AAV pharmaceutical formulations and methods thereof

Abstract

Aspects of the disclosure relate to pharmaceutical formulations of AAV vectors.

Description

38061.0011P1 AAV PHARMACEUTICAL FORMULATIONS AND METHODS THEREOF REFERENCE TO SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (38061_0011P1.xml; Size: 9,679 bytes;and Date of Creation: March 8, 2024) is herein incorporated by reference in its entirety. FIELD OF THE INVENTION

[0002] The present disclosure relates to pharmaceutical formulations for delivering AAV.BACKGROUND

[0003] Wild-type Adeno-associated viruses (AAV) are small non-enveloped viruses in the genusDependoparvovirus. AAV requires a co-infecting helper virus, such as adenovirus, to replicate. The AAV genome is approximately 4.7kB, which inverted terminal repeat (ITR) sequences that flank two genes, cap and rep. The cap gene encodes structural proteins that make up the capsid. The rep gene encodes replicase proteins that are required for AAV replication and packaging. Recombinant AAVs (rAAV) in which a coding sequence for a therapeutic agent replaces the cap and rep genes in the AAV genome can be used as a gene therapy vector.

[0004] Recombinant adeno-associated viruses (rAAVs) represent are used as gene deliveryplatforms and have promise for even wider use because of their wide range of tissue tropism, low immunogenicity, highly efficient and sustained gene transduction, and clinically proven track record in safety. The recombinant AAV particles having the recombinant genome encoding the therapeutic of interest and are stored in and delivered to a subject via pharmaceutical compositions. However, there is a need to develop pharmaceutical compositions comprising AAV particles for administration to humans having greater stability. SUMMARY

[0005] Aspects of the disclosure relate to the development of formulation for administration ofAAV gene therapy. The AAV formulations described herein exhibit stability upon storage, including for 12 months, 24 months, or 36 months at -70°C, such that the formulation maintains38061.0011P1 (e.g., within 1%, 5%, 10% or 20%) purity, potency and strength relative to the formulation at time zero.

[0006] Aspects of the disclosure provide pharmaceutical compositions comprising: a) adeno-associated virus (AAV) particles (including recombinant AAV comprising a genome comprising a transgene encoding a therapeutic product); b) about 5 mM to about 25 mM of a buffering agent; c) about 0.5 mM MgCl2to about 1.5mM MgCl2; d) about 50 mM to about 150 mM of a tonicity agent; and e) about 0.02% of a non-ionic surfactant, wherein the pH of said pharmaceutical composition is between about 5.0 and about 9.0. In embodiments, the tonicity agent is NaCl. In embodiments, the formulation contains from 0.02% to 0.2% poloxamer 188, including from 0.02% to 0.1%, from 0.02% to 0.05%, or from 0.05% to 0.2% and values within these ranges. In embodiments, the formulation does not exhibit toxicity in animal, including mouse or rat, toxicity models.

[0007] Further aspects of the disclosure relate pharmaceutical compositions comprising: a)recombinant AAV9 particles; b) 10 mM Tris; c) 1mM MgCl2; d) 150 mM NaCl; and e) 0.02% poloxamer 188, wherein said pharmaceutical formulation has a pH of about 8.0.

[0008] Further aspects of the disclosure relate to methods of producing the AAV formulationsdescribed herein and of storing the AAV formulations described herein such that the formulations maintain potency, strength and / or purity relative to the AAV formulation prior to storage. Also provided are methods of administering the recombinant AAV formulations described herein to a subject for treatment, prevention or amelioration of an indication that is treatable with the therapeutic encoded in the AAV genome. EMBODIMENTS

[0009] Embodiment 1. A pharmaceutical composition comprising: a) an adeno-associated virus(AAV) particle; b) about 5 mM to about 25 mM of a buffering agent; c) about 0.5 mM MgCl2to about 1.5mM MgCl2; d) about 50 mM to about 150 mM of a tonicity agent; and e) from about 0.02% to about 0.2% of a non-ionic surfactant, wherein the pH of said pharmaceutical composition is between about 5.0 and about 9.0.

[0010] Embodiment 2. The pharmaceutical composition according to embodiment 1, whereinsaid AAV is AAV9 or comprises a capsid protein that is at least 90%, 95%, or 99% identical to the capsid protein of SEQ ID NO:5.38061.0011P1

[0011] Embodiment 3. The pharmaceutical composition according to embodiment 2, whereinsaid AAV is AAV9.

[0012] Embodiment 4. The pharmaceutical composition according to any one of embodiments 1to 3, wherein said AAV particle is present at a concentration of about 1×1011vg / mL to about 1×1015vg / mL.

[0013] Embodiment 5. The pharmaceutical composition according to embodiment 4, whereinsaid AAV particle is present at a concentration of about 1×1014 vg / mL.

[0014] Embodiment 6. The pharmaceutical composition according to any one of embodiments 1to 5, wherein said buffering agent is 10 mM Tris.

[0015] Embodiment 7. The pharmaceutical composition according to any one of embodiments 1to 6, wherein said pharmaceutical composition comprises 1 mM MgCl2.

[0016] Embodiment 8. The pharmaceutical composition according to any one of embodiments 1to 7, wherein said tonicity agent is 150 mM NaCl.

[0017] Embodiment 9. The pharmaceutical composition according to any one of embodiments 1to 8, wherein said non-ionic surfactant is poloxamer 188.

[0018] Embodiment 10. The pharmaceutical composition according to any one of embodiments1 to 9, wherein said pharmaceutical composition is liquid at room temperature.

[0019] Embodiment 11. The pharmaceutical composition according to any one of embodiments1 to 10, wherein said pharmaceutical composition is stable at -80°C for at least 24 months, including having within 1%, 5%, 10% or 20% potency, strength and / or purity relative to the composition prior to storage.

[0020] Embodiment 12. The pharmaceutical composition according to any one of embodiments1 to 11 wherein the AAV particle comprises a genome comprising a transgene encoding a therapeutic protein or nucleic acid operably linked to a regulatory element and flanked by AAV ITR sequences.

[0021] Embodiment 13. The pharmaceutical composition according to any one of embodiments1 to 11, wherein said pharmaceutical composition is a unit dosage form.

[0022] Embodiment 14. The pharmaceutical composition according to any one of embodiments1 to 13, wherein said pharmaceutical composition in the unit dosage form has a volume of between about 2 mL and about 3 mL.38061.0011P1

[0023] Embodiment 15. The pharmaceutical composition according to any one of embodiments1 to 14, wherein said pharmaceutical composition has an osmolality is 150 to 450 mOsm / kg.

[0024] Embodiment 16. The pharmaceutical composition according to any one of embodiments1 to 15, wherein said pharmaceutical composition does not comprise a preservative.

[0025] Embodiment 17. A pharmaceutical composition comprising: a) an AAV9 particle; b) 10mM Tris; c) 1mM MgCl2; d) 150 mM NaCl; and e) 0.02% poloxamer 188, wherein said pharmaceutical formulation has a pH of about 8.0.

[0026] Embodiment 18. A unit dosage form comprising the pharmaceutical composition of anyone of embodiments 1-17 or 23-27.

[0027] Embodiment 19. A method of manufacturing the pharmaceutical composition of any oneof embodiments 1 to 17 or 23-27 comprising a) obtaining an AAV drug substance composition; and performing tangential flow filtration (TFF) to transfer the AAV drug substance composition into a buffer comprising: i) 10 mM Tris; ii) 1mM MgCl2; iii) 150 mM NaCl; and iv) 0.02% poloxamer 188.

[0028] Embodiment 20. A method of treating a subject in need thereof with an AAV genetherapy comprising administering the pharmaceutical composition of any one of embodiments 1 to 17 or 23-27to the subject.

[0029] Embodiment 21. The method of embodiment 20, wherein said step of administeringcomprises administering intravenously.

[0030] Embodiment 22. The method of embodiment 20, wherein said step of administeringcomprises administering intramuscular (IM), intraductal, intracerebroventricular (ICV), intravitreal, subretinal and / or suprachoroidal.

[0031] Embodiment 23. A pharmaceutical composition consisting essentially of: a) an AAV9particle; b) 10 mM Tris; c) 1mM MgCl2; d) 150 mM NaCl; and e) 0.02% poloxamer 188, wherein said pharmaceutical formulation has a pH of about 8.0.

[0032] Embodiment 24. A pharmaceutical composition consisting of: a) an AAV9 particle; b) 10mM Tris; c) 1mM MgCl2; d) 150 mM NaCl; and e) 0.02% poloxamer 188, wherein said pharmaceutical formulation has a pH of about 8.0.

[0033] Embodiment 25. A pharmaceutical composition consisting essentially of: a) an AAV9particle; b) 10 mM Tris; c) 1mM MgCl2; d) 150 mM NaCl; and e) 0.02% poloxamer 188, wherein said pharmaceutical formulation has a pH of about 8.0.38061.0011P1

[0034] Embodiment 26. A pharmaceutical composition consisting of: a) an AAV9 particle; b) 10mM Tris; c) 1mM MgCl2; d) 150 mM NaCl; and e) 0.02% poloxamer 188, wherein said pharmaceutical formulation has a pH of about 8.0.

[0035] Embodiment 27. The pharmaceutical composition of one of embodiments 23 to 26wherein the AAV9 particle is present at a concentration of about 1E13 vg / mL, 5E13 vg / mL, 1E14 vg / mL, or 5E14 vg / ml and comprises a genome comprising a transgene encoding a therapeutic product operably linked to a regulatory element. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is a bar graph showing GFP expression (CTII) of AAV formulations stored at25°C over 8 weeks.

[0037] FIGs. 2A and B are line graphs showing GFP expression (CTII) as a percentage (%) oftime zero (FIG.2A) and measured in vg / mL (FIG.2B) stored at -20°C at time zero (T=0), two weeks (T=2), four weeks (T=4) and eight weeks (T=8).

[0038] FIGs. 3A and B are line graphs showing GFP expression (CTII) as a percentage (%) oftime zero (FIG.3A) and measured in vg / mL (FIG.3B) stored at 5°C at time zero (T=0), two weeks (T=2), four weeks (T=4) and eight weeks (T=8).

[0039] FIGs. 4A-F are line graphs showing GFP expression of MOI standards Cell Counts andCT11 for F1, F2, and F3 at time zero (T=0).

[0040] FIGs. 5A-F are line graphs showing GFP expression of MOI standards Cell Counts andCT11 for F1, F2, and F3 after 3 days (T3day) at 40°C.

[0041] FIGs. 6A-F are line graphs showing GFP expression of MOI standards Cell Counts andCT11 for F1, F2, and F3 after 5 days (T5day) at 40°C.

[0042] FIGs. 7A-F are line graphs showing GFP expression of MOI standards Cell Counts andCT11 for F1, F2, and F3 after 2 weeks (T2week) at 25°C.

[0043] FIGs. 8A-F are line graphs showing GFP expression of MOI standards Cell Counts andCT11 for F1, F2, and F3 after 4 weeks (T4week) at 25°C.

[0044] FIGs. 9A-F are line graphs showing GFP expression of MOI standards Cell Counts andCT11 for F1, F2, and F3 after 8 weeks (T8week) at 25°C.38061.0011P1

[0045] FIGs. 10A and B are line graphs showing percentage (%) of AAV9 GFP expression(FIG.10A, cell count) and (FIG.10B, CTII) after 5 days relative to T0 at 40°C.

[0046] FIGs. 11A and B are line graphs showing percentage (%) of AAV9 GFP expression(FIG.10A, cell count) and (FIG.10B, CTII) after 8 weeks relative to T0 at 25°C. DETAILED DESCRIPTION Definitions

[0047] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. For example, “comprising an A, a B, or a C” contemplates and supports embodiments comprising two or more A, two or more B, and two or more C.

[0048] Unless defined otherwise, all technical and scientific terms have the same meaning ascommonly understood by one of ordinary skill in the art to which the embodiments pertain. The preferred materials and methods are described, but it is understood that any methods and materials similar or equivalent to those described can be used in the practice of embodiments. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In describing and claiming the present invention, the following terminology will be used.

[0049] "About" as used herein when referring to a measurable value such as an amount, atemporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the embodiments.

[0050] The term “AAV capsid” refers to the AAV protein shell, i.e., a capsid. It may or may notencapsidate a nucleic acid.

[0051] The terms “AAV particle” or “AAV virion” can be used interchangeably and refer to aninfectious non-replicative virus having an AAV protein shell, i.e., a capsid, encapsidating a nucleic acid. In some embodiments, the AAV particle is active for gene therapy, i.e., upon transduction of an appropriate target cell, a transgene (GOI) in the recombinant genome of the AAV particle is expressed in the target cell. Expression of the transgene can be measured by either protein expression or a functional assay, for example, as described herein.38061.0011P1

[0052] The terms “AAV particle composition” refers to a composition comprising AAVparticles and, optionally, other molecules, including contaminants from the manufacturing process, such as pDNA, hcDNA, and HCP. In some embodiments, the term AAV particle composition comprises adjuvants and / or excipients for administration to subjects and / or storage of the AAV particle composition.

[0053] "Identity" as used herein refers to the subunit sequence identity between two polymericmolecules particularly between two amino acid molecules, such as, between two polypeptide molecules, or two nucleic acid molecules, such as polynucleotides. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical. In the case of an insertion or deletion, identity is understood to realign those thereafter which would be identical and is considered to be not identical at the insertion or deletion.

[0054] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting atleast 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid level to the sequence used for comparison.

[0055] A “nucleic acid,” as used herein, is interchangeable with “polynucleotide” or “a specificsequence of nucleotide or “nucleotide sequences.” These terms refer to a discrete sequence that performs a specific function directly or indirectly in a cell. That function includes encoding a sequence of a gene that is transcribed into mRNA and translated into protein and regulating said transcription (i.e., as a promoter would) and / or translation (i.e., as microRNA would). A nucleic acid inherently has a sequence. Thereby, “a nucleic acid comprising SEQ ID NO.: X” can be used to contemplate and support “a nucleic acid comprising the sequence of SEQ ID NO.: X.”38061.0011P1 In recombinant molecular biology, discrete nucleic acids can be combined. In some embodiments, a nucleic acid that encodes a protein can be ligated to a promoter (which is a nucleic acid), and a cis-acting element of a viral vector (i.e., an inverted-terminal repeat (ITR), which is also a nucleic acid). For convenience, a “nucleic acid” might be used to refer to the discrete elements within the larger nucleic acid, which could be referred to as “a polynucleotide,” “an expression cassette” (i.e., a polynucleotide comprising a promoter and a nucleic acid that encodes a protein), or “a vector” (see definition below).

[0056] "Encoding" refers to the inherent property of a nucleic acid to serve as a template,whether directly (i.e., a sense strand) or indirectly (i.e., an antisense strand) for synthesis of peptide, polypeptides, proteins, or other nucleic acids (i.e., rRNA, tRNA, microRNA). A nucleic acid can “encode” whether it is the sense strand, antisense strand, or a double-stranded segment thereof. The sense strand directly encodes the rRNA, tRNA, microRNA, or mRNA. The mRNA then serves as the template for translation of a peptide, polypeptide, or protein. The anti-sense strand is generally considered to be the reverse complementary sequence and is sometimes called a “non-coding” strand in the art (although for present purposes “non-coding” is a misnomer because the non-coding strand still “encodes” the genetic information by perpetuating it during semi-conservative replication by acting as a template for the polymerization of a new, sense strand). Within semi-conservative replication two single strands in double-stranded nucleic acids are separated, and a new strand is polymerized from the information from each of the single- stranded nucleic acids (i.e., single-stranded template), regardless of whether one single-stranded template is the sense strand (e.g., that which is used to transcribe mRNA and thereby, or directly, encode the translate or protein) or the antisense strand. By perpetuating the genetic information, the antisense strand is still encoding the genetic information for, for example, a protein. Accordingly, “a nucleic acid encoding X”, includes sense and antisense sequences or strands whether X is a peptide, a polypeptide, or a protein or X is a sequence that encodes a rRNA, tRNA, microRNA, antisenseRNA, etc.

[0057] Further to which, “nucleic acid encoding X,” includes RNA, DNA, and combinationsthereof, since nucleic acids are synthesized from transcription, reverse-transcription, and replication, as naturally occurring processes and man-made processes (recombinant biology, molecular biology, etc.).38061.0011P1

[0058] With regard to an AAV particle or an AAV virion, the above-noted incorporation ofreverse complementary sequences and double-stranded segments into the definition of “a nucleic acid” and the above-noted use of “encoding” as including sense and antisense strands, is intended to incorporate the means by which the AAV vector can introduce an exogenous nucleic acid sequence that encodes nucleic acid or a protein into the cell. It is further intended to incorporate, in some embodiments, processes whereby said introduction results in the expression of said nucleic acid (i.e. miRNA or antisense RNA) or protein.

[0059] Take for example, a nucleic acid encoding a protein, and an AAV vector comprising anucleic acid encoding said protein. When a typical (i.e. naturally occurring) AAV vector encoding one sense or one antisense strand of the nucleic acid that encodes said protein enters the cell, the inverted-terminal repeats (ITRs) prime the synthesis of a sequence reverse complementary to the sense strand or antisense strand of the nucleic acid that encodes said protein. The polymerization thereby forms a segment of double-stranded DNA comprising the sense and antisense strands, regardless of whether the sense version or antisense version was first introduced to the cell. In this regard, the entire nucleic acid including ITRs and sense and antisense nucleic acids encoding a protein can be one single-stranded DNA, which loops upon itself to form a double-stranded segment, wherein the base-pairs the sense and antisense nucleic acids encoding the protein align.

[0060] From this segment of double-stranded DNA, transcription of mRNA and translation ofsaid protein is achieved from said sense strand of DNA, regardless of whether the AAV vector comprised only the sense strand or only the antisense strand when first entering the cell. In this regard, “an AAV vector comprising a nucleic acid encoding protein X” includes, contemplates, and supports embodiments in which the nucleic acid is the sense strand encoding protein X, the antisense strand encoding protein X, a double-stranded nucleic acid encoding protein X, and a single stranded nucleic acid comprising sense and antisense strands wherein the sense and antisense strands form a segment of double-stranded nucleic acid.

[0061] The term "operably linked" refers to functional linkage between a regulatory sequenceand a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid38061.0011P1 sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0062] The terms “final formulation,” and “drug product” as used herein can be usedinterchangeably and include compositions suitable for administration to mammals, e.g., humans. When the AAV particles of the present disclosure are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a liquid pharmaceutical composition containing, for example, a dose of from about 1E11 vg to about 1E18 vg. In embodiments, the pharmaceutical formulations comprise about 1E11 to about 1E18 vg / mL. In embodiments, the pharmaceutical formulations contain from about 0.005% to about 5% wt / volume of active ingredient in the composition, including about 0.005 to about 1% wt / vol, about 0.005 to about 0.5% wt / vol, about 0.01 to about 0.75% wt / vol, or about 0.1 to about 0.5% wt / vol, in combination with a pharmaceutically acceptable carrier. The term “drug substance” as used herein refers to a purified rAAV composition that needs only to be filtered and / or have added to it pharmaceutically acceptable excipients, diluents or carriers, to make the final formulation. “Pharmaceutical composition” refers to a composition comprising a drug product that is appropriate for administration to subjects, including human subjects in that excipients or contaminants are physiologically tolerable, on balance, do not detract from the therapeutic activity of the drug product or cause unduly adverse effects.

[0063] As used herein, the term "unit dose" or "unit dosage" refers to a physically discrete unitthat contains a predetermined quantity of active ingredient calculated to produce a desired therapeutic effect. The unit dose or unit dosage may be in the form of vial comprising a therapeutically effective amount of active ingredient for a single subject.

[0064] The term "pharmaceutically acceptable" refers to molecular entities and compositionsthat are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, that would cause a severe adverse reaction or side effect, when administered to a human, depending upon the active ingredient and indication to be treated. In embodiments, as used herein, the term "pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0065] The terms "promoter" and “regulatory sequence” as used herein, can be usedinterchangeably and are defined as a DNA sequence recognized by the synthetic machinery of38061.0011P1 the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. In some instances, this sequence may be the core promoter and in other instances, this sequence may also include, or be an enhancer alone and / or other regulatory elements which are required for expression of the gene product.

[0066] In certain instances the promoter may comprise enhancer elements, exons, and intronsfrom one or a variety of viruses and animals, and thereby the term “promoter” shall be understood to not be limited to being a non-expressed sequence, nor exclude a non-expressed sequence that is between expressed sequences (i.e. introns), nor be limited to exclude an enhancer alone so long as the combination of sequences used to construct the promoter are capable of initiating the specific transcription of a polynucleotide sequence. Other regulatory elements include polyadenylation signals and post-transcriptional regulatory elements, such as, for example, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0067] A “target gene” or “gene of interest” (“GOI”) refers to a nucleic acid encoding a targetprotein to be expressed within a target cell upon entry of the vector carrying the target gene into the cell under conditions such that the operably linked regulatory sequences promote expression of the GOI in the target cell.

[0068] A "vector" is a nucleic acid capable of delivering a target gene to the interior of a cell,and includes not only the expression-region (i.e. a promoter and a nucleic acid encoding a protein or even a nucleic acid), but also some cis-acting genetic component. The cis-acting genetic component provides for packaging within a virion, expression in a cell, replication in a cell, or a combination thereof.

[0069] By way of example, inverted-terminal repeats (ITRs) from adeno-associated viruses(AAVs) constitute a vector when adjoined to the nucleic acid encoding a target protein because the ITRs will provide for the nucleic acid encoding the target protein to be packaged within an AAV virion. ITRs also provide other cis-acting functions for expression of the nucleic acid encoding the target protein in the host cell upon entry of the vector into the host cell. Such cis- acting functions of ITRs include aiding in concatemer formation for genomic insertion; initiation of second strand formation in the case of a single-stranded (ss) AAV (ssAAV) vector; or initiation of replication and transcription in the case of ssAAV and self-complementary (sc) AAV (scAAV) vectors. In this regard, the AAV ITRs can be characterized based on the nucleic acid sequences providing such cis-acting functions from the serotypes of AAVs. That is, an ITR38061.0011P1 isolated from an AAV2 serotype can be known as an AAV2 ITR, even though the ITR generally does not contribute to the serotype of an AAV.

[0070] “Expression cassette” refers to a nucleic acid comprising a GOI operably linked toregulatory sequences sufficient for expression of the GOI in a target cell. An expression cassette includes a recombinant polynucleotide comprising a nucleic acid that controls expression (i.e. a promoter) and a nucleic acid that encodes the GOI. The GOI includes a nucleic acid that encodes a protein. Generally, the promoter is operatively linked to the nucleic acid that encodes the target protein (or other expression product, such as, but not limited to an siRNA or microRNA) in a manner that is capable of promoting expression of the protein upon entry of the vector into the target cell. The Expression Cassette may also include other regulatory elements such as polyadenylation (polyA) signal sequences and other expression elements such as enhancers, introns and post-transcriptional regulatory elements that promote or otherwise increase or decrease expression of the GOI in target cells.

[0071] Ranges: throughout this disclosure, various aspects of the invention can be presented in arange format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Pharmaceutical Compositions AAV

[0072] Aspects of the disclosure relate to pharmaceutical formulations for storing andadministering recombinant AAV particles for gene therapy. The Examples herein describe recombinant AAV formulations which exhibit enhanced storage stability, i.e., maintain strength, purity and potency during storage, and reduced aggregation as detailed herein. The pharmaceutical formulations disclosed herein are formulated for administration of AAV particles to deliver a functional copy of a transgene that expresses a protein or RNA to exert a therapeutic effect, such as restoring a cellular function.38061.0011P1

[0073] AAV refers to a replication-deficient (e.g., nonreplicating) Dependoparvovirus within theParvoviridae genus of viruses. AAV can be derived from a naturally occurring virus or can be recombinant. AAV can be packaged into capsids, which can be derived from naturally occurring capsid proteins or recombinant capsid proteins. The single-stranded DNA genome of AAV includes inverted terminal repeat (ITRs). ITRs are involved in the replication and encapsidation of the AAV genome, along with its integration in the host genome and its excision. Without wishing to be bound by any theory, AAV vectors can comprise one or more ITRs, including a 5’ ITR and / or a 3’ ITR, one or more promoters, one or more nucleic acid sequences encoding one or more proteins of interest, and / or additional posttranscriptional regulator elements. AAV vectors disclosed herein can be prepared using standard molecular biology techniques known to one of ordinary skill in the art, as described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (2012)), which is incorporated herein by reference in its entirety.

[0074] Adeno-associated virus (AAV) is a parvovirus which has use as a gene therapy vector.AAV is capable of infecting a variety of human cell types but do not replicate in human cells without expression of adenoviral accessory factors required for viral production. Accordingly, AAV has not been associated with disease in humans. The wild type AAV genome is a single stranded DNA of approximately 4.7 kb encoding the Rep or replication proteins and structural Cap or capsid proteins flanked by inverted terminal repeat (ITR) sequences which form hairpin structures that prime DNA replication upon transduction of the AAV. Recombinant AAV particles comprise a recombinant AAV genome in which an expression cassette, comprising the coding sequence for a gene of interest (GOI) is operably linked to regulatory sequences such as promoters, enhancers, and polyadenylation signals, such that the GOI is expressed in a target cell upon AAV particle transduction of that cell. The recombinant AAV genome has at least one ITR, and in embodiments, two ITRs which flank the expression cassette. In embodiments, one of the ITR sequences may be modified such that the packaged genome is a self-complementary (sc) genome rather than a single stranded (ss) genome.

[0075] There are multiple AAV serotypes, see, e.g., Gao, J. Virol. 78:6381 (2004), and theserotypes may have different tissue tropisms. The rAAV particles produced by the methods described herein can be any serotype or combination of serotypes. In embodiments, the capsid is from any AAV serotype, the Rep proteins are from any AAV serotype and the ITR sequences are38061.0011P1 from any AAV serotype, and the serotype of the capsid may differ from that of the Rep protein and / or the ITR sequences. In embodiments, the rAAV particles of the disclosure have a capsid (cap proteins, including VP1, VP2 and VP3) that is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh74, AAVrh10, or related engineered capsid. See for example, WO2013 / 158879, WO2015 / 013313, US 9,169,299, all of which are incorporated by reference in their entirety, for amino acid sequences of these capsids.

[0076] In embodiments, the disclosed pharmaceutical compositions comprise AAV capsids thatare an AAV8 capsid or an AAV9 capsid. In some embodiments, the AAV capsid protein comprises a VP1, a VP2, and a VP3. In embodiments, the disclosed pharmaceutical compositions comprise AAV capsids that have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to VP1, VP2, and / or VP3 of an AAV9 capsid (SEQ ID NO: 5). In embodiments, the disclosed pharmaceutical compositions comprise AAV capsids that have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to VP1, VP2, and / or VP3 of an AAV8 capsid (SEQ ID NO: 6).

[0077] The term “engineered capsid” or “engineered particle” as used herein, means an AAVhaving capsid protein which have been recombinantly engineered to include one or more amino acid substitutions, deletions or insertions relative to the amino acid sequence of the capsid proteins found in the wild type AAV. For example, engineered capsids may include, for example, PHP.B (WO 2015 / 038958 by Deverman et al.) and others (see, e.g, WO 2012 / 145601), which are incorporated herein by reference. Engineered capsids may confer enhanced or altered tissue tropism on a particular AAV serotype capsid.

[0078] In some embodiments, the rAAV particles described herein have ITRs from the sameserotype as the capsid or are “pseudotyped AAV” particles in which the ITR sequences are from a different serotype than the capsid. In embodiments, the ITR sequences are from AAV2. In some embodiments, the rAAV2, rAAV6, rAAV8 or rAAV9 particles are pseudotyped rAAV2, rAAV6, rAAV8, rAAV9, or related engineered particles. In some embodiments, the rAAV2, rAAV6, rAAV8 or rAAV9 particles are rAAV2 / 2, rAAV2 / 6, rAAV2 / 8, rAAV2 / 9, or related engineered pseudotyped particles. In some embodiments, the rAAV particles are rAAV8 or rAAV9 particles (e.g., rAAV2 / 8 or rAAV2 / 9 particles). In some embodiments, the rAAV particles are rAAV6 particles (e.g., rAAV2 / 6 particles). In some embodiments, the rAAV38061.0011P1 particles are rAAV9 particles (e.g., rAAV2 / 9 particles). Pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001).

[0079] AAV vectors of the present disclosure may comprise or be derived from any natural orrecombinant AAV serotype. In some embodiments, the AAV vector may utilize or be based on an AAV serotype described in WO 2017 / 201258A1, the contents of which are incorporated herein by reference in its entirety, such as, but not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42- 13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43- 23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2- 3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3- 11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV33.12 / hu.17, AAV33.4 / hu.15, AAV33.8 / hu.16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1 / hu.1, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39,38061.0011P1 AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV- PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA- 101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr- E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV38061.0011P1 CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1- 1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp- 8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, AAV-PHP.B (PHP.B), AAV-PHP.A (PHP.A), G2B-26, G2B-13, TH1.1-32 and / or TH1.1-35, and variants thereof. AAV vectors are described further in US 9,585,971, US 2017 / 0166926, and WO2020 / 160337, which are incorporated by reference herein in their entireties.

[0080] In embodiments, the AAV is an AAV9. In embodiments, the AAV is an AAV8.

[0081] In embodiments, the AAV is present at a concentration of about 1E11 vg / mL, about 5E11vg / mL, 1E12 vg / mL, about 5E12 vg / mL, about 1E13 vg / mL, about 5E13 vg / mL, about 1E14 vg / mL, about 5 E14 vg / mL, about 1E15 vg / mL, about 5E15 vg / mL, about 1E16 vg / mL, about 1E17 vg / mL, about 5E17 vg / mL, or about 1E18 vg / mL. AAV FORMULATIONS

[0082] Provided are formulations of recombinant AAV particles which have enhanced storagestability, including maintaining therapeutic activity, purity and strength and exhibiting reduced38061.0011P1 aggregation after storage, including after storage at -80°C for at least two years. The pharmaceutical compositions provided comprise, in addition to the AAV, a buffering agent, a Mg containing salt, a tonicity-modifying agent, and a non-ionic surfactant. In embodiments, the pharmaceutical compositions have pH of about 5.0 to about 9.0 and an osmolality of about 150 to about 450 mOsm / kg. The concentration of AAV particles , including AAV9 or AAV8 particles, may be about 1E13vg / ml, 5E13 vg / ml, 1E14 vg / ml or 5E14 vg / ml.

[0083] In some embodiments, the pharmaceutical composition comprises, in addition to AAVparticles, 10 mM Tris, 1 mM MgCl2, 150 mM NaCl, and 0.02% poloxamer 188, pH 8.0. The AAV, including AAV9 or AAV8 particles, may be about 1E13vg / ml, 5E13 vg / ml, 1E14 vg / ml or 5E14 vg / ml.. In some embodiments, the pharmaceutical composition consists essentially of AAV particles, 10 mM Tris, 1 mM MgCl2, 150 mM NaCl, and 0.02% poloxamer 188, pH 8.0. In some embodiments, the pharmaceutical composition consists of AAV particles, 10 mM Tris, 1 mM MgCl2, 150 mM NaCl, and 0.02% poloxamer 188, pH 8.0. The AAV, including AAV9 or AAV8 particles, may be about 1E13vg / ml, 5E13 vg / ml, 1E14 vg / ml or 5E14 vg / ml. Buffering Agent

[0084] The disclosed pharmaceutical formulations comprise at least one buffering agent. Inembodiments, the buffering agent is Tris HCl, Tris base, sodium phosphate, phosphate buffered saline (PBS), acetate, acetic acid, alanine, arginine, aspartic acid, boric acid, citric acid, glutamic acid, glycine, histidine, lysine, potassium phosphate, sodium acetate, sodium citrate, sodium succinate, succinic acid, tromethamine, HEPES and / or MOPS.

[0085] In embodiments, the buffering agent is present at a concentration between about 5 mMand about 100 mM, about 5 mM and about 50 mM, about 5 mM and about 25 mM, about 5 mM and about 15 mM, about 5 mM and about 10 mM.

[0086] In embodiments, the buffering agent is present at a concentration of about 5 mM, about10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM or about 100 mM.

[0087] The buffering agent is appropriate for a pH of about 5.0 to about 9.0, about 6.0 to about9.0, 6.0 to about 8.5, 6.5 to about 8.5, about 7.0 to about 8.0, about 7.5 to about 8.0, or about 7.5 to about 8.5.38061.0011P1 Magnesium-Containing Salts

[0088] The disclosed pharmaceutical formulations comprise at least one magnesium-containingsalt. In embodiments, the magnesium-containing salt is MgCl2. In embodiments, the magnesium- containing salt is MgSO4.

[0089] In embodiments, the magnesium-containing salt is present at a concentration of about 0.5mM MgCl2, about 1 mM MgCl2, about 1.5 mM MgCl2, or about 2 mM MgCl2. Tonicity Modifier

[0090] The disclosed pharmaceutical formulations comprise at least one tonicity modifier. Inembodiments, the tonicity modifier is sodium chloride (NaCl), sorbitol or trehalose. In embodiments, the tonicity modifier is dextrose, guanidine, magnesium chloride, maltose, mannitol, potassium chloride, sodium citrate, sodium phosphate, sodium sulfate, and / or sucrose.

[0091] In embodiments, the tonicity modifier is about 100 mM NaCl, about 125 mM NaCl,about 150 mM NaCl, about 175 mM NaCl or about 200 mM NaCl.

[0092] In embodiments, the tonicity modifier is about 2% sorbitol, about 2.5% sorbitol, about3% sorbitol, about 3.5% sorbitol, about 4% sorbitol, about 4.5% sorbitol, about 5% sorbitol, about 5.5% sorbitol, about 6% sorbitol, about 6.5% sorbitol or about 7% sorbitol. In embodiments, the formulation does not comprise sorbitol.

[0093] In embodiments, the tonicity modifier is about 2% trehalose, about 2.5% trehalose, about3% trehalose, about 3.5% trehalose, about 4% trehalose, about 4.5% trehalose, about 5% trehalose, about 5.5% trehalose, about 6% trehalose, about 6.5% trehalose or about 7% trehalose. In embodiments, the formulation does not comprise trehalose.

[0094] In embodiments, the pharmaceutical composition does not comprise a sugar (sucrose). Inembodiments, the pharmaceutical composition does not comprise a sugar substitute.

[0095] In embodiments, the tonicity modifier achieves an osmolality of about 150 to about 450mOsm / kg, about 175 to about 425 mOsm / kg, about 200 to about 400 mOsm / kg, about 225 to about 375 mOsm / kg, about 250 to about 350 mOsm / kg, about 275 to about 325 mOsm / kg or about 300 to about 400 mOsm / kg. Non-ionic Surfactant

[0096] In embodiments, the pharmaceutical composition comprises a non-ionic surfactant. Inembodiments, the non-ionic surfactant is poloxamer 188 (pluronic F68). In embodiments, the non-ionic surfactant is polysorbate 20 and / or polysorbate 80.38061.0011P1

[0097] In embodiments, the pharmaceutical composition comprises from about 0.02% to about0.20% non-ionic surfactant. In embodiments, the pharmaceutical composition comprises about 0.02%, about 0.03%, about 0.04% or about 0.05% about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.20% non-ionic surfactant.

[0098] In embodiments, the pharmaceutical composition comprises from about 0.02% to about0.20% poloxamer 188. In embodiments, the pharmaceutical composition comprises about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.20% poloxamer 188. In embodiments the concentration of poloxamer 188 is greater than 0.005%, greater than 0.01%, or greater than or including 0.02%, and up to 0.2% or 0.3% or 0.4% or 0.5%. In embodiments, the pharmaceutical composition comprises up to about 0.0460% or up to about 0.0663% poloxamer 188.

[0099] In embodiments, the pharmaceutical composition comprises from about 0.02% to about0.20% polysorbate 20. In embodiments, the pharmaceutical composition comprises about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.20% polysorbate 20.

[0100] In embodiments, the pharmaceutical composition comprises from about 0.02% toabout 0.20% polysorbate 80. In embodiments, the pharmaceutical composition comprises about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.10%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, or about 0.20% polysorbate 80.

[0101] In embodiments, the pharmaceutical composition comprises from about 0.02% toabout 0.05%, from about 0.02% to about 0.04%, or from about 0.02% to about 0.03% non-ionic surfactant. In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.05%, from about 0.02% to about 0.04%, or from about 0.02% to about 0.03% poloxamer 188. In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.05%, from about 0.02% to about 0.04%, or from about 0.02% to about 0.03% polysorbate 20.38061.0011P1 In embodiments, the pharmaceutical composition comprises from about 0.02% to about 0.05%, from about 0.02% to about 0.04%, or from about 0.02% to about 0.03% polysorbate 80. pH

[0102] In embodiments, the pharmaceutical composition has a pH of about 5.0, about5.5, about 6.0, about 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In embodiments, the pharmaceutical composition has a pH of about 8.0.

[0103] In embodiments, the pharmaceutical composition has a pH of about 5.0 to about9.0, about 6.0 to about 9.0, 6.0 to about 8.5, 6.5 to about 8.5, about 7.0 to about 8.0, about 7.5 to about 8.0, or about 7.5 to about 8.5.

[0104] In embodiments, the pharmaceutical composition has a pH of about 7.9 to about8.1, about 7.8 to about 8.2, about 7.8 to about 8.2, about 7.7 to about 8.3, about 7.6 to about 8.4, about 7.5 to about 8.5, about 7.4 to about 8.6, about 7.3 to about 8.7, about 7.2 to about 8.8, about 7.1 to about 8.9 or about 7.0 to about 9.0. In embodiments, the pharmaceutical composition has a pH of about 7.0 to about 8.0. Osmolality

[0105] In embodiments, the pharmaceutical composition has an osmolality of about 150to about 450 mOsm / kg, about 175 to about 425 mOsm / kg, about 200 to about 400 mOsm / kg, about 225 to about 375 mOsm / kg, about 250 to about 350 mOsm / kg, about 275 to about 325 mOsm / kg or about 300 to about 400 mOsm / kg.

[0106] In embodiments, the pharmaceutical composition has an osmolality of about 150mOsm / kg, about 175 mOsm / kg, about 200 mOsm / kg, about 225 mOsm / kg, about 250 mOsm / kg, about 275 mOsm / kg, about 300 mOsm / kg, about 325 mOsm / kg, about 350 mOsm / kg, about 375 mOsm / kg, about 400 mOsm / kg, about 425 mOsm / kg, or about 450 mOsm / kg. Preservatives

[0107] In embodiments, the pharmaceutical composition does not comprise apreservative. Unit dosage forms

[0108] In embodiments, the disclosed pharmaceutical composition is a unit dosage form.38061.0011P1

[0109] In embodiments, unit dosage form has a volume of about 1 mL, about 1.25 mL,about 1.5 mL, about 1.75 mL, about 2 mL, about 2.25 mL, about 2.5 mL, about 2.75 mL, or about 3 mL. In embodiments, unit dosage form has a volume of between about 1 mL and about 3 mL, between about 1.5 mL and about 2.5 mL, between about 2 mL and about 2.5 mL, or between about 2mL and about 3 mL. Storage

[0110] In embodiments, the pharmaceutical formulations can be stored at -80°C for morethan about 3 months, 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months without a change (including less than 1%, 5%, 10% or 20% reduction) in purity, strength or potency as measured using one of the methods described in the examples or any method known in the art for assessing stability, including purity, strength or potency of the AAV. Provided are formulations, including formulations having 1E13 vg / ml to 1E15 vg / ml, including about 1E14 vg / ml of a recombinant AAV particle that comprises a genome comprising a transgene encoding a therapeutic product, which exhibit storage stability. Storage stability of the formulation may be after storage at -70°C after 12 months, 24 months, 36 months or 48 months or after an accelerated storage for 2 weeks, 4 weeks or 8 weeks at -20°C, 5°C, 25°C or 40°C. Formulations may also be tested for storage stability under agitation conditions, for freeze-thaw cycles (e.g., 5 freeze-thaw cycles at -70°C or -20°C) or subjected to exposure to UV light. Stability is assessed by dynamic light scattering (DLS) to assess particle population, size and size distribution, FlowCAM, or assay for expression of the product encoded by the transgene within the AAV genome relative to either the formulation at time 0 or relative to a control formulation. In embodiments, AAV formulations after storage (including after storage for 12 months, 24 months, 36 months or 48 months at -70°C) maintain stability, including exhibit no more than a 1%, 5%, 10% or 20% reduction in purity, potency or strength relative to the formulation at time 0 or a reference standard.

[0111] In embodiments, the recombinant AAV formulations, including those withconcentrations of AAV of 1E13 vg / ml, 5E13 vg / ml, 1E14 vg / ml, or 5E14 vg / ml, exhibit limited toxicity in toxicology models in rodents or non-human primates.

[0112] Also provided are methods of storing the recombinant AAV formulations bystorage in formulations provided herein. Methods of making38061.0011P1

[0113] As discussed above, recombinant AAV can be made by any method known in theart. Further, the recombinant AAV can be made put into the final formulation buffer by any method known in the art.

[0114] In embodiments, a tangential flow filtration (TFF) process is used to transfer theAAV into the disclosed pharmaceutical formulations. In TFF, the bulk of the AAV particle composition flows tangentially across the surface of the filter, minimizing membrane fouling while maintaining a high filtration rate. TFF systems are known in the art and commercially available systems are sold by, for example, Millipore, Repligen, Sartorius and Pall.

[0115] In some embodiments, TFF, as discussed above, is used to concentrate the AAVparticle composition, remove impurities and transfer the AAV particle composition into the disclosed pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises AAV particles (including recombinant AAV9 particles at 1E13 vg / m:, 5E13 vg / mL, 1E14 vg / mL, or 5E14 vg / mL) and 10 mM Tris, 1 mM MgCl2, 150 mM NaCl, and 0.02% poloxamer 188, pH 8.0. In some embodiments, the pharmaceutical composition consisting essentially of AAV particles (including recombinant AAV9 particles at 1E13 vg / m:, 5E13 vg / mL, 1E14 vg / mL, or 5E14 vg / mL), and 10 mM Tris, 1 mM MgCl2, 150 mM NaCl, and 0.02% poloxamer 188, pH 8.0. In some embodiments, the pharmaceutical composition consists of AAV particles (including recombinant AAV9 particles at 1E13 vg / m:, 5E13 vg / mL, 1E14 vg / mL, or 5E14 vg / mL), and 10 mM Tris, 1 mM MgCl2, 150 mM NaCl, and 0.02% poloxamer 188, pH 8.0. Methods of Administering

[0116] As disclosed herein, polynucleotides encoding therapeutic proteins can bedelivered to a tissue or cell of interest in a viral vector. Vectors described herein can be used to deliver a nucleic acid encoding a protein of interest to a subject, including, e.g., delivery to specific organs or to the central nervous system (CNS) of a subject. In some embodiments, the protein of interest is a MiniShank3 protein, a PAX4 protein or a GALT protein.

[0117] In some embodiments, the recombinant AAV vector comprises a transgene and itsregulatory sequences, and optionally 5' and 3' ITRs. In some embodiments, the transgene and its regulatory sequences are flanked by the 5’ and 3’ ITR sequences. The transgene may comprise, as disclosed herein, one or more regions that encode a therapeutic protein. The transgene may also comprise a region encoding for another protein. The transgene may also comprise one or more expression control sequences (e.g., a poly-A tail). The transgene may be single stranded. In38061.0011P1 some embodiments, a recombinant AAV vector comprises at least AAV ITRs and a MiniShank3 transgene, a PAX4 transgene or a GALT transgene (encoding proteins having amino acid sequences of one of SEQ ID Nos 1-4).

[0118] As used in the present disclosure, “delivering” or “administering” a recombinantAAV vector can include any method known in the art for delivering or administering an AAV vector or a composition comprising an AAV vector to a subject. Administering can include but is not limited to direct administration of a recombinant AAV vector or a composition comprising the recombinant AAV vector, or peripheral administration via passive diffusion or convection- enhanced delivery (CED) to bypass the blood brain barrier as known in the art. Recombinant AAV vectors described herein can be administered in any composition that would be compatible with aspects of the disclosure.

[0119] Methods described herein comprise administering recombinant AAV vector insufficient amounts to transfect the cells of a desired tissue (e.g., brain) and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ, oral, inhalation, intraocular, intravenous including facial vein injection and retroorbital injection, intracerebroventricular (ICV), intramuscular (IM), intrathecal, intracranial, intraductal, subcutaneous, intradermal, intratumoral, and other parental routes of administration. In some embodiments, the recombinant AAV vector is delivered to the cells of a desired tissue (e.g., brain) via parenteral administration. In some embodiments, the recombinant AAV vector is delivered to the cells of a desired tissue (e.g., brain) via intravenous administration. In some embodiments, the recombinant AAV vector is delivered to the cells of a desired tissue (e.g., brain) via ICV administration. In some embodiments, the ICV administration can be unilateral administration. In some embodiments, the ICV administration can be bilateral administration.

[0120] In embodiments, the pharmaceutical compositions described herein are deliveredvia intramuscular (IM), intraductal, intracerebroventricular (ICV), intravitreal, subretinal and / or suprachoroidal administration.

[0121] Routes of administration may be combined, if desired. In some embodiments, thevector as disclosed herein is administered via intravenous administration, systemic administration, intracerebroventricular (ICV) administration, including bilateral or unilateral ICV38061.0011P1 administration, in utero administration, intrathecal administration, retro-orbital injection, or facial vein injection.

[0122] Methods provided herein, in some embodiments, comprise administering anddelivering an effective amount of a pharmaceutical composition comprising a recombinant AAV virion that comprises at least AAV ITRs and a MiniShank3 transgene, a PAX4 transgene or a GALT transgene, including those operably linked to promoters for appropriate expression of the transgene in the subject. In embodiments, the transgenes encode for miniSHANK3 (SEQ ID NO: 1 or SEQ ID NO: 2), PAX4 (SEQ ID NO: 3) or GALT (SEQ ID NO: 4). The pharmaceutical compositions described herein can be used to administer recombinant AAV virions.

[0123] The dose of the recombinant AAV vector or recombinant AAV virion comprisinga polynucleotide that encodes a therapeutic protein required to achieve a particular "therapeutic effect," e.g., the units of dose in absolute vector genomes (vg) or vector genomes per milliliter of pharmaceutical solution (vg / mL) will vary based on several factors including, but not limited to: the route of AAV administration, the level of gene expression required to achieve a therapeutic effect, the specific disorder being treated, and the stability of the gene product. One of skill in the art can readily determine a recombinant AAV vector or recombinant AAV virion dose range to treat a patient having a particular disorder based on the aforementioned factors, as well as other factors.

[0124] In some embodiments, an effective amount of a recombinant AAV vector or arecombinant AAV virion may be an amount sufficient to infect an animal or human subject or target a desired tissue. The effective amount will depend primarily on factors such as the species, age, gender, weight, health of the subject, and the tissue to be targeted, and may thus vary among subjects and tissues. The term “effective amount” or “amount effective” in the context of a composition or dose for administration to a subject refers to an amount of the composition or dose that produces one or more desired responses in the subject. In some embodiments, an effective amount of a composition disclosed herein may partially or fully rescue the effects of a mutated Shank3 gene and / or partially or fully restore loss of function of the Shank3 protein. An effective amount can involve reducing the level of an undesired response, although in some embodiments, it involves preventing an undesired response altogether. An effective amount can also involve delaying the occurrence of an undesired response. An effective amount can also be an amount that produces a desired therapeutic endpoint or a desired therapeutic result. In other38061.0011P1 embodiments, the amounts effective can involve enhancing the level of a desired response, such as a therapeutic endpoint or result. The achievement of any of the foregoing can be monitored by routine methods and the methods as disclosed in the present application. Effective amounts will depend, of course, on the particular subject being treated; the severity of a condition; the individual patient parameters including age, physical condition, size and weight; the duration of the treatment; the nature of concurrent therapy (if any); the specific route of administration and like factors. It should be appreciated that an effective amount as used herein does not need to be clinically effective.

[0125] A subject to be treated by methods described herein may be a human subject or anon-human subject. Non-human subjects include, for example: non-human primates; farm animals, such as cows, horses, goats, sheep, and pigs; pets, such as dogs and cats; and rodents.

[0126] A subject to be treated by methods described herein may be a subject having,suspected of having, or at risk for developing a neurodevelopmental disorder. In some embodiments, a subject has been diagnosed as having a neurodevelopmental disorder, while in other embodiments, a subject has not been diagnosed as having a neurodevelopmental disorder. In some embodiments, the subject is a human subject having, suspected of having, or at risk for developing an autism spectrum disorder (ASD). In some embodiments, the subject is a human subject having, suspected of having, or at risk for developing Phelan–McDermid syndrome. In some embodiments, the subject is a subject having reduced expression of the Shank3 gene relative to a control subject. In some embodiments, the expression of the Shank3 gene is reduced in the subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to a control subject. In some embodiments, the control subject is a subject that does not have, is not suspected of having, or is not at risk of having, a neurodevelopmental disorder. In some embodiments, the reduced expression of the Shank3 gene in a subject is caused by disruption of at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene comprises a deletion in at least one copy of the Shank3 gene. In some embodiments, the disruption of the Shank3 gene comprises one or more mutations within at least one copy of the Shank3 gene.

[0127] In some embodiments, the subject is a human subject who exhibits one or moresymptoms of an ASD. In some embodiments, the subject is a human subject who exhibits38061.0011P1 developmental delay. In some embodiments, the subject is a human subject who exhibits intellectual disability (ID). In some embodiments, the subject is a human subject who exhibits sleep disturbance. In some embodiments, the subject is a human subject who exhibits hypotonia. In some embodiments, the subject is a human subject who exhibits lack of speech. In some embodiments, the subject is a human subject who exhibits language delay. In some embodiments, the subject is a human subject who exhibits any symptoms or signs of an ASD.

[0128] In some embodiments, a subject is a human subject who is an adult. In someembodiments, the adult is older than 25 years of age. In some embodiments, the adult is not older than 25 years of age. In some embodiments, the adult is not older than 21 years of age. In some embodiments, the adult is not older than 18 years of age. In some embodiments, the adult is 16 years of age. In some embodiments, a subject is elderly (e.g., 65 years old or older). In some embodiments, the adult can be any age of adulthood that is suitable for the treatment disclosed herein.

[0129] In some embodiments, the subject is a human subject who is not an adult. In someembodiments, the human subject is not older than 16 years of age. In some embodiments, the human subject is not older than 10 years of age. In some embodiments, the human subject is 10 years of age or younger. In some embodiments, the human subject is a child or an infant. In some embodiments, the human subject is a toddler. In some embodiments, the human subject is at the fetal stage of development. In some embodiments, the human subject is at the prenatal stage of development.

[0130] For example, in some embodiments, the number of vector genomes (vg)administered to the subject is any value between about 6.0 x 1011vg and about 9.0 x 1013vg. In some embodiments, the number of vector genomes administered to the subject is any value between about 6.0 x1013vg / mL and about 9.0 x1013vg. In some embodiments, the number of vector genomes administered to the subject is any value between about 1 x 1010to about 1 x 1012vg. In certain embodiments, the effective amount of AAV is 1010, 1011, 1012, 1013, or 1014genome copies per kg. In certain embodiments, the effective amount of AAV is 1010, 1011, 1012, 1013, 1014, or 1015genome copies per subject. In some cases, a dosage between about 1011to 1013AAV genome copies is appropriate. In some embodiments, a dose of about 1.0 x 1013to about 1.0 x 1014vector genomes is administered to the subject. In some embodiments, the number of vector genomes administered to the subject can be any dose that is suitable for the38061.0011P1 treatments and methods disclosed herein. In some embodiments, the dose of vector genomes is administered by unilateral or bilateral ICV administration.

[0131] In some embodiments, the dose administered to the subject is about 1.4 × 1010vg / ml, about 1.4 × 1011vg / ml, about 1.4 × 1012vg / ml, about 1.4 × 1013vg / ml, about 1.4 × 1014vg / ml, about 1.4 × 1015vg / ml, or about 1.4 × 1016vg / ml. In some embodiments, the dose administered to the subject via unilateral ICV administration is about 1.4 × 1013vg / ml. In some embodiments, the dose administered to the subject via unilateral ICV administration is from about 1.4 × 1010vg / ml to about 1.4 × 1016vg / ml.

[0132] In some embodiments, a dose of recombinant AAV is administered to a subjectas a single dose. In some embodiments, a dose of recombinant AAV is administered to a subject as a single dose with the potential to be re-dosed at a later time.

[0133] Some aspects of the technology described herein may be understood further basedon the non-limiting illustrative embodiments described in the below Examples section. Any limitations of the embodiments described in the below Examples section are limitations only of the embodiments described in the below Examples section and are not limitations of any other embodiments described herein. EXAMPLES

[0134] In order that the invention described herein may be more fully understood, thefollowing examples are set forth. The examples described in this application are offered to illustrate the systems and methods provided herein and are not to be construed in any way as limiting their scope. Example 1: AAV9-GFP FORMULATION DEVELOPMENT

[0135] The stability of AAV9-GFP in various formulation conditions including buffer,pH, tonicity modifier, and surfactant were examined (visual appearance, pH, osmolality, micro flow imaging (FlowCAM), dynamic light scattering (DLS), green fluorescent protein (GFP) expression, and droplet digital PCR (ddPCR)). Table 1: AAV9-GFP Titers Titer by ddPCR Reported Titer by S l N b38061.0011P1 1 1.3E13 1.69E13 2 1.0E13 1.21E13on by ddPCR:1E12 vg / mL; 2) Fill volume: 0.6mL; and 3) Buffer concentration: 10mM.

[0137] The following parameters were examined in these formulations (Table 2):Table 2. Formulation Matrix for Accelerated Stability Study Form. Tonicity Buffer (1DS Poloxamer 188 No.0mM) pHModifier (v / mL) (F-68) (%)Formulation preparation

[0138] For surfactant confirmation study, AAV9-GFP (1.3E13 vg / mL), was diluted withformulation buffer (10 mM NaPO4, 150 mM NaCl, 1 mM MgCl2, 0.02% F-68 (poloxamer 188)) to 1E12 vg / mL and filled into vials at 0.6 mL fill volumes. For accelerated stability study, multiple lots of AAV9-GFP were removed from -70°C storage and thawed at room temperature. Once completely thawed, the multiple lots of AAV9-GFP were pooled. Formulations were prepared by dilution of 1:10.7 AAV9-GFP into each formulation buffer to 1E12 vg / mL. The detailed formulation matrix is described in Table 3. Table 3. Detailed Formulation Matrix for Accelerated Stability Study Residual Excipients* DS c. L) 238061.0011P1 Residual Excipients* Form. Buffer (10 Tonicity Sodium DS H NaCl MgCl2 F-68 (%) Conc. L) 2 2 2 2 2 2 22

[0039] samp es were m xed t oroug y and ster e tered under asept c cond t onsusing 0.2 µm sterile filters in a biosafety cabinet. Samples were filled into 2 cc sterile CZ vials at a 0.6 mL fill volume and were stoppered before removing from the biosafety cabinet. After sealing, samples were placed in their respective stability conditions and T0 analyses were performed. Stress studies

[0140] The candidate formulations were exposed to various stress conditions (Table 4) tocompare their relative stabilities. Formulations were stored at -70°C, -20°C, 5°C, 25°C, and 40°C for up to 8 weeks and were subjected to acute stresses such as agitation, freeze / thaw, and UV light exposure. Table 4: Stress conditions and analysis time points Stress Conditions Time Point(s)38061.0011P1 Stress Conditions Time Point(s)8 watt hrs / square meter of UVA light Photosensitivit UV li ht ex osure

[0141] The analytical methods used in this study are summarized in Table 5.Table 5: Formulation Development Accelerated Stability Analytical Methods Acute Stress Temperature Stress Time PointsAnalytical Agitation Freeze / UV T=0 2 wk 4 wk8 k

[00142] The following assays were used to analyze the individual formulations,sometimes both before and after stress:

[0143] Visual Appearance: Visual inspection was performed against a white and blackbackground. Digital photographs were acquired for all formulations at every time point.

[0144] pH: pH analysis was performed with a SympHony® pH Meter (VWR Scientific,catalog # SB70P), calibrated with three pH standard solutions (pH 4, 7, and 10) with a calibration slope of 95% or higher. All samples were equilibrated to room temperature prior to analysis.

[0145] Osmolality: Osmolality was measured at time zero using a Wescor Vapro 552Ovapor pressure osmometer.

[0146] FlowCAM: The FlowCAM particle imaging system combines optics, electronics,and fluidics for automated analysis of particles. The optical system is used to capture real-time images of the particles in the fluid as they pass through the flow cell. The imaging software provides the ability to assess particle size and morphology. All samples are degassed for 30 minutes at 635 torr (25 in Hg) to reduce air bubbles prior to analysis. All samples were analyzed neat.38061.0011P1

[0147] Dynamic Light Scattering (DLS): Changes in light intensity scattered byparticles are measured and information about particle population, size, and size distribution were collected using the DLS DynaPro Plate Reader. All samples were degassed by centrifugation at 3000 rpm for 5 min.25 µL of each sample was analyzed at ambient temperature with the instrument set to 10 acquisitions per sample at a rate of 2-3 seconds per acquisition for a total run time of 20-30 seconds per sample at ambient temperature. Average particle sizes less than 1 nm are considered too small for precise characterization and were not analyzed. Peaks with %Mass are reported. Particle distribution was categorized into peak 1 (1-10 nm) (not shown), peak 2 (10- 100 nm), peak 3 (100-1000), and peak 4 (1000-5000 nm).

[0148] GFP expression: The 293T cells at passage 3 were trypsinized and cell count permL was established using a hemocytometer. Cell suspension was centrifuged, and the pellet was resuspended with DMEM supplemented with 2% FBS (2%FBS / DMEM) at 2E5 cells per mL concentration.2E4 cells in 0.1 mL were dispensed into wells of a 96-well assay plate and transduced with either serially diluted control AAV9-GFP in F9 (-70oC) for a standard curve or 7.5 µL of formulation candidate AAV9-GFP in 0.1 mL of 2%FBS / DMEM. Cells were incubated in a CO2 chamber (37oC, 5% CO2, and 90%RH) for 48 hours prior to image acquisitions. Images are acquired using ImageXpress Pico at 48 hours post transduction and analyzed using provided software from Molecular Devices. Analysis protocol was as follows: 1) Cell count based on FITC, 2) Exposure, FITC: 200 ms and TL: 10 ms, 3) Threshold: 30, and 4) Cell size, Min: 5 µm, Max: 30 µm. Experimental Procedures Surfactant confirmation

[0149] AAV9-GFP (1.3E13 vg / mL), was diluted to 1E12 vg / mL with formulation buffer(10 mM NaPO4, 150 mM NaCl, 1 mM MgCl2, 0.02% poloxamer 188 (F-68)) and filled with a 0.6 mL fill volume. Vials were capped before removal from a biological safety cabinet (BSC) then sealed and labeled. Static set vials were kept at room temperature without agitation to serve as a static control. The agitation vials were agitated on an orbital shaker at 1,000 rpm for 4 hours at ambient temperature. The agitated samples were exposed to 5 consecutive freeze / thaw cycles after the agitation stress, while the static vials were stored at 5ºC. The samples were analyzed with the analytical methods as previously described.38061.0011P1

[0150] For comparison, AAV2-GFP was diluted to 1E12 vg / mL in the following buffers:10 mM NaPO4, 110 mM NaCl, 0.02% F-68, pH 7.4 and 10 mM NaPO4, 200 mM NaCl, 0.02% F-68, pH 7.4. Both diluted samples were analyzed by visual appearance and DLS analyses. Accelerated Stability Study Sample preparation for accelerated stability study

[0151] After the surfactant confirmation study, 0.005% and 0.02% of F-68 were added tothe testing formulation candidates (Table 3). AAV9-GFP lots were pooled based on ddPCR titers (Table 6) and AAV9-GFP formulations were prepared by dilution of 1:10.7 AAV9-GFP into each formulation buffer. Final concentrations of excipients described in Table 3. Table 6: AAV9-GFP Pool Preparation and Final Concentration SamplesConc.(vg / mL) Vol. (mL) Total vg in Total Vol. Final Conc.Filling for Accelerated Study

[0152] Each sterile-filtered formulation was filled into the final containers at 0.6 mL fillvolumes. After filling, the containers were stoppered before removal from the biological safety cabinet (BSC) then sealed and labeled. The samples were then stored at the designated temperatures (Table 7.2) or exposed to acute stress conditions, such as agitation, freeze / thaw, and UV light exposure (Table 7.1). T0 analyses were performed. Table 7.1: Number of Vials for Acute Stresses per Formulation Acute Stresses Number of Vials38061.0011P1 Table 7.2: Number of Vials for Time Zero and Temperature Storage per Formulation Number of Vials Temps (ºC) T0 T=2 week T=4 week T=8 week ExtraSa

[0153] The agitation samples were vortexed on a bench-top shaker at 1,000 rpm for 4hours. An identical set of samples was kept at room temperature, without agitation for comparison. The freeze / thaw samples were exposed to 5 consecutive cycles of freeze / thaw from -70°C to ambient temperature. The UV light stressed samples were exposed to a total of 8 watts- hrs / m2UVA light. For negative controls, an identical set of samples was incubated in the same UV exposure chamber but was shielded from UV light exposure by covering the samples with foil. Results and Discussion Surfactant Confirmation

[0154] To evaluate the effect of 0.02% of poloxamer 188 (F68) in formulations forstorage of AAV, particularly AAV9, AAV9-GFP at 1E12 vg / mL with 0.6 mL fill volume in a 2 cc vial was exposed to 4 hours of agitation followed by 5 cycles of freeze / thaw. AAV9-GFP was clear, colorless, and with light particulates upon agitation and comparable to a static control. AAV9-GFP showed comparable numbers of subvisible particles concentrations by FlowCAM (Table 8) and mean radii by DLS analyses (Table 9) to a static control. In contrast, AAV2-GFP in both 110 mM NaCl and 200 mM NaCl buffers showed large mean radii and were multimodal (Table 9). These results suggest that DLS analysis can differentiate changes in mean radius. GFP expression assay using cell counts and cell total integrated intensity (CTII) (Table 10) did not show significant differences between the samples exposed to agitation and repeated freeze / thaw compared to static control. These results suggest that 0.02% of F68 is sufficient to stabilize AAV9-GFP under agitation and repeated freeze / thaw stresses. The lower concentration of F-68, 0.005%, was chosen for incorporation into the formulation matrix (Tables 2 and 3). Table 8. Results of FlowCAM Analysis38061.0011P1 Sample Cell Counts CTII Treatments ID. AAV9-GFP STDEV AAV9-GFP STDEV 1010101010. y Mean Peak 2 Peak 3 Samples Radius %Pd Radius Radius ity .0.1.1.0.6.1Table 10. Results of GFP Expression Analysis Sample Cell Counts CTII TreatmentsAccelerated Stability Study Osmolality of Formulations

[0155] Osmolality of formulations containing salt and sorbitol at time zero (T0) were~320 and ~360 mOsm / kg, respectively (Table 11). As shown in Table 3, there were residual excipients due to formulation preparation by dilution.38061.0011P1 Table 11. Results of Osmolality at T0 Ave. Osmolality* Form. No. (mOsm / kg) STDEVa y ga s g a o

[0156] Formulations F1 and F9 exposed to agitation were clear, colorless, and had lightparticulates, while the rest of the formulations were clear, colorless, and free of particles. All static control formulations were clear, colorless, and free of particles as summarized in Table 12. Table 12. Summary of Visual Appearance After Agitation Stress Form. No.Static Agitatedsssssss

[0157] Subvisible particle analysis by FlowCAM showed that agitated formulations F2,F6, and F7 exhibited an increased number of particles compared to their respective static controls (Table 13), but overall concentrations of subvisible particles are insignificant. Table 13. FlowCAM Results After Agitation Stress Particles / mL waterStatic38061.0011P1 >25 µm 0 0 0 0 0 10 0 0 0 0 Agitation[, , 5,F7, and F9) had mean radii of approximately ~16 nm. In contrast, the mean radii of formulations F2 and F6 were ~120 nm and formulations F4 and F8 were ~22 nm (Table 14). Formulations F2, F4, F6, and F8 containing sorbitol were multimodal. Formulations F1, F3, F5, F7, and F9 did not show significant differences in mean radii after agitation, while formulations F2, F4, F6, and F8 showed smaller mean radii than those of respective static formulations. Table 14. DLS Results After Agitation Stress Peak2 Peak3 Peak4 Mean Stress Form Radius Radius ass (I) 5538061.0011P1

[0159] GFP expression analysis showed that agitated formulations F1, F3, F5, and F7showed higher percentages of static formulations compared to formulations F2, F4, F6, and F8 (Table 15). These results obtained from agitation stress study suggest that salt is a protective excipient for AAV9-GFP. Table 15. GFP Expression Results After Agitation Stress Form. Stress Cell Counts CTII CTII No Condition AAV9-GFP STDEV AAV9-GFP STDEV % f St ticStability Against Freeze / Thaw

[0160] All formulations after 5 cycles of freeze / thaw were clear, colorless, and free ofparticles (Table 16). Table 16. Summary of Visual Appearance After Freeze / Thaw Stress Form. No. Freeze-Thaw38061.0011P1 F8 Clear, colorless, and free of particlesF9 Clear, colorless, and free of particlesw showed that formulations F1, F3,and F7 had greater numbers of particles, though these particulate concentrations are relatively low (Table 17). Table 17. FlowCAM Results After Repeated Freeze / Thaw Stress Particles / mL Water Freeze / ThawF1 F2 F3 F4 F5 F6 F7 F8 F9ana ys s a er cyc es o reeze aw s owe a e mean ra oformulations containing salt (F1, F3, F5, F7, and F9) were ~16 nm. In contrast, the mean radii of formulations F2 and F6 were ~110 nm and formulations F4 and F8 were ~22 nm (Table 18). Table 18. DLS Results After Repeated Freeze-Thaw Stress Peak2 Peak3 Peak4 Mean Radius Radius Radius ss

[0163] GFP expression analysis showed that formulations F2, F4, F6, and F8 showedhigher GFP expression compared to formulations F1, F3, F5, and F7 (Table 19).38061.0011P1 Table 19. GFP Expression Results After Repeated Freeze / Thaw Stress Cell Counts CTII Form AAV9-GFPSTDEVAAV9-GFPa y aga ns g exposure

[0164] Following exposure to UV light, all formulations showed no differences inappearance and all were clear, colorless, and free of particles regardless of UV light exposure (Table 20). Table 20. Summary of Visual Appearance After UV Light Exposure Stress Form. No. UV OFF UV ONF1 Clear colorless and free ofClear colorless and free of

[0165] DLS analysis after UV light exposure did not show significant changes (Table21). Formulations containing salt (F1, F3, F7, and F9) had mean radii of approximately ~16 nm, apart from formulation F5 which has a mean radius of ~35 nm when exposed to UV and ~16 nm mean radius when not exposed to UV light. In addition, formulations F2, F4, F6, and F8 containing sorbitol were multimodal.38061.0011P1 Table 21. DLS Results After UV Light Exposure Stress Cell Counts CTII CTII Form. No. UV Condition AAV9-GFP STDEV AAV9-GFP STDEV % of UVTable 22. GFP Expression Results after UV Light Exposure Stress Form. UVCell Counts CTII CTIIV38061.0011P1 Form. UVCell Counts CTII CTIINo. Condition AAV9-GFP STDEV AAV9-GFP STDEV % of UV.with sorbitol or 0.02% F68 with salt (F5 and F7) appeared to provide better protection to AAV9- GFP from UV light exposure (Table 22). These results obtained from UV light exposure stress study suggest that appropriate combinations of F68 and either sorbitol or salt are necessary for AAV9-GFP's stability. Stability During Temperature Storage

[0167] To evaluate the stability of AAV9-GFP in various formulations at differentstorage temperatures over a period of 8 weeks, formulations were stored at designated temperatures and analyzed by various analytical methods.

[0168] Visual appearance of all formulations at T0 were clear, colorless, and free ofparticles, and remained the same when stored at -70°C, -20°C, 5°C, and 25°C for 8 weeks and at 40°C for 4 weeks not shown). No formulations showed visible changes over 8 weeks of storage regardless of temperatures.

[0169] The pH of formulations F3 and F7 showed bigger deviations from their target pH8.0 by ~0.3 units after 8 weeks of storage at -20°C, 5°C, and 25°C (Table 23). Formulations with phosphate buffer F1, F2, F5, and F6 appeared to show less deviations from their target pH 7.0 (Table 23). Table 23. pH Changes Over Time Code T=2weeks T=4weeks T=8weeks T038061.0011P1 Code T=2weeks T=4weeks T=8weeks T0 -5°C 25°C 40°C -20°C 5°C 25°C 40°C -70°C -20°C 5°C 25°CbyFlowCAM analyses showed no significant changes over 8 weeks regardless of formulations. Results of intermediate time point, T=4weeks, are shown in Table 26. Table 24. FlowCAM Results at T0 Particles / mL Water F1 F2 F3 F4 F5 F6 F7 F8 F9Table 25. FlowCAM Results at T=8weeks Particles / 38061.0011P1 Table 26. FlowCAM Results at T=4weeks Particles / Temp.Water F1 F2 F3 F4 F5 F6 F7 F8 F9

[0000] S ana yses s owed t at ormu at ons conta n ng sorb to were mu t moda atT0 and remained multimodal over 8 weeks of storage regardless of temperature (Tables 27 and 28). Formulations containing salt (F1, F3, F5, F7, and F9) showed mean radii of ~16 nm at T0, while formulations containing sorbitol such as F2 and F6 showed mean radii between ~130-150 nm and formulations 4 and F8 showed mean radii of ~25 nm. Mean radii of all formulations stored at -70°C and -20°C for 8 weeks did not show significant changes compared to T0. However, formulations F3 and F5 stored at -20°C for 8 weeks became multimodal. Formulations F1, F3, F5, F7, and F9 stored at 5°C for 8 weeks showed mean radii of ~16 nm, like their respective T0. Formulations F4, F6, and F8 stored at 5°C for 8 weeks showed smaller mean radii than those measured at T0, while F2V mean radius did not change. Formulation F2 stored at 25°C for 8 weeks showed a smaller mean radius compared to those stored at -70°C, -20°C, and - 5°C for 8 weeks. Formulations F1, F3, and F5 at 25°C and F9 at -70°C for 8 weeks showed inconsistent %Pd from triplicate analysis (Table 28). In addition, all formulations stored at 40°C for 4 weeks showed multimodal (Table 30). Results of intermediate time points, T=2weeks and T=4weeks, are shown in Tables 29 and 30. Results of Dynamic Light Scattering (DLS) analysis over 8 weeks suggest that formulations containing salt (F1, F3, F5, and F7) stored at -70°C and 5°C appeared to be stable.38061.0011P1 Table 27. DLS Results at T0 Peak2 Peak3 Peak4 Mean Form. Radius Radius Radius Radius %Pd %Pd %Mass %Pd %Mass %Pd %Mass AAA742AAa e . esu s a = wee s Peak2 Peak3 Peak4 Mean Radius Radius Radius as (I) 138061.0011P1 Peak2 Peak3 Peak4 Mean Radius Radius Radius Radiu %Pd %Mas %Pd %Mas %Pd %Mas (I)Table 29. DLS Results at T=2weeks Mean Peak2 Peak3 Peak4 Radiu Radiu Radiu Radi a I) AAAAAAAAA1AAAAAAAA38061.0011P1 Mean Peak2 Peak3 Peak4 Radiu Radiu Radiu Radi s s s us a I) AAAAAA68551AAA844ATable 30. DLS Results at T=4weeks Peak2 Peak3 Peak4 Mean R di R di ass 6AAAAAA7A6AAA538061.0011P1 Peak2 Peak3 Peak4 Mean Radius Radius Form Radius ass 2AAAAAA9A9820

[0172] To evaluate the biological activity of formulations, GFP expression analyses wereperformed. As shown in Table 31, formulations containing sorbitol (F2, F4, F6, and F8) at T0 showed slightly higher AAV titers compared to respective formulations containing salt (F1, F3, F5, and F7). It was not clear how GFP expression correlated with DLS results of multimodal and mean radii (Tables 27 and 31). As shown in Tables 32 and 34, biological activities of formulations appeared to inversely correlate with storage temperatures. Formulations at 40°C were stored up to 4 weeks. GFP expression analyses showed that formulations at 40°C for 2 weeks lost almost all biological activities regardless of formulation (Table 33). Formulations stored at 25°C for 8 weeks showed loss of biological activities by 70% or more compared to respective T0 (Table 32 and FIG.1). Stabilities of formulations appeared to be ranked from the most stable to the least as follows: F9, F4 and F8, F3 and F7, F1 and F5, and F2 and F6. F9 (CTRL) formulation appeared to show better stability than F5 formulation, which is similar in38061.0011P1 composition to F9 (CTRL) apart from MgCl2(FIG.1).

[0173] All formulations stored at -70°C for 8 weeks showed comparable GFPexpressions to respective T0 and appeared to be stable. Another frozen storage at -20°C for 8 weeks, formulations F2 and F6 showed loss of biological activities by ~20% compared to respective T0, possibly due to changes in pH and concentrations of excipients and / or AAV9-GFPduring storage (Tables 31, 32, 34, and FIG. 2). AAV9-GFP titers and equations of linearregression using AAV titers (FIG. 2B) at each time point are detailed in Table 35 for formulations stored at -20°C. Formulation F3 and F8 after 8 weeks of storage at -20°C appeared to be maintaining AAV9-GFP titers compared to respective T0. All formulations except F3 stored at 5°C for 8 weeks showed decreased titers compared to respective T0 (Tables 31, 32, 34and 36 and FIG.3). Formulations F2 and F6 after 8 weeks of storage at 5°C showed loss of biological activities by ~20% compared to respective T0 (Tables 31, 32, 34, and 36). AAV9- GFP titers and equations of linear regression using AAV titers (FIG.3B) at each time point are detailed in Table 36 for formulations stored at 5°C. Formulation F3 appeared to be the most stable, followed by formulations F1 and F9. Table 31. GFP Expression Results at T0 Form.Cell Counts CTIIAAV9-GFP AAV9-GFP38061.0011P1 Table 32. GFP Expression Results of Cell Counts at T=8weeks rm. -° ° ° °Fo 70C -20C 5C 25C No. AAV9- AAV9- AAV9- AAV9- GFP GFP V 09 09 09 09080910 0809Table 33. GFP Expression Analysis Results at T=2weeks -20°C 5°C 25°C 40°Cll F V 09090909090908080038061.0011P1 For AAV9- AAV9- AAV9- AAV9-GFP m GFP STDEV GFP STDEV GFP STDEV(vg / mL)(vg / mL)STDEVL L 090909090907090907. p -70°C -20°C 5°C 25°CForm. AAV9-GFP AAV9-GFP V 090809090809090909Table 35. GFP Expression Results of Formulations Stored at -20°C Over 8 Weeks Form. No.AAV9-GFP (CTII, vg / mL) Titers,T0 T 2 k T 4 k T 8 k E ti21121122238061.0011P1 Table 36. GFP Expression Results of Formulations Stored at 5°C Over 8 Weeks Form. No.AAV9-GFP (CTII, vg / mL) Titers,T0 T=2wks T=4wks T=8wks Equations111111111111121212t provide maximalstability to AAV9-GFP. To develop optimal formulations, pH, buffer, tonicity modifier, and F68 concentration were evaluated. Based on surfactant confirmation results, 0.02% of F68 appeared to stabilize AAV9-GFP during agitation and repeated freeze / thaw stresses.

[0175] The osmolalities of formulations containing salt and sorbitol were ~320mOsm / Kg and ~360 mOsm / Kg, respectively. Acute stress studies showed no significant changes in visual appearance, subvisible particle concentrations by FlowCAM, and mean radii and %Pd by DLS analyses. Agitation stress study showed that salt in the formulations appeared to be stabilizing AAV9-GFP. However, sorbitol appeared to be stabilizing AAV9-GFP when exposed to 5 cycles of freeze / thaw. UV light exposure study showed that combination of 0.005% F68 and sorbitol and combination of 0.02% F68 and salt appeared to stabilize AAV9-GFP.

[0176] Temperature storage studies showed no significant changes in visual appearances,pH, subvisible particle concentrations by FlowCAM, and mean radii by DLS analyses (except F2 and F6) compared to time 0. However, more formulations showed multimodal as storage temperature increased. It has been noted that DLS analyses of all formulations containing sorbitol were multimodal at T0 and remained throughout the study. GFP expression analyses showed that formulations' biological activities were inversely correlated with the storage temperature. All formulations at -70°C for 8 weeks appeared to be stable. Formulations F2 and F6 after 8 weeks of storage at -20°C lost biological activities by ~20%. But formulations F3 and F8 appeared to be stable at -20°C for 8 weeks. In addition, formulation F3 (F3) is the leading formulation for refrigerated storage (5°C) among those evaluated.38061.0011P1 Example 2: AAV9-GFP FORMULATION OPTIMIZATION

[0177] The short-term stability of AAV9-GFP at 1E14 vg / mL in liquid formulationscontaining either no sugar, 2% sorbitol, or 2% trehalose was evaluated. Formulations were examined under static storage conditions at frozen (¬70ºC), controlled ambient (25ºC), and accelerated (40ºC) temperatures for up to eight (8) weeks. As a stability-indicating assay, GFP expression assays were performed at each timepoint. Experimental Design

[0178] The active pharmaceutical ingredient (API) examined was AAV9-GFP. Thematerial used was comprised of the following: AAV9-GFP at a concentration of 1.68E14 vg / mL in a formulation comprising 10 mM Tris, 150 mM NaCl, 0.02% (w / v) poloxamer 188, 1 mM MgCl2, pH 8.0. Formulation Parameters

[0179] The stability of AAV9-GFP was monitored in three (3) formulations. Table 37describes the matrix of formulations that were monitored in the stability study. Formulations were prepared using the materials and techniques described below. Table 37: Formulation Matrix for Accelerated Stability Study Form. AAV9-GFP SorbiMgCl2 Surfactant NoConcentration tol Trehalose Buffer pH NaCl(%w / v) erFormulation Preparations Formulation Optimization Study for AAV9-GFP

[0180] Approximately 10.2 mL of AAV9-GFP at 1.68E14 vg / mL was diluted to 1E14vg / mL in each formulation buffer to make the final formulations listed in Table 37. F2 and F3 formulations were spiked with Sorbitol or Trehalose, respectively, to make the final 2% sugar in the final formulations. In an aseptic biological safety cabinet (BSC), the formulations were then sterile filtered through 0.2 μm syringe filters. For each formulation, twenty-five 2 mL CZ vials were filled with a 0.6 mL fill volume and 12 vials were filled at a 0.09 mL fill volume. All vials38061.0011P1 were then stoppered, crimped, and labeled. Vials were then stored at frozen (-70°C), refrigerated (5°C), controlled ambient (25°C), and accelerated (40°C) storage temperatures based on the study design. Stress Conditions Formulation Optimization Stress Conditions

[0181] The Formulation Optimization Study for AAV9-GFP included incubation offormulations at frozen (-70°C), refrigerated (5°C), controlled ambient (25°C), and accelerated (40°C) storage temperatures. The temperature storage portion of the study was performed over an eight-week period. Table 38 summarizes stress conditions that were used for stability evaluation. Table 38: Summary of Formulation Optimization Stress Conditions Stress Conditions Time Point(s)5°C 0Analytical Methods Table 39: Analytical Methods for Formulation Optimization Study Temperature Stress Time Points Analytical MethodsT0 3 da s 5 da s 2 wk 4 wk 8 wkResults and Discussion

[0182] GFP Expression was measured as described above in Example 1. At eachtimepoint, GFP expression assay was conducted on all samples. At all timepoints, the38061.0011P1 multiplicity of infection (MOI) standard using aliquots of AAV9-GFP stored at -70°C showed good linearity for both cell count and cell total integrated intensity (CTII) readouts (R2>0.99). ). All MOI graphs can be found in FIGs.4-9 Only sample data within the MOI standard ranges were used. The calculated AAV9-GFP concentrations of formulations F1–F3 for both cell count and CTII readouts can be found in Table 40.

[0183] There were no significant differences between formulations F1–F3 at T0 foreither cell count or CTII readouts and all formulations were similar to the target concentration (1E14 vg / mL). After 3 days at 40°C, the AAV9-GFP concentration decreased 72–79% by cell count and 69–75% via CTII compared to T0 values. The AAV9-GFP concentration decreased 88–91% by cell count and 87–88% by CTII after 5 days storage at 40°C when compared to T0 values. At 25°C, the calculated AAV9-GFP concentration by cell count decreased 50–60% after 2 weeks, 80–83% after 4 weeks, and 95–96% after 8 weeks when compared to T0 values. This trend was reflected in the CTII-based AAV9-GFP concentrations which saw a decrease of 46– 54% at 2 weeks, 77–78% at 4 weeks, and 94% after 8 weeks storage at 25°C when compared to T0 values. After 8 weeks at -70°C, there was no significant change in calculated AAV9-GFP concentration from T0 values for either cell count or CTII readouts. Overall, all formulations displayed similar trends of decreasing AAV9-GFP concentrations during storage at both 40°C (FIG.10) and 25°C (FIG.11).38061.0011P1 Table 40: Formulation Optimization Study Calculated AAV9-GFP Concentration Results Calculated AAV9-GFP Concentration (vg / mL) Time Point Form. No.Cell Count CTIIAverage STDEV ΔT0 Average STDEV ΔT0

[0184] The stabilities of AAV9-GFP at 1E14 vg / mL in three (3) different liquidformulations were examined. The three (3) liquid formulations contained either a sugar (sorbitol or trehalose) or no sugar (Table 37). The formulations were stored at frozen (-70ºC), controlled ambient (25ºC), and accelerated (40ºC) temperatures for up to eight (8) weeks. Over the course of the storage, the stability of AAV9-GFP was evaluated by GFP expression assays.

[0185] The multiplicity of infection (MOI) standard showed good linearity for both cellcount and CTII readouts (R2>0.99) at all timepoints. At T0, there were no significant differences among formulations F1–F3 for either cell count or CTII readouts and all formulations were comparable to the target concentration of 1E14 vg / mL. At 40°C, the AAV9-GFP concentration decreased approximately 74% after 3 days and ~89% after 5 days compared to T0 values. At 25°C, the calculated AAV9-GFP concentration decreased ~52% after 2 weeks, ~80% after 4 weeks, and ~95% after 8 weeks when compared to T0 values. After 8 weeks at -70°C, there was no significant change in calculated AAV9-GFP concentration from T0 values for either cell count or CTII readouts.

[0186] Overall, similar trends of decreasing AAV9-GFP concentrations during storage at38061.0011P1 both 40°C and 25°C were observed for all formulations. The three liquid formulations performed equivalently to each other throughout the duration of the study. Example 3: SHANK3 AAV Formulation

[0187] Deletions or mutations involving SHANK3 account for 0.5-1% of all autismspectrum disorder (ASD) and about 2% of ASD patients with intellectual disability (ID). However, there is no effective treatment for ASD and / or ID. To treat this disorder, an AAV therapy was developed. The formulation is as follows: AAV2 / 9-miniSHANK3(~6.5E13vg / ml), 10 mM Tris, 1 mM magnesium chloride (MgCl2), 150 mM sodium chloride (NaCl) and 0.02% poloxamer 188, pH 8.0. The formulation was stable at -80°C for 24 months with no significant decrease in purity, strength or potency (data not shown). Example 4: GALT AAV Formulation

[0188] Galactosemia results from the inability to metabolize galactose, and type 1galactosemia specifically is caused by pathogenic variants in the GALT gene. To treat this disorder, an AAV therapy was developed. The formulation is as follows: AAV2 / 9-hGalt(~2.4E13 vg / mL), 10 mM Tris, 1 mM magnesium chloride (MgCl2), 150 mM sodium chloride(NaCl) and 0.02% poloxamer 188, pH 8.0. The formulation was stable at -80°C for 24 months with no significant decrease in purity, strength or potency (data not shown). Example 5: PAX4 AAV Formulation

[0189] Mutations in PAX4 are associated with diabetes, and delivery of intact PAX4would provide therapeutic benefits for T1D and late stage T2D patients. To treat this disorder, an AAV therapy is being developed. The formulation is as follows: AAV2 / 9-hPAX4 , 10 mM Tris, 1 mM magnesium chloride (MgCl2), 150 mM sodium chloride (NaCl) and 0.02% poloxamer 188, pH 8.0.38061.0011P1 Table 41. Therapeutic Protein Sequences and Capsid Sequences SEQ ID Description Sequence GT SD VE KP VN SP SE DS PT LS GG PG PA GT SD VE KP VN SP SE DS PT LS GG SG LP GR GL RG RV AL LC RP QP AS QA RH AN EV38061.0011P1 SEQ ID Description Sequence KY RL DS GG KQ PK GC TY QG LA TN KI QY RP

[0190] Furthermore, the disclosure encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists (e.g., in Markush group format), each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the38061.0011P1 disclosure, or aspects of the disclosure, is / are referred to as comprising particular elements and / or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included in such ranges unless otherwise specified. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub–range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0191] This application refers to various issued patents, published patent applications,journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0192] Those skilled in the art will recognize or be able to ascertain using no more thanroutine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the disclosure, as defined in the following claims.

Claims

38061.0011P1 CLAIMS What is claimed is:

1. A pharmaceutical composition comprising:a) an adeno-associated virus (AAV) particle;b) about 5 mM to about 25 mM of a buffering agent;c) about 0.5 mM MgCl2 to about 1.5mM MgCl2;d) about 50 mM to about 150 mM of a tonicity agent; ande) from about 0.02% to about 0.2% of a non-ionic surfactantwherein the pH of said pharmaceutical composition is between about 5.0 and about 9.

0.

2. The pharmaceutical composition according to claim 1, wherein said AAV is AAV9 or avariant thereof.

3. The pharmaceutical composition according to claim 2, wherein said AAV is AAV9.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein saidAAV is present at a concentration of about 1×1011vg / mL to about 1×1015vg / mL.

5. The pharmaceutical composition according to claim 4, wherein said AAV is present at aconcentration of about 1×1014vg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein saidbuffering agent is 10 mM Tris.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein saidpharmaceutical composition comprises 1 mM MgCl2.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein saidtonicity agent is 150 mM NaCl.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein said non-ionic surfactant is poloxamer 188.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein saidpharmaceutical composition is liquid at room temperature.38061.0011P111. The pharmaceutical composition according to any one of claims 1 to 10, wherein saidpharmaceutical composition is stable at -80°C for at least 24 months.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein saidpharmaceutical composition is a unit dosage form.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein saidpharmaceutical composition in the unit dosage form has a volume of between about 2 mL and about 3 mL.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein saidpharmaceutical composition has an osmolality is 150 to 450 mOsm / kg.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein saidpharmaceutical composition does not comprise a preservative.

16. A pharmaceutical composition comprising:a) an AAV9 particle;b) 10 mM Tris;c) 1mM MgCl2;d) 150 mM NaCl; ande) 0.02% poloxamer 188wherein said pharmaceutical formulation has a pH of about 8.0.

17. A unit dosage form comprising the pharmaceutical composition of any one of claims 1 to16.

18. A method of manufacturing the pharmaceutical composition of any one of claims 1 to 17comprising a) obtaining an AAV drug substance composition; andb) performing tangential flow filtration (TFF) to transfer the AAV drugsubstance composition into a buffer comprising: i) 10 mM Tris;38061.0011P1 ii) 1mM MgCl2;iii) 150 mM NaCl; andiv) 0.02% poloxamer 188.

19. A method of treating a subject in need thereof with an AAV gene therapy comprisingadministering the pharmaceutical composition of any one of claims 1 to 16 to the subject.

20. The method of claim 19, wherein said step of administering comprises administeringintravenously.

21. The method of claim 19, wherein said step of administering comprises administeringintramuscular (IM), intraductal, intracerebroventricular (ICV), intravitreal, subretinal and / or suprachoroidal.