperature, storage at about 37°C, or repeated freeze-thaw cycles. In certain embodiments, the composition is substantially stable after storage for up to an extended period of time. For example, storage can be for less than 24 hours, about 1-2 days, about 2-5 days, about 5-7 days, about 1-2 weeks, about 2-4 weeks, up to 1 month, or more than 1 month. In certain embodiments, the composition may be substantially stable after storage at 4° C. for up to one month, at room temperature for up to one month, or at 37° C. for up to two weeks or up to one month. In other embodiments, the composition is substantially stable after one or more freeze-thaw cycles. For example, the composition is substantially stable after 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or more than 25 repeated freeze-thaw cycles.

[0031] Stability can also be assessed by aggregation as determined by size-exclusion high-performance liquid chromatography (SE-HPLC). For example, the rAAV in the formulations described herein may exhibit an increase in aggregation of less than 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% after exposure to conditions such as those described above (e.g., storage at 4°C, room temperature, 37°C, or repeated freeze-thaw cycles). Reverse-phase HPLC (RP-HPLC) can also be used to compare the occurrence rates of different species before and after stress. For example, the rAAV in the formulations described herein may exhibit an increase in RP-HPLC species of less than 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% after exposure to conditions such as those described above (e.g., storage at 4°C, storage at room temperature, or repeated freeze-thaw cycles). In vitro potency may also be used to assess rAAV stability. For example, the rAAV in the formulations described herein may exhibit a decrease in in vitro potency of less than 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% after exposure to conditions such as those described above (e.g., storage at 4°C, storage at room temperature, or repeated freeze-thaw cycles).

[0032] A recombinant AAV vector comprises a heterologous nucleic acid. The heterologous nucleic acid may comprise or be in the form of an "expression cassette," which refers to a polynucleotide comprising one or more regulatory elements operably linked to a coding sequence (i.e., a polynucleotide sequence encoding an RNA or peptide of interest). A recombinant AAV vector may comprise any heterologous nucleic acid of interest, including a transgene encoding a peptide or protein of interest. The transgenes referred to herein generally contain no introns or no more than one intron, although this is not required. The transgene may be derived from a cDNA sequence rather than a genomic sequence. In some cases, the transgene encodes an ion channel, a neurotransmitter regulator, a transcription factor, or a subunit, variant, or functional fragment of any of the foregoing. Examples of ion channels include voltage-gated and ligand-gated ion channels. Voltage-gated ion channels include sodium channels, calcium channels, potassium channels, and proton channels. In some embodiments, the transgene encodes SCN1A. In some cases, the transgene encodes a subunit of a voltage-gated sodium channel, e.g., a sodium ion channel alpha subunit, a sodium ion channel beta subunit, or a variant or functional fragment thereof. An example of a voltage-gated sodium channel subunit is SCNIA (NM_001165963.1).

[0033] In some embodiments, the transgene encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SCN1A, or a functional fragment thereof. In this regard, the transgene optionally encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:6.

[0034] Another example of a heterologous nucleic acid of interest encodes a transcription factor, which can be a transcription activator or a transcription repressor. Transcription factors contain a DNA-binding domain and a transcriptional regulatory domain. The DNA-binding domain binds to a transcription factor binding site in target DNA. The transcriptional regulatory domain (TMD) contains a binding site for other proteins that promote or repress transcription of the target nucleic acid sequence. The TMD can contact the transcription machinery (e.g., RNA polymerase) either directly or through other proteins (known as coactivators or comodulators). The transcription factor can be wild-type (i.e., unmodified) or can be a non-naturally occurring transcription factor, such as a transcription factor that has been engineered such that the DNA-binding domain is operably linked to a transcriptional regulatory domain to which the DNA-binding domain is not naturally linked (e.g., from a different transcription factor or a different species). In various embodiments, the heterologous nucleic acid encodes a transcription factor that modulates (e.g., enhances) the expression of SCN1A.

[0035] Examples of DNA-binding domains include zinc finger, helix-turn-helix, leucine zipper (e.g., bZIP), helix-loop-helix, and beta-scaffold Cas9, Cas family proteins, dCas9, dCas family proteins, or transcription activator-like effectors (TALEs). In some cases, the transgene is a DNA-binding protein that includes an inactivated DNA cleavage region. In some cases, the transgene includes a gene-editing protein, such as a Cas protein, Cas9. The heterologous nucleic acid can encode multiple copies of the same DNA-binding domain or can include multiple DNA-binding domains of different sequences. For example, various embodiments of the present disclosure provide heterologous nucleic acids that include 2 to 10 DNA-binding domains, such as zinc fingers (e.g., 3 to 8 zinc fingers, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers).

[0036] Examples of suitable DNA-binding domains are those having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 7 to 28. In exemplary embodiments, the DNA-binding domain comprises the sequence of any one of SEQ ID NOs: 7 to 28. See also International Patent Publication No. 2020 / 243651 (incorporated herein by reference).

[0037] The TMD and DNA-binding domain (DBD) can be derived from different proteins. An engineered TF can include two or more TMDs, and two or more TMDs can be derived from (e.g., isolated from) different proteins compared to other TMDs in the protein. In various aspects, the TMD is a transactivation domain that enhances or upregulates expression. Examples of transactivation domains include, for example, VP64 (SEQ ID NO: 29), VPR (SEQ ID NO: 30), VP16, VP128, p65, p300, CBP / p300-interacting transactivator 2 (CITED2) (SEQ ID NO: 31 or 32), CBP / p300-interacting transactivator 4 (CITED4) (SEQ ID NO: 33 or 34), EGR1 (SEQ ID NO: 35), or EGR3 (SEQ ID NO: 36). See also International Patent Publication No. WO 2019 / 109051, incorporated herein by reference in its entirety, particularly for disclosure of transactivation domains and DNA-binding domains. Any suitable arrangement of one or more DNA-binding domains and one or more transcriptional regulatory domains is contemplated. For example, a non-naturally occurring transcription factor optionally comprises a DNA-binding domain (DBD) operably linked to at least two transcriptional regulatory domains (TMDs) in the following manner: TMD1-TMD2-DBD, DBD-TMD3-TMD4, or TMD1-TMD2-DBD-TMD3-TMD4. In certain embodiments, TMD1, TMD2, TMD3, and TMD4 are independently selected from the following: VP16, VP64, Viper, CITED2, CITED4, and CREB3, or functional fragments of any of the foregoing. Optionally, TMD1 and TMD2 are the same TMD. Optionally, TMD3 and TMD4 are the same TMD. In various aspects, TMD1, TMD2, TMD3, and TMD4 are the same TMD. In various embodiments, a linker is present between two or more of the TMDs. Examples of suitable linkers include, but are not limited to, GS, GGSGGGSG (SEQ ID NO: 37), or GGSGGGSGGGSG (SEQ ID NO: 38).In a representative example, the heterologous nucleic acid encodes a transcription factor-like effector DNA binding domain fused to a zinc finger DNA binding domain (optionally comprising multiple zinc fingers) or a transcription regulatory domain (e.g., VP16 or VP64), and the DNA binding domain-transcription regulatory domain fusion comprises one or more linkers selected from GGSGGGSG (SEQ ID NO: 37) or GGSGGGSGGGSG (SEQ ID NO: 38).

[0038] Native transcription factors can be active in most cell types. Transcription factors can also be tissue-specific, such as those from muscle cells (e.g., MyoD and muscle enhancer factor 2 (MEF2)) or neuronal cells (e.g., nuclear factor 1C (NF1C), nuclear factor 1X (NF1X), brain-1 (Brn-1), or brain-2 (Brn-2)). Transcription factors can also be ligand-dependent. Ligand-dependent transcription factors contain additional domains that are bound by ligands, resulting in up- or down-regulation of gene expression. Steroid hormone receptors and nuclear receptors are examples of ligand-dependent transcription factors. Other examples of ligand-dependent transcription factors are metal-responsive transcription factors, which regulate, for example, metal (iron, zinc, or copper) homeostasis.

[0039] Examples of transcription factors include AF-4 transcription factor, androgen receptor transcription factor, AP-2 transcription factor, ARID transcription factor, bHLH transcription factor, C / EBP transcription factor, CBF transcription factor, CG-1 transcription factor, COE transcription factor, COUP transcription factor, CP2 transcription factor, CSD transcription factor, CSL transcription factor, CTF / NFI transcription factor, CUT transcription factor, DM transcription factor, E2F transcription factor, EAF2 transcription factor, Ecdystd receptor transcription factor, ETS transcription factor, forkhead transcription factor, GCM transcription factor, GCR transcription factor, GTF2I transcription factor, HMG transcription factor, HMGI / HMGY transcription factor, homeobox transcription factor, HSF transcription factor, HTH transcription factor, IRF transcription factor, MBD transcription factor, MH1 transcription factor, MYB transcription factor, NDT80 / PhoG transcription factor, NF-YA transcription factor, NF-YB / C transcription factor, Nrf1 transcription factor, and nuclear orphan receptor transcription factor , estrogen receptor transcription factors, P53 transcription factors, PAX transcription factors, PC4 transcription factors, POU transcription factors, PPAR receptor transcription factors, PREB transcription factors, progesterone receptor transcription factors, Prox1 transcription factors, retinoic acid receptor transcription factors, RFX transcription factors, RHD transcription factors, ROR receptor transcription factors, Runt transcription factors, SAND transcription factors, SPZ1 transcription factors, SRF transcription factors, STAT transcription factors, T-box transcription factors, TEA transcription factors, TF-bZIP transcription factors, TF-Otx transcription factors, THAP transcription factors, thyroid hormone receptor transcription factors, TSC22 transcription factors, Tub transcription factors, ZBTB transcription factors, zf-BED transcription factors, zf-C2H2 transcription factors, zf-C2HC transcription factors, zf-GATA transcription factors, zf-LITAF-like transcription factors, zf-MIZ transcription factors, and zf-NF-X1 transcription factors. Metal-responsive transcription factors include, but are not limited to, Aft1, Aft2, Fep1, SREA, Urbs1, Ace1, Amt1, Srf1, Mac1, Cuf1, GRISEA, Crr1, Zap1, and metal-responsive element-binding transcription factor-1 (MTF-1), which responds to heavy metals such as copper to induce the expression of metallothionein and other genes involved in metal homeostasis.See, for example, Rutherford and Bird, Eukaryot Cell. 2004 Feb;3(1):1-13, and Wang et al., Biol Chem. 2004 Jul;385(7):623-32.

[0040] The transcription factor can be any of the transcription factors disclosed herein or can include any component of the reference transcription factors referenced herein (e.g., the DNA-binding domain or transcription regulatory domain of the reference transcription factor). In exemplary aspects of the present disclosure, the heterologous nucleic acid encodes a transcription factor that upregulates SCN1A production and is any of the engineered transcription factors described in International Patent Publication No. WO 2020 / 243651 (incorporated herein by reference in its entirety). For example, in exemplary embodiments, an engineered transcription factor comprises a DNA-binding domain comprising a zinc finger motif having the following structure: LEPGEKP-[YKCPECGKSFSXHQRTHTGEKP]n-YKCPECGKSFSXHQRTH-TGKKTS (SEQ ID NO: 39), where n is an integer between 1 and 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, and each X is independently a recognition sequence (e.g., a recognition helix) capable of binding to 3 bp of a target sequence. In exemplary embodiments, n is 3, 6, or 9. In particularly preferred embodiments, n is 6. In various embodiments, each X can independently have the same amino acid sequence or a different amino acid sequence compared to other X sequences within the DNA-binding domain. In exemplary embodiments, each X is a sequence comprising 7 amino acids that is designed to interact with 3 bp of a target binding site of interest using the Zinger Finger Design Tool from Scripps on the World Wide Web (scripps.edu / barbas / zfdesign / zfdesignhome.php). The engineered transcription factor optionally further comprises a VP64 transcriptional regulatory domain. In some examples, the transcription factor may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 40-80.

[0041] The heterologous nucleic acid sequence optionally includes a promoter that drives expression of the nucleic acid. The promoter may be native or non-native to the nucleic acid sequence to which it is operably linked, and may be native or non-native to a particular host cell. In various aspects, the promoter may be a constitutive promoter, a tissue-specific promoter, or an inducible promoter. Examples of constitutive promoters include herpes simplex virus (HSV), thymidine kinase (TK), Rous sarcoma virus (RSV), simian virus 40 (SV40), mouse mammary tumor virus (MMTV), AdE1A, and cytomegalovirus (CMV) promoters. Additional examples of constitutive promoters include the GAD2 promoter, human synapsin promoter, CBA promoter, minCMV promoter, TATA box, supercore promoter, or EF1a promoter. Examples of inducible promoters include, but are not limited to, those from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone-inducible genes such as estrogen gene promoters. Another example of an inducible promoter is the tet promoter, which is responsive to tetracycline. In various embodiments, the heterologous nucleic acid comprises a CMV promoter. In certain embodiments, the heterologous nucleic acid comprises a Syn1 promoter.

[0042] Optionally, the heterologous nucleic acid comprises one or more additional regulatory elements (optionally in addition to a promoter), such as sequences associated with transcription initiation or termination, enhancer sequences, and efficient RNA processing signals. Exemplary regulatory elements include, for example, introns, enhancers, UTRs, stability elements, WPRE sequences, Kozak consensus sequences, post-translational response elements, microRNA binding sites, polyadenylation (polyA) signal sequences, or combinations thereof. Regulatory elements can function to regulate gene expression at the transcriptional, post-transcriptional, or translational stages of gene expression. At the RNA level, regulation can occur at the level of translation (e.g., stability elements that stabilize mRNA for translation), RNA cleavage, RNA splicing, and / or transcription termination.

[0043] The regulatory element contained in the heterologous nucleic acid can be a cell-type-selective regulatory element, such as a regulatory element that drives expression in a central nervous system cell type. Optionally, the regulatory element selectively drives expression in GABAergic cells. GABAergic cells are inhibitory neurons that produce gamma-aminobutyric acid. GABAergic cells can be identified by the expression of glutamic acid decarboxylase 2 (GAD2). Other markers of GABAergic cells include GAD1, NKX2.1, DLX1, DLX5, SST, PV, and VIP. The regulatory element can selectively drive expression in GABAergic cells that express parvalbumin ("PV cells") to a greater extent than in other cell types (e.g., other CNS cell types, such as non-GABAergic neurons (e.g., non-PV GABAergic neurons)). Examples of non-PV CNS cells include excitatory neurons, dopaminergic neurons, astrocytes, microglia, motor neurons, and vascular cells. Non-GABAergic neurons also include cells that do not express one or more of GAD2, GAD1, NKX2.1, DLX1, DLX5, SST, and VIP. In some cases, non-PV GABAergic neurons include, but are not limited to, calretinin (CR), somatostatin (SOM), cholecystokinin (CCK), CR+SOM, CR+neuropeptide Y (NPY), CR+vasoactive intestinal polypeptide (VIP), SOM+NPY, SOM+VIP, VIP+choline acetyltransferase (ChAT), CCK+NPY, CR+SOM+NPY, and CR+SOM+VIP-expressing cells.

[0044] In some embodiments in which the recombinant AAV vector comprises a regulatory element, the regulatory element may comprise one or more sequences set forth in SEQ ID NOs: 81-112. In some examples, the recombinant AAV vector comprises: (i) a heterologous nucleic acid comprising one or more of SEQ ID NOs: 81-112; (ii) a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 81-112; (iii) a functional fragment of any of the sequences in (i) or (ii); or (iv) a combination of any of the sequences in (i), (ii), and / or (iii). In some cases, sequence identity is measured by BLAST. The regulatory element may be positioned upstream or downstream of the transgene. Regulatory elements are described, for example, in International Patent Publication No. WO 2018 / 187363, which is incorporated herein by reference in its entirety, particularly with respect to regulatory elements and sequences.

[0045] In certain embodiments, the recombinant AAV vector comprises a nucleotide sequence operably linked to a regulatory element, wherein the regulatory element results in at least a two-fold increase in transgene expression compared to expression of the transgene when operably linked to a CMV promoter. In certain embodiments, the promoter sequence provides at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, or 75-fold increased expression of the transgene sequence in a mammalian cell compared to the expression level of the same transgene sequence from a CMV promoter in the same type of mammalian cell. 0-90 fold, 20-80 fold, 20-70 fold, 20-60 fold, 30-90 fold, 30-80 fold, 30-70 fold, 30-60 fold, 40-90 fold, 40-80 fold, 40-70 fold, 40-60 fold, 50-90 fold, 50-80 fold, 50-70 fold, 50-60 fold, 60-90 fold, 60-80 fold, 60-70 fold, 70-90 fold, 70-80 fold, or 80-90 fold higher expression is obtained. In certain embodiments, the promoter sequence drives expression of the transgene sequence in a high percentage of neural cells, e.g., at least 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more, that contain the vector. wherein at least 20-90%, 20-80%, 20-70%, 30-90%, 30-80%, 30-70%, 40-90%, 40-80%, 40-70%, 50-90%, 50-80%, 50-70%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 80-100%, 80-95%, 80-90%, 90-100%, or 90-95% of the GABAergic cells express the transgene.In certain embodiments, the promoter sequence drives expression of the transgene in a high percentage of glial cells, e.g., at least 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more of the glial cells containing the vector. or at least 20-90%, 20-80%, 20-70%, 30-90%, 30-80%, 30-70%, 40-90%, 40-80%, 40-70%, 50-90%, 50-80%, 50-70%, 60-90%, 60-80%, 60-70%, 70-90%, 70-80%, 80-100%, 80-95%, 80-90%, 90-100%, or 90-95% of the oligodendrocytes express the transgene.

[0046] In some embodiments, the AAV expression cassette comprises a 120 bp or less human-derived regulatory element operably linked to a transgene of at least 3 kb, wherein the regulatory element results in at least 2-fold increased transgene expression compared to expression of the transgene when operably linked to a CMV promoter. In some cases, the increased transgene expression is at least 50-fold. In some cases, the increased transgene expression is at least 100-fold. In some cases, the increased transgene expression occurs in at least two different cell types (e.g., excitatory neurons and inhibitory neurons). In some cases, the increased transgene expression occurs in at least three different cell types (e.g., excitatory neurons, inhibitory neurons, and liver cells).

[0047] In some cases, such increased expression of the transgene in a cell or in vivo is relative to expression of the transgene without the regulatory element, where expression of the transgene with the regulatory element is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, or greater than expression of the transgene without the regulatory element or compared to transgene expression with a negative control (e.g., buffer alone, vector alone, or a vector comprising a sequence known to have no expression activity). at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, at least 1010-fold, at least 1020-fold, at least 1030-fold, at least 1040-fold, or at least 1050-fold.

[0048] In certain embodiments, the heterologous nucleic acid further comprises a polyA signal sequence. Suitable polyA signal sequences include, for example, an artificial polyA (PA75) having a length of approximately 75 bp (see, e.g., International Patent Publication No. 2018 / 126116), bovine growth hormone polyA, SV40 early polyA signal, SV40 late polyA signal, rabbit beta globin polyA, HSV thymidine kinase polyA, protamine gene polyA, adenovirus 5 Elb polyA, growth hormone polyA, or PBGD polyA. In an exemplary embodiment, the polyA sequence is hGH polyA (SEQ ID NO: 113) or synthetic polyA (SEQ ID NO: 114). See also International Patent Publication No. 2019 / 109051 (incorporated herein by reference in its entirety). Typically, the polyA signal sequence is operably linked to the coding nucleic acid sequence.

[0049] The recombinant AAV vector may comprise inverted terminal repeats (ITRs) and may be derived from the same serotype as the capsid of the viral particle, or may be derived from a different serotype (e.g., AAV2 ITRs and AAV9 capsid proteins, AAV2 ITRs and AAV8 capsid proteins, etc.). In a representative embodiment, the recombinant AAV vector comprises an AAV2 ITR.

[0050] In various aspects of the present disclosure, the composition exhibits properties suitable for direct administration to the central nervous system (e.g., direct administration to the brain or cerebrospinal fluid). In some embodiments, the composition exhibits a conductivity of about 15.0 to about 17.0 mS / cm. "Conductivity" is the ability of an aqueous solution to conduct an electric current between two electrodes. Generally, electrical conductivity or specific conductivity is a measure of the ability to conduct an electric current generated via ion transport. As the amount of ions in a substance increases, the conductivity increases. Conductivity can be measured, for example, using a commercially available conductivity meter.

[0051] The present disclosure further provides a method for delivering a heterologous nucleic acid of interest to a host cell, the method comprising administering a composition of the present disclosure to a subject, e.g., a human subject. In this regard, the present disclosure further provides a method for treating a medical condition in a subject, such as a human subject, comprising administering a composition to a subject in need thereof. In various embodiments, the medical condition is a neurological or neurodegenerative disorder. In some cases, the subject suffers from (or is at risk of suffering from) a psychiatric disorder, an autism spectrum disorder, epilepsy (e.g., Dravet syndrome), or neurodegeneration (e.g., neurodegeneration associated with Alzheimer's disease or Parkinson's disease). Exemplary mutations include mutations in SCN1A.

[0052] Optionally, the composition is administered to a subject suffering from (or at risk of suffering from) a neurological condition associated with dysfunction of PV neurons. Disorders associated with dysfunctional PV neurons (such as disorders resulting from loss of function mutations in SCN1A or Nav1.1) include, but are not limited to, Dravet syndrome, Ohtahara syndrome, epilepsy, early infantile epileptic encephalopathy 6 (EIEE6), familial febrile seizures 3A (FEB3A), intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC), migraine, familial hemiplegia 3 (FHM3), Panayiotopoulos syndrome, familial atrial fibrillation 13 (ATFB13), generalized epilepsy with febrile seizures plus type 1 (gefs+type1), Brugada syndrome, nonspecific cardiac conduction disorder, generalized epilepsy with febrile seizures plus, benign familial infantile seizures, early infantile epileptic encephalopathy 11 (EIEE11), benign familial infantile epilepsy, neurodegeneration, tauopathy, and Alzheimer's disease. In some Alzheimer's disease patients, the production of amyloid beta (Aβ) can affect neuronal excitability, leading to seizures and causing downregulation of Nav1.1 sodium channels in PV neurons.

[0053] In some embodiments of the present disclosure, the neurological disorder is Dravet syndrome. The majority of cases of Dravet syndrome are associated with mutations in the SCN1A and / or SCN2A genes. Mutations or abnormalities in SCN1A are also associated with seizure disorders, epilepsy, autism, familial hemiplegic migraine type 3 (FHM3), genetic epilepsy with febrile seizures plus (GEFS+), and the effectiveness of certain anti-seizure medications. For example, the ICS5N+5G>A mutation in SCN1A is associated with the maximum safe dose of the anti-seizure drugs phenytoin and carbamazepine. Symptoms associated with Dravet syndrome include seizures, memory impairment, developmental delay, poor muscle tone, and / or cognitive impairment. Administration of a composition described herein can result in the improvement of one or more symptoms associated with any of the disorders described herein, or the prevention or delay of the onset of one or more symptoms. With respect to Dravet syndrome, "treatment" includes, for example, a reduction in the number, duration, and / or intensity of seizures.

[0054] The compositions provided herein can be administered to a subject via parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intraparenchymal, intrathecal, intracisternal, intraventricular, or intraperitoneal administration. In various embodiments, the compositions are optionally administered directly to the CNS (such as directly into the cerebrospinal fluid (CSF)) by intraparenchymal, intrathecal, intracisternal, or intraventricular injection. Methods of administering any of the compositions disclosed herein are discussed in more detail below.

[0055] The present disclosure contemplates methods of administering the compositions disclosed herein to a primate (e.g., a human), including intracerebroventricular (ICV) administration of the composition. Also described herein are compositions and methods for expressing a gene of interest, or a biologically active variant and / or fragment thereof, comprising administering to a primate a therapeutically effective amount of a composition comprising an adeno-associated viral vector encoding the gene of interest, wherein the route of administration is selected from the group consisting of intravenous administration, intrathecal administration, intracerebroventricular administration, intraparenchymal administration, or a combination thereof. Further provided herein are compositions and methods for inhibiting or treating one or more symptoms associated with a neurological disorder in a primate in need thereof, wherein the method comprises administering to the primate a composition comprising AAV, wherein the route of administration is selected from the group consisting of intravenous administration, intrathecal administration, intracerebroventricular administration, intraparenchymal administration, or a combination thereof.

[0056] In some embodiments, the present disclosure provides a method of administering a composition disclosed herein to a subject (e.g., a primate) via intrathecal or intraventricular administration. In the case of intrathecal administration, the vector of the present invention is delivered to the intrathecal space, which is located around the spinal cord and filled with cerebrospinal fluid. This space is surrounded by two membranes: the arachnoid mater and the dura mater. The intrathecal space is the space below the arachnoid mater, i.e., the inner layer of the two membranes; therefore, intrathecal administration refers to administration into the subarachnoid space. Both the space around the brain and the space around the spinal cord are filled with CSF, and the ventricles in the brain are also filled with CSF. The ventricles, pericerebral space, and intrathecal space are generally connected to form a continuous space through which CSF circulates. Therefore, intraventricular and intrathecal administration are contemplated as methods of administering any of the compositions disclosed herein to the CSF.

[0057] In some embodiments, the present disclosure provides a method of administering any of the compositions disclosed herein to a subject (e.g., a primate). In some aspects, the composition is delivered to the CNS. In some aspects, the composition is delivered to the cerebrospinal fluid. In some aspects, the composition is administered to the brain parenchyma. In some aspects, the composition is delivered to the primate by intraventricular administration.

[0058] In some embodiments, the composition is delivered to a subject (e.g., a primate) by intravenous administration. In some embodiments, the composition is delivered to a subject (e.g., a primate) by intrathecal administration, e.g., intrathecal cisterna magna or intrathecal lumbar administration. In some embodiments, the composition is delivered to a subarachnoid cisterna, e.g., the cisterna magna. In some embodiments, the composition is delivered to the lumbar subarachnoid space that surrounds spinal nerves. In some embodiments, the composition is delivered to a subject (e.g., a primate) by intraparenchymal administration. Widespread distribution of the compositions described herein within the central nervous system can be achieved by intraparenchymal, intrathecal, or intracerebroventricular administration.

[0059] In some aspects, any of the compositions disclosed herein is administered to a subject (e.g., a primate) in combination with an imaging agent, e.g., gadolinium or gadoteridol. In other aspects, the vector is not administered in combination with an imaging agent, e.g., gadolinium or gadoteridol.

[0060] In some aspects, any of the compositions disclosed herein are administered to any one or more chambers of the brain via intracerebroventricular (ICV) administration. In some aspects, the composition is administered unilaterally to one chamber, e.g., the left or right ventricle, via ICV administration. In some aspects, the composition is administered unilaterally to the left ventricle via ICV administration. In some embodiments, the composition is administered unilaterally to the right ventricle via ICV administration. In some aspects, the composition is administered bilaterally, e.g., to both the left and right ventricles, via ICV administration. In some embodiments, the composition is administered to only one chamber of the brain, e.g., the left ventricle, via ICV administration. In some aspects, the composition is administered to only the left ventricle via ICV administration. In some aspects, the composition is administered to only the right ventricle via ICV administration. In some aspects, the composition is administered to only the third chamber via ICV administration. In some aspects, the composition is administered to only the fourth chamber via ICV administration. In some embodiments, the composition is administered via ICV administration to two or more chambers of the brain, for example, the left ventricle, the right ventricle, and the third ventricle. In some embodiments, the composition is administered via ICV administration, for example, to the left ventricle and the right ventricle at the same time. In some embodiments, the composition is administered via ICV administration, for example, to the left ventricle and the right ventricle at different times sequentially. In some embodiments, each dose of the composition is administered via ICV administration at least 24 hours apart.

[0061] In some aspects, the disclosure provides a method of administering a composition to a primate, comprising intracerebroventricular (ICV) administration of a composition to the primate, wherein the composition comprises a vector capable of expressing a transgene, wherein the ICV administration results in at least 1.25-fold increased transgene expression in the central nervous system (CNS) compared to expression of the transgene when the composition is administered by any other route of administration. In certain aspects, the ICV administration results in at least 1.5-fold, 1.75-fold, 2-fold, or 3-fold increased expression of the transgene sequence in the central nervous system (CNS) compared to expression of the transgene when the composition is administered by any other route of administration. In some embodiments, ICV administration results in greater than 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, or 75-fold, or at least 20-90-fold, 20-80-fold, 20-70-fold, 20-60-fold, 30-90-fold, 30-80-fold, 30-70-fold, 30-60-fold, 40-90-fold, 40-80-fold, 40-70-fold, 40-60-fold, 50-90-fold, 50-80-fold, 50-70-fold, 50-60-fold, 60-90-fold, 60-80-fold, 60-70-fold, 70-90-fold, 70-80-fold, 80-90-fold. In some embodiments, ICV administration results in gene transfer throughout the brain. In certain embodiments, gene transfer occurs in the frontal cortex, parietal cortex, temporal cortex, hippocampus, medulla, and occipital cortex. In certain embodiments, gene transfer is dose-dependent.

[0062] In certain aspects, the composition comprises a vector further comprising a cell-type-selective regulatory element. In certain aspects, the regulatory element is selectively expressed in the brain. In certain aspects, the regulatory element is selectively expressed in the frontal cortex, parietal cortex, temporal cortex, hippocampus, medulla, and occipital cortex. In certain embodiments, the regulatory element is selectively expressed in the spinal cord. In certain aspects, the regulatory element is selectively expressed in the spinal cord and dorsal root ganglia. In certain aspects, the regulatory element is selectively expressed in neuronal cells. In certain aspects, the neuronal cells are selected from the group consisting of unipolar, bipolar, multipolar, or pseudounipolar neurons. In certain aspects, the neuronal cells are GABAergic neurons. In certain aspects, the regulatory element is selectively expressed in glial cells. In certain aspects, the glial cells are selected from the group consisting of astrocytes, oligodendrocytes, ependymal cells, Schwann cells, and satellite cells. In certain aspects, the regulatory element is selectively expressed in non-neuronal cells.

[0063] The present disclosure contemplates methods of administering a composition disclosed herein to a subject (e.g., a primate) by multiple routes of administration. For example, the present disclosure provides methods of administering a composition disclosed herein by one route of administration (e.g., intraventricular administration) and the same composition by another route of administration (e.g., intravenous administration). The present disclosure further provides methods of administering a composition disclosed herein by intraventricular administration and the same composition also by intravenous administration. In some aspects, the present disclosure provides methods of administering a composition disclosed herein by intrathecal administration and the same composition also by intravenous administration. In some aspects, the present disclosure provides methods of administering a composition disclosed herein by one route of administration (e.g., intraventricular administration) and an additional therapeutic agent (e.g., any of the additional therapeutic agents disclosed herein) by another route of administration (e.g., intravenous administration). In some aspects, methods of administering a composition disclosed herein by intraventricular administration and an additional therapeutic agent by intravenous administration. In some aspects, the present disclosure provides methods of administering a composition disclosed herein by intrathecal administration and an additional therapeutic agent by intravenous administration. In some aspects, the present disclosure provides methods of administering a composition disclosed herein by intravenous administration and an additional therapeutic agent by intraventricular administration. In some aspects, the present disclosure provides methods of administering a composition disclosed herein by intravenous administration and an additional therapeutic agent by intrathecal administration. In some aspects, the intrathecal administration comprises intrathecal cisternal administration. In some aspects, the intrathecal administration comprises intrathecal lumbar administration. In some aspects, the route of administration is any one or combination of intravenous, intrathecal, intraventricular, or intraparenchymal administration.

[0064] In some embodiments, the route of administration is any one or combination of subcutaneous, intramuscular, intraarterial, intraperitoneal, or intracranial administration.

[0065] In some aspects, administering comprises administering via injection. In some aspects, administering comprises administering via a cannula. In some aspects, the composition is administered as a bolus, e.g., as a single injection. In some embodiments, the composition is administered continuously, e.g., by infusion using a syringe pump.

[0066] In some aspects, intraventricular (ICV) administration involves inserting a cannula through a hole in the skull, through brain tissue, and into a CSF-filled ventricle of the brain. In some embodiments, a single cannula is inserted (e.g., into either of two lateral ventricles). In some aspects, two cannulas may be inserted (into both lateral ventricles). In some aspects, the cannula may be connected to a controlled device, such as a syringe or infusion pump for single administration, or an Ommaya reservoir. In some aspects, the present disclosure provides for administration of any of the vectors disclosed herein into one or more lateral ventricles of a subject. Due to concerns about neurovascular injury and intracranial hemorrhage, repeated "chipping" of ventricles is not routinely performed. An exception to this rule may be in premature infants, who often have very large ventricles, thin cortical mantles, and open fontanelles during pathological conditions, lowering the cumulative risk of repeated chips in this population.

[0067] Intrathecal intracisternal injection is less frequently performed in humans due to the cisterna magna's proximity to vital brain tissue. However, in some embodiments, an intrathecal injection device (e.g., a Medtronic device) can be inserted into the lumbar subarachnoid space, followed by a catheter extending upward toward the skull for administration. In some aspects, intrathecal administration in humans involves surgically inserting a catheter near the L4 / L5 interspace and administering either (i) a bolus dose (via a syringe or Ommaya reservoir), (ii) a short-term infusion (via a pump), or (iii) a long-term infusion (via an implantable programmable pump system, e.g., Synchromed II, Medtronic, where the pump is placed in a subcutaneous pocket somewhere in the body, such as the abdominal region). See, e.g., Hamza M, et al. Neuromodulation, 2015;18(7):636-48).

[0068] In some embodiments, the intrathecal administration of any of the compositions disclosed herein comprises administering the composition into the lumbar cistern by lumbar puncture. In some embodiments, spinal tipping can be performed at the bedside under sterile conditions using a local anesthetic. In some embodiments, the spinal needle is advanced through the interlaminar space in the lower lumbar spine into the thecal sac. In some embodiments, access to the lumbar cistern is confirmed when CSF is obtained. See, for example, Cook AM, et al. Pharmacotherapy. 2009; 29(7):832-45.

[0069] In some aspects, the compositions disclosed herein are administered to a subject (e.g., a primate) by injecting the composition through a spinal needle. This technique is frequently used for the administration of chemotherapy. The advantages of this technique include its relatively low risk and the ability to perform it at the bedside under local anesthesia. The disadvantage of this technique is that a separate puncture must be performed each time a dose is administered, resulting in the cumulative risk of introducing infection, developing a skin-CSF fistula, damaging nerve roots, and causing intraspinal bleeding. In some embodiments, to avoid this problem, a temporary indwelling catheter can be placed using a similar technique with a larger Tuohy needle.

[0070] In some embodiments, the compositions disclosed herein can be administered to a subject (e.g., a primate) by advancing a catheter through the center of a needle into the subject's thecal sac, after which the needle is withdrawn. In some embodiments, the catheter is then advanced subcutaneously through the skin, providing sterile access for the planned dose of the selected intrathecal drug. Disadvantages of this technique include the risk of infection due to prolonged catheter placement and catheter malfunction due to blockage, kinking, or displacement. However, these drawbacks can be mitigated by removing or replacing the catheter after several days (e.g., 1-4 days).

[0071] In some embodiments, the compositions disclosed herein are administered via a catheter-based device. In some embodiments, a permanent catheter-based device is implanted. In some embodiments, a temporary catheter-based device is implanted. In some embodiments, a catheter connected to a subcutaneous reservoir (e.g., an Ommaya reservoir) is implanted for permanent access. In some embodiments, the catheter is connected to the Ommaya reservoir. The Ommaya reservoir can be repeatedly accessed at the bedside by sterile puncture through the scalp into the reservoir using a 25-gauge needle. In some embodiments, several milliliters of CSF are withdrawn before injecting the therapeutic agent. Contamination and infection of the Ommaya reservoir are a risk, but may be less likely than other methods of accessing the intraventricular compartment (approximately 10% of patients ultimately have bacterially contaminated CSF). In case series reporting infectious complications with the Ommaya reservoir, infection rates often appear higher due to the longer duration of implantation (often exceeding one year) compared to other, more temporary access devices. Other rare complications that can occur with the Ommaya reservoir include leukoencephalopathy, white matter necrosis, and intracerebral hemorrhage.

[0072] In situations requiring limited access to the CSF space, a ventriculostomy can be placed. In this technique, a catheter is threaded under the skin away from the burr hole. The catheter is typically connected to a sterile collection chamber. The catheter allows for aseptic access as needed for administration of any of the vectors disclosed herein. In some embodiments, the compositions described herein can be administered by injecting a solution into the most proximal port of the ventriculostomy and flushing the solution into the brain with a small amount of saline (3-5 mL). After this instillation, the ventriculostomy tubing is typically clamped for at least 15 minutes to allow the injected solution to equilibrate in the CSF before drainage is resumed. Patients with persistently elevated intracranial pressure may not tolerate abrupt cessation of CSF drainage, so clamping of the ventriculostomy should be performed carefully and the patient closely monitored. The ventriculostomy is ideal for conditions requiring a limited period for CSF drainage or intraventricular administration of any of the vectors disclosed herein.

[0073] In some aspects, the disclosure provides a method of administering a composition disclosed herein to a subject, wherein the subject is a primate. In some aspects, the primate is a human. In some aspects, the primate is a non-human primate. In some aspects, the non-human primate is an Old World monkey, an orangutan, a gorilla, a chimpanzee, a cynomolgus monkey, a rhesus monkey, or a pig-tailed macaque.

[0074] The present disclosure contemplates methods of treating a subject (e.g., a human or a primate such as a cynomolgus monkey) in need of treatment, comprising administering to the subject any of the nucleic acids, vectors, viral particles, and / or compositions disclosed herein.

[0075] In certain embodiments, the compositions provided herein comprise an "effective amount" or "therapeutically effective amount" of an active agent (e.g., rAAV). As used herein, such amount refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.

[0076] The dosage of the compositions of the present disclosure depends on factors including the route of administration, the disease being treated, and the subject's physical characteristics (e.g., age, weight, general health). The dosage can be adjusted to provide a desired therapeutic response. Typically, the dosage can be an amount that effectively treats the disease without inducing significant toxicity. In one embodiment, the AAV vectors provided herein are administered to patients for the treatment of neurological diseases (e.g., including Dravet syndrome) at doses of 5×10 10 ~1×10 14 The AAV vector can be administered in an amount or dose in the range of gc / kg (genome copies per kilogram of patient body weight (gc / kg)). In a more specific embodiment, the AAV vector is administered in an amount of about 5×10 10 gc / kg ~ approx. 1×10 13 gc / kg, or approximately 1 x 10 11 ~Approx. 1×10 15 gc / kg, or approximately 1 x 10 11 ~Approx. 1×10 14 gc / kg, or approximately 1 x 10 11 ~Approx. 1×10 13 gc / kg, or approximately 1 x 10 11 ~Approx. 1×10 12 gc / kg, or approximately 1 x 10 12 ~Approx. 1×10 14 gc / kg, or approximately 1 x 10 12 ~Approx. 1×10 13 gc / kg, or approximately 5 x 10 11 gc / kg, 1 × 10 12 gc / kg, 1.5 × 10 12 gc / kg, 2.0 × 10 12 gc / kg, 2.5 × 10 12 gc / kg, 3 × 10 12 gc / kg, 3.5 × 10 12 gc / kg, 4 × 10 12 gc / kg, 4.5 × 10 12 gc / kg, 5 × 10 12 gc / kg, 5.5 × 10 12 gc / kg, 6 × 10 12 gc / kg, 6.5 × 10 12 gc / kg, 7 × 10 12gc / kg, 7.5 × 10 12 gc / kg, 8 × 10 12 gc / kg, 8.5 × 10 12 gc / kg, 9 × 10 12 gc / kg, 9.5 × 10 12 gc / kg, l x 10 13 gc / kg, 1.5 × 10 13 gc / kg, 2.0 × 10 13 gc / kg, 2.5 × 10 13 gc / kg, 3 × 10 13 gc / kg, 3.5 × 10 13 gc / kg, 4 × 10 13 gc / kg, 4.5 × 10 13 gc / kg, 5 × 10 13 gc / kg, 5.5 × 10 13 gc / kg, 6 × 10 13 gc / kg, 6.5 × 10 13 gc / kg, 7 × 10 13 gc / kg, 7.5 × 10 13 gc / kg, 8 × 10 13 gc / kg, 8.5 × 10 13 gc / kg, 9 × 10 13 gc / kg, or 9.5 x 10 13 In another aspect, the AAV vectors provided herein can be administered to a patient for the treatment of neurological disorders (including, for example, Dravet syndrome) in an amount or dose that is dependent on the volume of the patient's CSF. The AAV vectors provided herein (e.g., provided in the disclosed compositions) can be administered to a patient in an amount that is within the range of 5×10 11 ~1×10 12 The AAV vector may be administered in an amount or dose in the range of gc / ml of estimated CSF volume (genomic copies per ml of estimated CSF volume in the patient (gc / ml)). In a more specific embodiment, the AAV vector is administered in an amount or dose in the range of about 5×10 10 gc / ml ~ approx. 1×10 13 gc / ml, or approximately 1 x 10 11 ~Approx. 1×10 15 gc / ml, or approximately 1 x 10 11 ~Approx. 1×10 14 gc / ml, or approximately 1 x 10 11 ~Approx. 1×1013 gc / ml, or approximately 1 x 10 11 ~Approx. 1×10 12 gc / ml, or approximately 1 x 10 11 ~Approx. 1×10 14 gc / ml, or approximately 1 x 10 11 ~Approx. 1×10 13 gc / ml, or approximately 5 x 10 11 gc / ml ~ approx. 1×10 12 gc / ml, or approximately 3 x 10 11 gc / ml ~ approx. 2.0×10 12 gc / ml, or approximately 2.5 x 10 11 gc / ml ~ approx. 3×10 12 In some embodiments, the AAV provided herein is administered in an amount falling within the range of about 5×10 11 gc / ml, or approximately 1 x 10 12 The composition may be administered in an amount of gc / ml. The gc / kg or gc / ml may be determined, for example, by qPCR or digital droplet PCR (ddPCR) (see, e.g., M. Lock et al., Hum Gene Ther Methods. 2014 Apr;25(2):115-25). In another aspect, the compositions comprising the AAV vectors provided herein are administered to patients for the treatment of neurological disorders (including, e.g., Dravet syndrome) at doses of 1 x 10 9 ~1×10 11 The composition can be administered in an amount or dose in the range of 1×10 iu / kg (infectious units (iu) of vector / kg body weight of subject or patient). In certain embodiments, the composition can be formed into unit doses as needed. Such a single dosage unit can be about 1×10 9 gc ~ approx. 1×10 16 gc, approx. 1×10 10 gc ~ approx. 1×10 15 gc, approx. 1×10 2 gc ~ approx. 1×10 15 gc, approx. 5×10 13 gc~approx. 5×10 14 gc, approx. 5×10 13 gc~approx. 1.4×10 14 gc, approx. 9×10 13 gc ~ approx. 2×10 14 gc, or approximately 1 × 10 14gc~approx. 1.5×10 14 It may contain gc.

[0077] The compositions of the present disclosure may be administered to a subject in need thereof one or more times (e.g., 1 to 10 or more times) for example, daily, weekly, monthly, semi-annually, yearly, or as medically indicated. In exemplary embodiments, a single administration is sufficient. The compositions, in various aspects of the present disclosure, are suitable for use in human subjects and are administered by intraventricular administration. In various aspects, the compositions are suitable for use in human subjects and are administered by intraventricular, intravenous, intrathecal, intraparenchymal, or combinations thereof. In various aspects, the compositions are delivered via a peripheral vein by bolus injection. In various aspects, the compositions are delivered via a peripheral vein by infusion over about 10 minutes (±5 minutes), about 20 minutes (±5 minutes), about 30 minutes (±5 minutes), about 60 minutes (±5 minutes), or about 90 minutes (±10 minutes). In various aspects, the compositions are delivered to the CSF by bolus injection. In various embodiments, the composition is delivered to the CSF by infusion over about 10 minutes (±5 minutes), over about 20 minutes (±5 minutes), over about 30 minutes (±5 minutes), over about 60 minutes (±5 minutes), or over about 90 minutes (±10 minutes).

[0078] The present disclosure further provides kits comprising the compositions described herein and instructions for use. The compositions, in various aspects of the disclosure, are provided as sterile compositions for administration to a subject. In this regard, the compositions may be "pharmaceutical compositions," i.e., compositions suitable for administration to a subject, such as a human.

[0079] Optionally, the composition is present in a delivery device, a container for storage or transport or administration, or a container suitable for use in drug substance or drug product manufacturing. The kit can include a container that is a single-use container (i.e., a container that holds enough increments to ensure that, in addition to the single-dose formulation, a complete single dose can be administered to a patient from the container, but is not so redundant that the container can be used to administer a second dose) or a multi-use container (e.g., a vial, syringe, or infusion bag). The container can be a drug delivery device (e.g., a syringe), or a container for storage or transport or administration (e.g., a vial or bag).

[0080] Alternatively, a kit may include one or more containers containing the composition and instructions for use in manufacturing or preparing a drug substance or drug product. Indeed, the composition may be used at any step in the manufacturing process before filling into a final storage container (e.g., a vial, syringe, or infusion bag). The present disclosure contemplates a container suitable for use in manufacturing prior to filling into a final container intended for distribution, the container containing a composition (as described herein) comprising a recombinant adeno-associated virus (AAV) vector containing a heterologous nucleic acid, sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, and a non-ionic surfactant at a pH of 7.2-7.4. For example, a kit may include one or more containers (e.g., a vial, syringe, or infusion bag) suitable for use in a filtration system or a system for filling parts of a drug delivery system, the container containing a composition described herein. The kits described herein may include separate containers containing one or more of the composition components (rAAV vector, sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, and / or non-ionic surfactant). For example, the kit may include a container containing sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, and a non-ionic surfactant, and a separate container containing rAAV.

[0081] The present disclosure further contemplates a method of making a pharmaceutical composition, the method comprising: (i) combining a mixture comprising: (a) sodium chloride, optionally from about 145 mM to about 150 mM sodium chloride; (b) potassium chloride, optionally from about 1.5 mM to about 4.5 mM potassium chloride; (c) magnesium chloride, optionally from about 0.05 mM to about 1 mM magnesium chloride; (d) a phosphate buffer (e.g., sodium phosphate), optionally present in an amount sufficient to provide from about 0.5 mM to about 2 mM (e.g., about 1 mM) phosphate; and (e) a non-ionic surfactant (e.g., a poloxamer such as Poloxamer 188), optionally present at a concentration ranging from about 0.001% to about 0.01% (w / v); and (ii) an rAAV vector. Optionally, the resulting pharmaceutical composition has a concentration of about 5×10 13 vg / mL ~ approx. 1×10 14 vg / mL (e.g., approximately 8 × 10 13 vg / mL) of the recombinant AAV vector. Optionally, the resulting pharmaceutical composition also comprises a pH of 7.2 to 7.4.

[0082] Generally, "sequence identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity to a reference sequence (e.g., a nucleic acid sequence or an amino acid sequence) can be calculated by dividing the number of exact matches between two optimally aligned sequences by the length of the reference sequence and multiplying by 100. When determining the number of matches for sequence identity, conservative substitutions are not considered matches. It will be understood that if the length of a first sequence (A) is not equal to the length of a second sequence (B), the percent identity of the A:B sequence will differ from the percent identity of the B:A sequence. Sequence alignment, such as for purposes of assessing percent identity, can be performed using any suitable alignment algorithm or program, including, but not limited to, the Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle aligner available on the World Wide Web at ebi.ac.uk / Tools / psa / emboss_needle / ), the BLAST algorithm (see, for example, the BLAST alignment tool available on the World Wide Web at blast.ncbi.nlm.nih.gov / Blast.cgi), the Smith-Waterman algorithm (see, for example, the EMBOSS Water aligner available on the World Wide Web at ebi.ac.uk / Tools / psa / emboss_water / ), and the Clustal Omega alignment program (see, for example, the World Wide Web at clustal.org / omega / and F. Sievers et al., Mol Sys Biol. 7:539 (2011)). Optimal alignment can be assessed using any suitable parameters for the selected algorithm, including default parameters.The BLAST program is based on the alignment method of Karlin and Ahschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), as discussed in Ahschul, et al., J. Mol. Biol. 215:403-410 (1990), Karlin and Ahschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993), and Ahschul et al., Nucleic Acids Res. 25:3389-3402 (1997). [Example]

[0083] The following examples are presented merely to illustrate the present invention and are not intended to limit its scope.

[0084] Example 1: Recombinant AAV Sample Preparation Recombinant AAV (rAAV) for the studies described herein was produced in HEK-293 T cells by cotransfection of three plasmids: (i) a vector expressing the AAV Rep and Cap genes, (ii) the pALD-X80 adenovirus helper plasmid (Aldevron), and (iii) a plasmid containing the AAV genome to be packaged. The transgene encoded by the AAV genome plasmid is an engineered transcriptional activator (eTF) containing a DNA-binding domain with known DNA-binding specificity (described in International Patent Publication No. 2019 / 109051, entitled "Engineered DNA Binding Proteins," incorporated herein by reference in its entirety). The rAAV capsid serotype is rAAV9. After post-transfection incubation, cells and supernatants were harvested and processed for rAAV concentration and diafiltration using a Millipore-Sigma Amicon Ultra-15 centrifuge unit (Millipore-Sigma P / N UFC910024) with a 100 kilodalton (kD) molecular weight cutoff (MWCO) at 3000 × g. The viral genomes / mL (vg / mL) of each concentrated sample was approximated using UV absorbance. A buffer exchange was performed on the diafiltration-concentrated samples (approximately 10-fold), after which the samples were sterile filtered. The final concentration was reconfirmed by UV and subsequently tested via ddPCR as described by Lock et al. (Hum. Gene Ther. 2010;21:1273-1285) using forward and reverse PCR primers and a hybridization probe specific for the packaged rAAV genome.

[0085] Example 2: Assay A series of assays were used to assess the stability of rAAV in multiple sample formulations at time point 0 (t=0) and across multiple stress conditions. The assays and attributes they were designed to measure are provided below.

[0086] Size exclusion chromatography Size-exclusion chromatography (SEC or SE-HPLC) is a liquid chromatography technique that separates species in a sample based on molecular size. rAAV samples were analyzed under non-dissociating conditions by injection onto a Sepax SRT SEC-1000 column using a mobile phase of 2x DPBS and 10% ethanol. Species were eluted in order of decreasing molecular size (i.e., species eluted from largest to smallest). Species eluted from the column were analyzed at two wavelengths: 260 nm, which detects rAAV genomic DNA (vg), and 280 nm, which detects rAAV capsids (cp). SEC can detect intact rAAV monomers as well as species larger or smaller than rAAV monomers. These are generally referred to as "high molecular weight species" (HMWS) and "low molecular weight species" (LMWS). HMWS include aggregates of rAAV / rAAV components, while LMWS include empty rAAV capsids, unencapsidated viral genomes, and their subparts. HMWS and LMWS are considered non-functional in this assay and therefore represent undesired species in the sample. Therefore, SEC provides a sensitive method to monitor virus stability under different stress conditions.

[0087] Reversed-phase HLPC Reverse-phase HPLC (RP-HPLC) is a liquid chromatography technique that dissociates and separates proteins in order of increasing hydrophobicity. Samples were injected onto a C3 column in an acetonitrile gradient in the presence of the ion-pairing agent trifluoroacetic acid (TFA, 0.2%). The relative concentrations of rAAV capsid proteins VP1, VP2, and VP3 were quantified in each sample using the area under the curve with UV detection at 280 nm.

[0088] Polydispersity and external DNA The UNcle system from UNchained Labs was used to assess particle polydispersity (i.e., the distribution of particle sizes within a sample) as well as the amount of external DNA (i.e., DNA not packaged into rAAV capsids). Specific assays were performed during thermal transitions (e.g., 15°C to 95°C) or at specific holding temperatures, as indicated.

[0089] The UNcle system uses dynamic light scattering (DLS) to measure the polydispersity of particles in a sample, representing how much material is present in different sized "slices" of the sample. In DLS, the original distribution is an intensity distribution that shows how much light is scattered from various sized "slices" or "bins." The mean size and standard deviation from that mean can be obtained directly from the distribution statistics. The (absolute) standard deviation (or "half-width") of the distribution can be compared to the mean to obtain the relative polydispersity (standard deviation / mean). Historically, instead of requiring a distribution, a simpler forced monoexponential fitting scheme (cumulative method) has been used to find the overall mean size (by intensity) and overall polydispersity (a normalized second cumulative quantity). For a theoretical Gaussian distribution, the overall polydispersity would be the relative polydispersity of the distribution. Traditionally, this overall polydispersity has also been converted to the overall polydispersity index (PDI), which is the square of the light scattering polydispersity. A perfectly homogeneous sample would have a PDI of 0.0. A PDI of 0.1 or less is generally considered monodisperse, a PDI of 0.1 to 0.4 is considered moderately polydisperse, and a PDI above 0.4 is considered broadly polydisperse.

[0090] External fluorescence in the presence of Sybr Gold measures the level of external, non-encapsidated DNA in the sample, which can be in the form of non-packaged DNA present in the sample (DNA co-purified with the rAAV during the manufacturing process) as well as packaged DNA released from rAAV capsids in the sample, e.g., during storage, retention, and / or stress conditions.

[0091] Example 3: Sample analysis In this example, heat stress was applied to different rAAV sample formulations, followed by analyses that monitored several physical and biochemical properties indicative of rAAV stability (described in Example 2).

[0092] Analysis was performed on 11 different rAAV sample formulations (Table 1), all stored at -70°C or below. Samples were subjected to different stress conditions and analyzed using one or more of the assays described in Example 2. Stress conditions included freeze / thaw (FT) cycles (from -70°C or below to room temperature (RT) for the indicated number of times) and incubation at RT or 37°C for the indicated number of days. As shown in Table 1, the sample formulations differed with respect to the presence / amount of base buffer and three different components: Kolliphor® P188 (BASF), MgCl2, and trehalose. Samples 10 and 11 had higher AAV titers than samples 1-9. [Table 1]

[0093] Assessment of the presence of viral genomes in each sample by ddPCR analysis showed no discernible differences between samples under either t=0 or stress conditions, indicating that genomic DNA was not degraded (data not shown). Note that this assay does not provide any information regarding the infectivity of rAAV present in the sample or whether the genomic DNA is encapsidated.

[0094] Size exclusion chromatography The results of the SEC analysis are shown in Figures 1 and 2. As shown in these figures, the percent loss of vg, as represented by the loss of absorbance at 260 nm (Figure 1), and the percent loss of cp, as represented by the loss of absorbance at 280 nm (Figure 2), were most pronounced in Samples 1 and 3 when stressed at 37°C. Both of these samples contain lower amounts of P188 (0.001%).

[0095] Sample 1, the Buffer 1-based formulation, showed the most instability, with vg losses of 5.5% and 7.9% at 37°C on days 3 and 7, respectively, and cp losses of 6.0% and 8.6% at 37°C on days 3 and 7, respectively.

[0096] The buffer 2 based sample, Sample 3, performed slightly better than Sample 1, showing a 6.5% loss of vg and cp after 7 days at 37°C.

[0097] Samples 5, 9, 6, and 10 exhibited the highest stability of the samples tested in this assay and had the least loss of vg and cp. Samples 5 and 9 contained MgCl, while samples 6 and 10 contained both MgCl and trehalose. 6xFT stress conditions, as well as 7-day incubation at RT, had little or no apparent effect on cp or vg loss in any of the samples tested.

[0098] Reversed-phase HLPC Figure 3 shows RP-HPLC results for a representative rAAV sample at t=0, 17 days of incubation at RT, and 7 days of incubation at 37°C. At t=0 and 17 days of incubation at RT, five peaks (P1–P4 and P6) are observed. At the more stressful 37°C incubation, a sixth peak (P5) becomes apparent (the shoulder behind P4 in Figure 3). This peak was shown to increase in size as a function of time (data not shown). P1 and P3 also increase under heat stress conditions, while P2, P4, and P6 decrease. P2 and P3 are approximately equimolar and therefore likely represent VP1 and VP2, while P4, the most abundant and highest abundant, likely represents VP3 (given their expected ratio in the AAV capsid). The apparent increase in P1 and P3 under heat stress conditions may represent alterations in VP1 and VP2 (e.g., deamidated and / or oxidized forms) that co-elute with their unaltered counterparts.

[0099] Samples were subjected to each of the above stress conditions and quantified by RP-HPLC for P1–P6. The fold change (increase or decrease) of each peak was determined compared to their zero-time point counterparts. For each sample and treatment, the fold changes of P1, P3, and P5 relative to t=0 are shown in Figures 4, 5, and 6, respectively. As shown in these figures, there were no significant changes in P1, P3, or P5 after 7 days of treatment at 6×FT or RT, indicating stability in all tested formulations through six freeze-thaw cycles or 7 days at room temperature. After 14 days at room temperature, P1 and P2 showed greater increases in samples 1, 2, 8, 9, and 10 than in samples 3–7, indicating that the Buffer 2-based formulation was more stable at room temperature. Under higher heat stress conditions (3 days at 37°C), all formulations showed increases in P1 and P2, with the greatest increases in the Buffer 1-based formulations (1 and 2), followed by the Tris-based formulations (8–11). After 3 days at 37°C, P5 remained unchanged in the Buffer 2-based formulations (3–7) and increased in both the Buffer 1-based formulations (1 and 2) and three of the four Buffer 3-based formulations (9–11). Under the highest heat stress conditions tested (7 days at 37°C), all formulations showed increases in P1, P3, and P5. The Buffer 1-based formulations showed the greatest increases in all three peaks, indicating that these formulations had the lowest stability at 37°C. For P3 and P5, the lowest increases were observed in the Buffer 2-based formulations, while higher increases were observed in all Buffer 3-based formulations. For P1, the lowest increases were observed in the Buffer 2-based formulations, Formulations 6 and 4. These results indicated that rAAV was more stable in the Buffer 2-based formulations (3–7) than in the Buffer 1-based (1 and 2) or Buffer 3-based (8–11) formulations.

[0100] DLS analysis using the UNcle system was used to determine the particle size (or Z-average) and polydispersity index (PDI) for each sample as a function of holding and stress conditions. In general, all samples showed relatively uniform Z-average at t = 0 and under all stress conditions at 15 °C (data not shown). Although there was some variation in PDI at 15 °C, all samples had a PDI of less than 0.1 (data not shown). However, differences between samples were observed when examined at 95 °C.

[0101] As shown in Figure 7, the Z-averages for Samples 5 and 6 were the lowest of all formulations tested, with very narrow statistical variability, indicating that they were the most stable formulations in this study. Samples 1-4 and 7-11 formed larger aggregates or oligomers than Samples 5 and 6 when stressed, and Buffer 3-based Samples 8 and 9 exhibited the lowest stability under stress conditions. As shown in Figure 8, Samples 5 and 6 had favorable statistical variability in PDI. Only Sample 1 had significantly narrow statistical variability in PDI. However, Sample 1's significantly higher Z-average indicated that it was a less effective formulation for rAAV stability. In summary, the Buffer 2-based sample with 0.005% P188 and MgCl2 performed best in this assay.

[0102] Based on the results of the above assays, Buffer 2-based formulations were superior to both Buffer 1- and Buffer 3-based formulations, with Samples 5 and 6 demonstrating the highest level of heat stress stability for rAAV. Formulation Samples 5 and 6 demonstrated high levels of stability in SEC and RP-HPLC analyses and narrow statistical variations under stress conditions in the PDI assay. Both of these sample formulations contain 0.005% P188 and 0.8 mM MgCl.

[0103] Example 4: Comparative study of two rAAV formulations Two rAAV formulations were prepared for further testing. The formulation components of Formulation 1 and Formulation 2 are provided in Table 2. Both formulations contained the same base buffer (1 mM sodium phosphate, 148 mM sodium chloride, 3 mM potassium chloride, 0.8 mM magnesium chloride, pH 7.30). The difference between Formulation 1 and Formulation 2 was the addition of 0.05% w / v trehalose in Formulation 2. Both formulations (Formulation 1 and Formulation 2) were evaluated for vector genome titer by digital droplet polymerase chain reaction (ddPCR), aggregation by size-exclusion high-performance liquid chromatography (SE-HPLC), characterization by reverse-phase high-performance liquid chromatography (RP-HPLC), in vitro potency, thermal transition analysis by UNcle and dynamic light scattering (DLS), and subvisible particle (SVP) analysis. The conditions tested are summarized in Table 3. [Table 2] [Table 3]

[0104] Vector genome titer Vector genome titers were measured by UV absorbance and ddPCR. Initial vector concentrations were determined by UV absorbance to achieve final target concentrations. After initial concentration measurements, vector genome titers were determined for the formulation degradation conditions using ddPCR. ddPCR analysis produced genome titer data that was consistent across both formulations and within assay variability. There were no discernible differences in their respective performance across the two formulations and across various stress and retention conditions. For the degradation conditions listed in the table, vector genome titer results by ddPCR are graphed in Figure 9. Concentrations ranged from 1.3E14 to 1.5E14 vg / mL.

[0105] agglomeration Aggregation of rAAV in the two formulations was measured by size-exclusion high-performance liquid chromatography (SE-HPLC) after a series of different sample treatments (storage at -70°C for 1 month, storage at 4°C for 1 week or 1 month, storage at room temperature for 1 month, storage at 37°C for 2 weeks or 1 month, and 10 freeze-thaw cycles). Low molecular weight species (LMWS) were not observed, and therefore, comparisons were made between the percent high molecular weight species (HMWS) at A260 and A280 nm. The HMWS% at selected degradation conditions is graphed in Figure 10. Formulations 1 and 2 showed little or no increase in HMWS during the different storage conditions, and both formulations peaked at 3% or less HMWS after 10 freeze-thaw cycles. These results indicate good stability of rAAV in both formulations.

[0106] Characterization by reversed-phase HPLC (RP-HPLC) RP-HPLC was also used as a characterization method for the two tested formulations. Five peaks (P1-P4 and P6) were observed under non-heat stress conditions, and a sixth peak (P5) was evident under heat stress at 37°C, as shown in Figure 13, and increased in magnitude over time. In addition, peaks 1 (Figure 11) and 3 (Figure 12) increased as a function of heat stress, while peaks 2, 4, and 6 decreased. Peak 5 appears as a shoulder peak shifted behind peak 4. Peak 4, represented by VP3, is the most abundant.

[0107] Overall, the heat-stressed samples showed the largest fold changes relative to T=0 in peaks 1, 3, and 5. Formulation 2 showed larger fold changes among the identified peaks in the heat-stressed samples held at 2 weeks and 1 month.

[0108] In vitro efficacy In vitro potency was analyzed for room temperature / ambient stability, freeze-thaw, and heat stress conditions. Ten freeze-thaw cycles had little effect on potency. Formulations subjected to heat stress conditions showed a decrease in potency. Potency results for the degradation conditions listed in Table 3 are shown in Figure 14. Both formulations showed similar levels of potency under the different conditions.

[0109] Thermal transition analysis and dynamic light scattering (DLS) The UNcle system (Unchained Labs) is a system that allows multiple applications to characterize capsid particle stability as a function of formulation and disassembly conditions. DLS applications can be used to measure particle size, T, as a function of temperature. agg The aggregation temperature and polydispersity of capsid particles as a function of temperature were measured at both 15°C and 95°C. For monodisperse samples, the Z-average (nm) is recommended by the ISO standard for DLS analysis, and the measurement is independent of distribution bias, which can vary depending on the size class of the fitted model. However, the polydispersity index (PDI) is also displayed as a measure of the degree of monodispersity of the sample, whereas the Z-average (nm) is the overall size distribution of the sample. The size and polydispersity before the thermal gradient are indicators of sample quality at the start of the experiment, while the post-heat DLS confirms the degree of aggregation observed between the two formulations at their respective degradation conditions.

[0110] In another application, similar to temperature-controlled differential scanning fluorometry (DSF), a thermal transition assay was performed to assess the Tm (melting temperature) of capsid particles, measured using the DNA fluorescent stain Sybr Gold, as DNA leaks from the capsid during a thermal gradient. The assay is used to assess multiple Tm stability points and external fluorescence of capsid particles in the same assay. External fluorescence measures the initial fluorescence intensity of residual unencapsidated DNA present outside the capsid as a byproduct of the rAAV manufacturing process, vector genome concentration, formulation, and degradation conditions. rAAV in formulations exhibited two distinct biphasic Tm events. The first was a T onset or T m1This represents the first instance in which a detectable genome leaks from the capsid as a function of thermolability during a thermal gradient to 95°C. m2 where the entire capsid integration is lost and the entire genome is released into solution.

[0111] Z-average (T agg ) and PDI results are graphed in Figures 15 and 16. As shown in Figure 15, the Z-averages of Formulations 1 and 2 and the T based on their respective degradation conditions at both 15°C and 95°C. agg demonstrates similar aggregation between the formulations. The final measured value of polydispersity at 95°C was subtracted from the initial value at 15°C to normalize the degree of polydispersity observed from the start and end of the thermal gradient to compare between the formulations and degradation conditions. As shown in Figure 16, the results were similar for the two formulations.

[0112] For the degradation conditions listed in Table 3, external fluorescence, T onset / T m1 , and T m2 Evaluation of the thermal transition analysis using the results is shown in Figures 17, 18, and 19, respectively. External fluorescence was highest after 10 freeze-thaw cycles, summarizing the observation of increased high molecular weight species by SE-HPLC. Additionally, between the two formulations, there appears to be a higher level of fluorescence in Formulation 2 at 37°C, although the formulations appeared similar at other degradation conditions. onset / T m1 , and T m2 A difference of 2°C is considered significant in Tm. Both formulations behaved similarly when assessing degradation conditions according to this metric.

[0113] Subvisible Particle (SVP) Characterization SVPs were characterized using a Horizon system (Halo Labs, Burlingame, CA), which measures subvisible and visible particles at low volumes by using background membrane imaging (BMI) technology. First, an image is captured before the sample is added to the membrane, and then aggregates too large to pass through the membrane are captured and retained on the membrane for sizing and count distribution.

[0114] SVP analysis, as graphed in Figures 20 and 21, showed that particles larger than 2 μm were the most frequently observed subvisible particles across all samples and conditions, followed by 10 μm and 25 μm particles. The actual increase in SVP formation was more evident under 10 freeze-thaw cycles, where particles larger than 2 μm and larger than 10 μm increased compared to the other conditions. Formulation 2 generally had lower SVP content for particles larger than 2 μm compared to Formulation 1, specifically as a function of degradation under conditions below -70°C, 4°C, and 37°C. In Formulation 1, there was only a slight increase in particles larger than 25 μm as a function of the 10 freeze-thaw conditions, indicating that trehalose in Formulation 2 may have particular advantages as a cryoprotectant, reducing larger particle formation.

[0115] Formulation degradation considerations Overall, the differences between the two formulations were very slight, indicating that the specifically specified pH and ionic strength of the base formulation were sufficient to prevent significant aggregation.

[0116] Example 5: Comparative testing of additional rAAV formulations Further testing was performed using the formulations in Table 4. The different formulations in Table 4 were evaluated as described above. As shown in Figure 22, ddPCR analysis produced consistent genome titer data across all candidate buffer conditions, with no discernible loss of titer as a function of stress. Thermal transition analysis (T) by UNcle of formulation samples onset / T m1, Figure 23, and external fluorescence, Figure 24) showed similar results for samples 7 and 8, showing no advantage of higher poloxamer 188 concentrations in terms of genomic titer. Similar results were also observed for samples 3 and 5, showing no advantage to higher poloxamer 188 concentrations in terms of genomic titer. [Table 4]

[0117] Example 6: Poloxamer loss during processing An experiment was conducted to evaluate the loss of poloxamer 188 under manufacturing conditions. rAAV was produced as in Example 1. The rAAV material was concentrated to a target concentration using tangential flow filtration (TFF). Once the TFF filtrate reached the secondary concentration target, a 1% volume (v / v) of P188 loading buffer was added to the final TFF concentrate material to target a concentration of 0.005% P188 in the material. The conditioned material was passed through a 0.2 μm low-particle-removal filter. An aliquot of the final conditioned TFF concentrate was analyzed by ddPCR to determine the rAAV vector genome titer in the TFF concentrate.

[0118] The adjusted TFF concentrate was diluted with formulation dilution buffer to a target concentration of 8.0E13 vg / mL to produce the drug substance (DS), which was then filtered through a 0.2 μm filter into a 125 mL polycarbonate final container. The ddPCR test results from the TFF-B retentate sample were used to calculate the volume of formulation dilution buffer needed to reach the final DS target concentration. The required buffer was added to the adjusted TFF concentrate, then mixed and filtered through a 0.2 μm filter into the final container. The DS was frozen.

[0119] The drug product (DP) manufacturing process began with thawing the DS container in a 150 mL polycarbonate container. Once the DS was fully thawed, a potency sample was collected for testing by digital droplet polymerase chain reaction (ddPCR). The thawed DS was maintained at 2-8°C between testing intervals. The post-thaw DS potency value was used to determine the calculated volume of formulation dilution buffer to dilute 8.0E13 vg / mL of DS. Prior to dilution, the thawed DS was removed from 2-8°C conditions, returned to ambient conditions, and mixed. The entire contents of the thawed DS were transferred to a 250 mL polycarbonate bottle, and the calculated volume of formulation dilution buffer was added. All diluted DS was mixed again before proceeding to sterile filtration. Filtration occurred from the DS bottle into a sterile single-use fill bag.

[0120] The filtered bulk DP was filled under aseptic conditions using semi-automated filling equipment into sterile 2 mL Crystal Zenith® (CZ, cyclic olefin polymer) vials supplied by West Pharmaceutical Services (133MMX960). The concentration of poloxamer 188 in the 2 mL vials was measured and the results are shown in Table 5. Poloxamer was measured using HPLC-ELSD (High Performance Liquid Chromatography Evaporative Light Scattering Detector).

[0121] The results described herein demonstrate that manufacturing processes associated with the production of rAAV DP, such as those described herein, can result in the loss of surfactant (here, poloxamer). As shown in Table 5, the final poloxamer concentrations after processing steps ranged from 0.0034% to 0.0044%, representing approximately 60% to approximately 88% poloxamer recovery (i.e., approximately 12% to approximately 40% loss of surfactant during processing). [Table 5]

[0122] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0123] In the context of describing the present disclosure (particularly in the context of the claims that follow), the use of the terms "a," "an," and "the" and similar referents should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context, and the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The term "or" should be understood to include items in the alternative or together unless the context clearly requires otherwise. The term "and / or" should be understood to include each item in a list (individually), any combination of items in a list, and all items in a list together. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise stated. The present disclosure contemplates embodiments described as "comprising" a feature to include embodiments "consisting of" or "consisting essentially of" the feature. The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more, as is customary in the art. Alternatively, "about" can mean within a range of up to 10%, up to 5%, or up to 1% of a given value.

[0124] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated herein as if it were individually recited herein. In any of the ranges described herein, the endpoints of the range are included within the range. However, the description also contemplates the same range as excluding the lower and / or higher endpoints.

[0125] The steps of all methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as" and "optionally") provided herein is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] Table 6-8 Table 6-9 Table 6-10 Table 6-11 Table 6-12 Table 6-13 Table 6-14 Table 6-15 Table 6-16 Table 6-17 Table 6-18 Table 6-19 Table 6-20 Table 6-21 Table 6-22 Table 6-23 Table 6-24 Table 6-25 Table 6-26 Table 6-27 Table 6-28 Table 6-29 Table 6-30 Table 6-31 Table 6-32 Table 6-33 Table 6-34 Table 6-35 Table 6-36 Table 6-37 Table 6-38 Table 6-39 Table 6-40 Table 6-41 Table 7-1 Table 7-2

Claims

1. A composition comprising a recombinant adeno-associated virus (AAV) vector containing a heterologous nucleic acid, sodium chloride, potassium chloride, magnesium chloride, phosphate buffer, and a non-ionic surfactant at a pH of 7.2 to 7.

4.

2. 10. The composition of claim 1, exhibiting a conductivity of 15.0 to 17.0 mS / cm.

3. 3. The composition of claim 1, wherein the non-ionic surfactant is present at a concentration of about 0.001% to about 0.02% (w / V).

4. The composition of any one of claims 1 to 3, wherein the nonionic surfactant is a poloxamer.

5. The composition of claim 4, wherein the nonionic surfactant is poloxamer 188.

6. 6. The composition of claim 5, wherein the non-ionic surfactant is poloxamer 188 present at a concentration of about 0.001% (w / V) to about 0.005% (w / V).

7. 7. The composition of claim 6, wherein the non-ionic surfactant is poloxamer 188 present at a concentration of about 0.005% (w / V).

8. 8. The composition of claim 1, comprising about 145 mM to about 150 mM sodium chloride.

9. 9. The composition of any one of claims 1 to 8, comprising about 1.5 mM to about 4.5 mM potassium chloride.

10. 10. The composition of any one of claims 1 to 9, comprising about 0.05 mM to about 1 mM magnesium chloride.

11. 11. The composition of any one of claims 1 to 10, comprising about 148 mM sodium chloride, about 3 mM potassium chloride, and about 0.8 mM magnesium chloride.

12. 12. The composition of any one of claims 1 to 11, comprising a phosphate buffer in an amount sufficient to provide about 0.5 mM to about 2 mM phosphate.

13. 13. The composition of claim 12, comprising a phosphate buffer in an amount sufficient to provide about 1 mM phosphate.

14. The composition of any one of claims 1 to 13, wherein the phosphate buffer is sodium phosphate.

15. 10. The composition of claim 1, wherein the composition comprises 1 mM phosphate, 148 mM NaCl, 3 mM KCl, 0.8 mM magnesium chloride, and 0.005% poloxamer 188.

16. 10. The composition of claim 1, wherein the composition comprises 1 mM phosphate, 148 mM NaCl, 3 mM KCl, 0.8 mM magnesium chloride, and 0.01% poloxamer 188.

17. 17. The composition of claim 15 or 16, wherein the composition further comprises 0.05% trehalose.

18. Approximately 5×10 １３ vg / mL ~ approx. 1.5 x 10 １４ 18. The composition of any one of claims 1 to 17, comprising vg / mL of an AAV vector.

19. Approximately 8×10 １３ 20. The composition of claim 18, comprising an AAV vector at 1000 ng / mL.

20. The composition of any one of claims 1 to 17, wherein the composition does not contain calcium chloride.

21. The composition of any one of claims 1 to 20, wherein the AAV vector comprises a regulatory element that is active in neuronal cells.

22. 22. The composition of claim 21, wherein the regulatory element active in neurons is active in GABAergic neurons.

23. 20. The composition of claim 19, wherein the AAV vector comprises the sequence of any one of SEQ ID NOs: 81-112.

24. The composition of any one of claims 1 to 23, wherein the AAV vector comprises a therapeutic transgene.

25. 25. The composition of claim 24, wherein the therapeutic transgene is associated with a neurological disease or disorder.

26. 26. The composition of claim 25, wherein the therapeutic transgene is selected from (i) SCN1A, or (ii) a transcription factor that activates SCN1A.

27. The composition of any one of claims 1 to 26, wherein the AAV vector comprises the sequence of SEQ ID NO:

115.

28. 28. A method of treating a neurological disorder in a subject, said method comprising administering the composition of any one of claims 1 to 27 directly to the central nervous system of a subject in need thereof.

29. 29. The method of claim 28, wherein the method comprises administering the composition to the subject via intraventricular injection.

30. 30. The method of claim 28 or 29, wherein the neurological disorder is Dravet syndrome or epilepsy.

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