Methods for purification of adeno associated virus particles by anion exchange chromatography

Abstract

Provided herein are methods for purifying adeno-associated virus particles by anion exchange mixed mode chromatography using a pH buffer.

Description

METHODS FOR PURIFICATION OF ADENO ASSOCIATED VIRUS PARTICLES BY ANION EXCHANGE CHROMATOGRAPHYCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 569,613, filed March 25, 2024, which is entirely incorporated herein by referenceBACKGROUND

[0002] The use of recombinant adeno-associated viruses (rAAV)-based vectors as gene therapy products has been advanced. Different naturally occurring and recombinant Adeno-Associated Vims (AAV)-based vector serotypes target different tissues, organs, and cells. The ability of AAV to transduce non-dividing and dividing cells, and the resulting long-term transgene expression observed in clinical trials may help realizing the potential of the gene therapy approach. As part of the production of rAAV, purification of rAAV is needed.

[0003] rAAV particles may comprise an AAV capsid composed of about 60 capsid protein subunits, enclosing a single-stranded DNA genome of about 4.7 kilobases (kb). AAV or rAAV particles are produced in packaging host cell cultures and the co-expression of AAV rep cap genes and gene of interest (GOI) are required to replicate and package the recombinant genome into the viral particle. Genes for genome replication, capsid formation and genome packaging can be expressed from transfected plasmids, integrated into the host cell genome or introduced to the cell by recombinant viruses. Then cells are lysed to release rAAV particles for collection. However, the cell lysate contains various cellular components that must be separated from the rAAV particles.BRIEF SUMMARY

[0004] Currently, there are needs in the development of effective methods for separating genome-containing AAV vector particles (full) from genome-deficient AAV particles (empty capsids). The full rAAV particles have the desired transgene packaged while the empty rAAV capsids lack the desired transgene. The method of purifying a lysate after Affinity chromatography comprising both full and empty rAAV particles of the present disclosure solves the needs thereof.

[0005] In one aspect, disclosed herein is a method of purifying full adeno-associated virus (AAV) capsids from an AAV fraction comprising empty AAV capsids and full AAV capsids on a preparative scale, comprising:(a) loading an AAV fraction comprising empty AAV capsids and full AAV capsids onto a preparative chromatography;(b) applying an aqueous mobile phase to the preparative chromatography, wherein the aqueous mobile phase comprises a pH gradient from about pH 10 to about pH 2.7 and a gradient length of at least 3 column volumes, and(c) collecting fractions comprising the full AAV capsids.

[0006] In some embodiments, the preparative chromatography comprises a plurality of quaternary ammonium ligands or diethylaminoethyl group on (i) porous or non-porous particles, (ii) monolith, (iii) membrane, or (iv) hydroxyapatite. In some embodiments, the AAV is natural AAVs or any engineered AAVs. In some embodiments, the AAV is AAV1, AAV2, AAV2 7m8, AAV3, AAV4, AAV5, AAV 6, AAV7, AAVS, AAV9, AAV10, AAVrhlO, AAVRh74, AAV2i8, Anc80L85, or MyoAAV. In some embodiments, steps (b) and (c) are conducted at a temperature from about 2 °C to about 25 °C. In some embodiments, steps (b) and (c) are conducted at a temperature from about 16 °C to about 22 °C. In some embodiments, the aqueous mobile phase comprises a first buffer and a second buffer. In some embodiments, the first buffer is ammonium hydroxide / ammonium chloride buffer, ammonium hydroxide / ammonium acetate buffer, borate buffer, bicarbonate / carbonate buffer, phosphate buffer, HEPES (4-(2- hy droxy ethyl)- 1 -piperazineethane sulfonic acid) buffer, HEPPSO (4-(2- hydroxylehtyl)piperazine- 1 -(2-hydroxypropane sulfonic acid)) buffer, AMPD (2-amino-2- methyl-l,2-propanediol) buffer, CABS (4-cyclohexylamino-l-butane sulfonic acid) buffer, CAPS (N-cy cl ohexylo-3 -aminopropane sulfonic acid) buffer, CAPSO (3-cyclohexylamino-2- hydroxyl-1 -propane sulfonic acid) buffer, carbonate buffer, tri s(hy roxymethyl)aminom ethane (Tris) buffer, or l,3-bis(tris(hydroxymethyl)methylamino)propane (bis-tris propane or BTP) buffer. In some embodiments, the first buffer comprises bis-tris propane from about 15 mM to about 25 mM, magnesium chloride from about 1 mM to about 3 mM, and pluronic from about 0.0005% to about 0.002% (v / v). In some embodiments, the first buffer comprises bis-tris propane at about 20 mM, magnesium chloride at about 2 mM, and pluronic at about 0.001% (v / v). In some embodiments, the first buffer is at about pH 9.0. In some embodiments, the second buffer is acetate buffer, citrate buffer, succinate buffer, propionate buffer, or dimethyl glutarate buffer. In some embodiments, the second buffer comprises acetate from about 15 mM to about 25 mM, magnesium chloride from about 1 mM to about 3 mM, and pluronic from about 0.0005% to about 0.002% (v / v). In some embodiments, the second buffer comprises acetate at about 20 mM, magnesium chloride at about 2 mM, and pluronic at about 0.001% (v / v). In some embodiments, the second buffer is at about pH 5.0. In some embodiments, the pH gradient is from about 9.0 to about 5.0. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 0% second buffer to about 100% second buffer. In some embodiments, the acetate in the second buffer is sodium acetate, potassium acetate, orammonium acetate. In some embodiments, the acetate is sodium acetate. In some embodiments, the gradient length is from about 3 to about 250 column volumes. In some embodiments, the method provide good separation of the full capsids from the empty capsids. In some embodiments, the combined collected fractions of full capsids comprise low percentage of empty capsids. In some embodiments, an average percentage of empty capsids in total capsids in the final collected fractions is no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 7%, no more than 5%, no more than 3%. In some embodiments, the average percentage of empty capsids in total capsids in the final collected fractions is no more than 1%BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and the disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0008] FIG. 1 shows a sample AEX chromatogram generated using a traditional salt gradient in elution.

[0009] FIG. 2 shows a sample analytical ultracentrifugation (AUC) diagram characterizing rAAV particles distribution in the product obtained by the salt gradient process.

[0010] FIG. 3 shows a sample AEX chromatogram generated using a pH gradient in elution.

[0011] FIG. 4 shows a sample analytical ultracentrifugation (AUC) diagram characterizing rAAV particles distribution in the product obtained by the pH gradient process.DETAILED DESCRIPTION[0012[ Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.Definitions

[0013] As used in the specification and claims, the singular forms “a”, “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “rAAV particle” includes one or more rAAV particles.

[0014] The term “about” or “approximately” refers to a particular value within the acceptable error range determined by a person of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e , the limitations of the measurement system For example, according to the practice in the art, “about” can mean within 1 or more than 1 standard deviation. Alternatively, “about” can mean a range of up to 20 %, up to 10 %, up to 5 %, or up to 1 % of a given value Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, up to 5-fold, or up to 2-fold, of a value. Where particular values can be described in the application and claims, unless otherwise stated the term “about” meaning up to an acceptable error range for the particular value should be assumed.

[0015] As used herein, in the case of a polynucleotide, the term “sequence” generally refers to the sequence of nucleotides in the polynucleotide in the direction from the 5’ end to the 3’ end, wherein the nucleotides adjacent to each other in the sequence are in the polynucleotide It is continuous in the primary structure. The sequence can also be a linear sequence of a part of a polynucleotide known to contain additional nucleotides in one or two directions

[0016] As used herein, the terms “identity,” “homology,” or “sequence identity” generally refer to the similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When using program such as Emboss Needle or BestFit to determine the sequence identity, homology, or similarity between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix may be selected, such as blosum45 or BLOSUM80, to optimize the identity, similarity, or homology score. Preferably, homologous polynucleotides are those that hybridize under stringent conditions as defined herein and have at least 70 %, preferably at least 80 %, more preferably at least 90 %, more preferably at least 95 %, more preferably at least 97 %, more preferably at least 98 %, and even more preferably at least 99 % sequence identity. When a sequence of comparable length is optimally aligned, homologous polypeptide preferably has at least 80 %, at least 90 %, at least 95 %, at least 97 %, at least 98 % sequence identity, or at least 99 % sequence identity.

[0017] With regard to the polypeptide or polynucleotide herein, the “percent sequence identity (%)” is defined as the percentage of amino acid residues or nucleotides in the query sequence that are identical to the amino acid residues or nucleotides of the second, reference polypeptide / polynucleotide sequence or part thereof calculated after aligning the sequences and introducing gaps if necessary to obtain the maximum sequence identity percentage, and notremoving any conservative substitutions that are regarded as part of sequence. The alignment aimed at determining the percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, such as using publicly available computer software, such as the BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including the full length of the sequences being compared is eligible for any algorithms needed to obtain maximal alignment. The percent identity can be measured over the length of the entire defined polypeptide / polynucleotide sequence, or can be measured over a shorter length, for example, the length of a fragment taken from a larger, defined polypeptide / polynucleotide sequence, such as A fragment of at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, or at least 200 consecutive residues / nucleotides. These lengths are exemplary only, and it should be understood that the forms herein shown in the drawings, or the sequence supported in the Sequence Listing can be used to describe any fragment length thereon may be measured with a percentage of the length.

[0018] The proteins described herein may have one or more modifications relative to the reference sequence. The modification may be deletion, insertion or addition, or substitution or substitution of amino acid residues. “Deletion” refers to a change in amino acid sequence due to the lack of one or more amino acid residues “Insertion” or “addition” refers to an amino acid sequence change that results in the addition of one or more amino acid residues compared to a reference sequence. “Substitution” or “substitution” refers to the replacement of one or more amino acids with different amino acids. In the present disclosure, the mutation of the polypeptide relative to the reference sequence can be determined by comparing the polypeptide with the reference sequence. The optimal alignment of sequences for comparison can be performed according to any known method in the art.

[0019] As used herein, the term “extracted” refers to the isolation and / or separation of cellular or other components that are associated with, in nature, polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof under normal circumstances Those skilled in the art should understand that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not need to be “isolated” to be distinguished from their naturally occurring counterparts. In addition, “concentrated”, “isolated” or “diluted” polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are distinguishable from their naturally occurring counterparts because of their concentration or number of molecules per unit volume is greater than (“concentrated”) or less than its naturally occurring counterpart (“isolated”). Enrichment can be measured based on absolute amounts,such as the weight of solution per unit volume, or it can be measured relative to the second, potentially reference species present in the source mixture.

[0020] The terms “polynucleotide”, “nucleic acid”, “nucleotide” and “oligonucleotide” are used interchangeably. They refer to polymeric forms of nucleotides of any length (whether they ae deoxyribonucleotides or ribonucleotides) or their analogs. A polynucleotide can have any three- dimensional structure and can perform any known or unknown function. The following are nonlimiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, loci determined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, isolated plasmids, vectors, any DNA isolated sequence, any RNA sequence, nucleic acid probes, primers, or a synthetic oligonucleotide DNA. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example, by conjugation with a labeling component When referring to a DNA / RNA, A can mean adenine, C can mean cytosine, G can mean guanine, T can mean thymine, U can mean uracil. U and T can be used interchangeably when referring to a DNA or an RNA

[0021] When applied to polynucleotides, “recombinant” means that the polynucleotide is the product of cloning, restriction digestion, ligation, other procedures that produce constructs different from those found in nature, or any combinations thereof. When applied to polypeptides, “recombinant” means that the polypeptide is the expressed / translated product of a recombinant polynucleotide.

[0022] The terms “gene” or “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame, the open reading frame capable of encoding a particular protein after transcription and translation. Gene or gene fragment may be a gene group, the cDNA, or synthetic, as long as the polynuclear nucleotide comprises a sequence having at least one open reading frame, the open reading frame may cover the entire coding region or a section thereof.

[0023] The term “variant” as used herein generally refers to a polynucleotide or polypeptide having a sequence substantially similar to a reference polynucleotide or polypeptide. Procedures for the introduction of nucleotide and amino acid changes in a polynucleotide, protein or polypeptide (see, e.g., Sambrook et al. (1989)). In the case of a polynucleotide, a variant can have deletions, substitutions, additions of one or more nucleotides at the 5' end, 3' end, and / or one or more internal sites in comparison to the reference polynucleotide. Similarities and / ordifferences in sequences between a variant and the reference polynucleotide can be detected using conventional techniques known in the art, for example polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis. Generally, a variant of a polynucleotide, including, but not limited to, a DNA, can have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polynucleotide as determined by sequence alignment programs In the case of a polypeptide, a variant can have deletions, substitutions, additions of one or more amino acids in comparison to the reference polypeptide. Similarities and / or differences in sequences between a variant and the reference polypeptide can be detected using conventional techniques known in the art, for example Western blot. Generally, a variant of a polypeptide, can have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polypeptide as determined by sequence alignment programs.

[0024] As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNAs, and / or the process by which transcribed mRNAs (also referred to as “transcripts”) is subsequently translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

[0025] As used herein, the term “vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction, infection, or transfection, so that the genetic material it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from Pl ; bacteriophages such as lambda phage or Ml 3 phage body and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillae Polyoma vacuole virus (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences,enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site.

[0026] As used herein, the term “AAV virion” or “AAV viral particle” generally refers to a viral particle composed of at least one AAV capsid protein and an encapsulated AAV polynucleotide.

[0027] As used herein, the term “rAAV” generally refers to a recombinant adeno-associated virus. The term “recombinant,” as applied to a polynucleotide, generally refers to that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that collectively result in a construct that is distinct from a polynucleotide found in nature A recombinant virus is a viral particle comprising a recombinant polynucleotide. This term respectively includes replicates of the original polynucleotide construct and progeny of the original virus construct.

[0028] As used herein, the term “host cell” refers to a cell that can be used to be introduced with a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, or yeast or fungal cells such as Aspergillus, or insect cells such as Drosophila S2 cells or Sf9 cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, BHK cells, HEK293 cells, HEK293 derivatives 293T cells, HeLa cells, or human cells.

[0029] While various embodiments of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.Recombinant AAV Vector

[0030] Adeno-associated virus (AAV) belong to the parvovirus, a single strand DNA (ssDNA) virus. The full-length genome of the AAV contains approximately 4.7 kilobases (kb), comprising inverted terminal repeats (ITR) DNA sequences at both ends of the virus encompassing two open reading frames (ORF) called rep and cap.

[0031] The “AAV inverted terminal repeat (ITR)” sequence is a sequence of about 145 nucleotides that exists at both ends of the natural single-stranded AAV genome. ITR is required for the efficient replication of the genome nucleic acid sequences of the symmetrical AAV particles, which can be used as a viral DNA synthesis origin of replication and are necessary structural components for the recombinant AAV vector.

[0032] As used herein, the term “packaging” generally refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle

[0033] AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. The “rep” gene contains polynucleotide sequences encoding four rep proteins rep78, rep68, rep52, and rep40 required for the life cycle of AAV. The “cap” gene contains polynucleotide sequences encoding the AAV capsid proteins VP1, VP2, and VP3 proteins The AAV capsid proteins VP1, VP2 and VP3 are capable to form a 60-subunit symmetrical AAV capsid through interaction between them. AAV rep and cap are referred to herein as AA V “packaging genes.”

[0034] AAV can effectively infect dividing and non-dividing human cells, and its genome can persist extra chromosomally in an episomal form or integrate into the cellular genome Most importantly, although AAV already exists in humans, current research believes that AAV is not related to any disease. Based on its high safety, low immunogenicity, broad host range, ability to mediate stable long-term expression of exogenous genes in vivo AAV has become the most promising vector system in gene therapy.

[0035] To date, more than 13 natural AAV serotypes has been identified according to the tissues or different cell types they infect Capsid engineering has the capability to generate millions or billions of rAAV variants with different tropism. Further, according to the TABLE 1 below, different AAVs have been developed as advantageous vector systems for transfection of specific cell typesTABLE 1; Examples of AAV Serotypes and the Target Tissues Used in Gene Therapy

[0036] The methods described herein are suited to any rAAV serotype, including but not limited to: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-11, AAV-12, AAV-13, AAV-14, AAV-15 and AAV-16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8,AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV HSC12, AAV.HSC13, AAV.HSC14, AAV HSC15, and AAV.HSC16, and derivatives, modifications, or pseudotypes thereof.

[0037] The term “recombinant AAV vector (rAAV vector)” as used herein refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences not derived from AAV) flanked by two AAV ITR sequences. When present in host cells expressing AAV rep and cap proteins, the rAAV vector can replicate and be packaged into AAV virus particles The rAAV vector includes an rAAV polynucleotide (e g , a single stranded polynucleotide encoding rAAV (ss-rAAV); a double stranded polynucleotide encoding rAAV (ds-rAAV), such as, for example, plasmids encoding rAAV; and the like). If an AAV virion includes a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, such as, for example, a transgene to be delivered to a target cell, etc.), it is generally referred to as a “recombinant AAV (rAAV) virion” or an “rAAV viral particle.”

[0038] The term “recombinant AAV (rAAV) virus” or “rAAV viral particle” as used herein refers to rAAV vector encapsidated by at least one AAV capsid protein into AAV viral particles. Currently used host cells for rAAV viral particle production is derived from mammalian cell types, such as human embryonic kidney 293 (HEK293) cells, 293T cells, human fibrosarcoma (HT-1080) cells, differentiated hepatocyte-derived carcinoma (Huh-7) cells, PER.C6 cells, COS cells, murine myeloma (NS0) cells, HeLa cells, Baby Hamster Kidney (BHK) cells, KB cells, and other mammalian cell lines.

[0039] The rAAV virus particles can be produced in the mammalian cell culture system by providing the rAAV plasmid to the mammalian cell. However, the productivity of the virus of most of the above mammalian cell culture systems is insufficient in meeting the requirements of clinical trials and commercial scale production. To this end, an rAAV virus particle production system using insect cells such as Sf9 cells has been developed However, to produce AAV in insect cells, some modifications must be made to obtain the correct stoichiometric ratio of the AAV capsid protein.

[0040] As used herein, the term “empty capsid” or “empty particle” generally refers to a virion that comprises at least one AAV capsid but may lack in whole or in part of the polynucleotide (artificial genome, e.g., an rAAV vector). Empty capsids do not include an intact rAAV vector comprising a heterologous polynucleotide (e g., a transgene).

[0041] Baculovims is a double-stranded circular DNA virus, belonging to Baculoviridae vims family, and has a genome size of 90 kb-230 kb. Baculoviruses are parasites exclusive to arthropods and known to infect more than 600 species of insects. Using Autographa CalifornicaMulticapsid Nuclear Polyhedrosis Virus (AcMNPV), Smith et al. successfully expressed human beta-interferon in the Sf9 cell line in 1983, developing the first baculovirus expression system (Mol. Cell Biol., 1983, 3: 2156-2165). Since then, the baculovirus expression system has been continuously improved and developed, and it has become a very widely used eukaryotic expression system. In 2002, Urabe et al. showed that the baculovirus-infected Sf9 insect cell can support AAV replication, using three recombinant baculoviruses carrying AAV's rep gene, cap gene, and ITR to co-infect the Sf9 cells and successfully prepared rAAV virus particles (Hum. Gene Ther. 2002;13:1935-1943). On this basis, researchers have successively developed systems that are more suitable for large-scale preparation of rAAV virus particles

[0042] In some embodiments, the rAAV vector used to carry the gene of interest in the rAAV virus particle may also include one or more “expression control elements”. The term “expression control element” or “expression regulatory element” as used herein refers to a nucleic acid sequence that affects the expression of an operably linked polynucleotide, including polynucleotide sequences that promote the transcription and translation of heterologous polynucleotides. The expression control elements that can be used in the present disclosure include, but are not limited to, promoters, enhancers, intron splicing signals, poly A sequences, or inverted terminal repeats (ITR).

[0043] A “promoter” is a DNA sequence located adjacent to a heterologous polynucleotide sequence encoding a target product, which is usually operably linked to an adjacent sequence, such as a heterologous polynucleotide. Compared to the amount expressed in the absence of a promoter, a promoter generally increases the amount of heterologous polynucleotide expression.

[0044] Examples of promoter include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG (composite of the CMV enhancer the chicken beta actin promoter (CBA) and the rabbit beta globin intron.), NSE (neuronal specific enolase), synapsin or NeuN promoters, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), SFFV promoter, rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. Other promoters can be of human origin or from other species, including from mice. Common promoters include, e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, [beta] -actin, rat insulin promoter, the phosphoglycerate kinase promoter, the human alpha- 1 antitrypsin (AAT or hAAT) promoter, the transthyretin (TTR) promoter, the TBG promoter and other (hybrid) liver-specific promoters, the desmin promoter and similar muscle-specific promoters, the EFl -alpha promoter, the CAG promoter and other constitutive promoters, hybrid promoters with multi-tissuespecificity, promoters specific for neurons like synapsin and glyceraldehyde-3 -phosphate dehydrogenase promoter, all of which are promoters well known and readily available to those of skill in the art, can be used to obtain high-level expression of the coding sequence of interest. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, also find use herein. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif ).

[0045] Another regulatory element in the rAAV vector is a polyadenylation (poly A) sequence. In some embodiments, the polyadenylation sequence is selected from the group consisting of a modified SV40 late poly-A (SV40pAmL), SV40 early poly-A (SV40pAE), SV40 late poly-A (SV40pAL), rabbit beta-globin poly-A (rbGlobpA), modified synthetic poly-A (mspA), human growth hormone poly-A (hGHpA), and bovine growth hormone poly-A (bGHpA). In some embodiments, the polyadenylation sequence comprises a sequence having at least 90% identity to a modified SV40 late poly-A (SV40pAmL), SV40 early poly-A (SV40pAE), SV40 late poly- A (SV40pAL), rabbit beta-globin poly-A (rbGlobpA), modified synthetic poly-A (mspA), human growth hormone poly-A (hGHpA), and bovine growth hormone poly-A (bGHpA).

[0046] An “enhancer” is a sequence that enhances the activity of a promoter. Different from the promoter, an enhancer does not have the promoter activity, and may generally depend on its location relative to the promoter (i e , upstream or downstream of the promoter) Non-limiting examples of enhancer elements (or portions thereof) that can be used in the present disclosure include baculovirus enhancers and enhancer elements found in insect cells.

[0047] A “stuffer sequence” refers to a nucleotide sequence of a larger nucleic acid molecule (such as, but not limited, to a vector), and is usually to create a desired gap or separation between two nucleic acid features (such as, but not limited, between two coding gene sequences) or to extend the nucleic acid molecule a desired length. The stuffer sequence does not contain protein coding information and may have unknown or synthetic origin, not related to other nucleic acid sequences within the larger nucleic acid molecule, or any combination thereof

[0048] As used herein, the term “intron” generally refers to a DNA molecule that may be isolated or identified from a gene and may be defined generally as a region spliced out during messenger RNA (mRNA) processing prior to translation. Alternately, an intron may be a synthetically produced or manipulated DNA element. An intron may contain enhancer elements that effect the transcription of operably linked genes. An intron may be used as a regulatory element for modulating expression of an operably linked transcribable DNA molecule. A construct may comprise an intron, and the intron may or may not be heterologous with respect to the transcribable DNA molecule.Compositions of rAAV

[0049] In some embodiments, the rAAV is a self-complementary adeno-associated virus (scAAV) vector or a single-stranded AAV (ssAAV) vector. In some embodiments, the rAAV protein is an adeno-associated virus (AAV) capsid protein. In some embodiments, the AAV capsid protein is an AAV3 capsid, an AAV5 capsid, an AAV6 capsid, an AAV8 capsid, an AAV9 capsid, an AAV-DJ capsid, a KPI capsid, an LK03 capsid, an NP59 capsid, or fragments or variants thereof, or fragments or variants thereof.

[0050] In some embodiments, the recombinant rAAV protein comprises an AAV cap protein In some embodiments, the AAV cap protein can be any structural protein known in the art that can form a functional AAV capsid (i.e., packaging DNA and infecting target cells). In some embodiments, the cap protein includes VP1, VP2, and VP3. In some embodiments, the cap protein does not need to comprise all of VP1, VP2, and VP3, as long as it can produce a functional AAV capsid. In some embodiments, the cap protein includes VP1 and VP2. In some embodiments, the cap protein comprises VP1 and VP3. In some embodiments, the cap protein includes VP2 and VP3. In some embodiments, the case, the cap protein comprises VP1. In some embodiments, the cap protein includes VP2. In some embodiments, the cap protein includes VP3.

[0051] VP1, VP2, or VP3 may be derived from any AAV serotype In some embodiments, the VP1 may be derived from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV2 variants, AAV serotype 3 (AAV3, including serotypes 3A and 3B), AAV serotype 4 (AAV4), the AAV serotype 5 (AAV5), the AAV serotype 6 (AAV6), the AAV serotype 7 (AAV7), the AAV serotype 8 (AAV8), the AAV serotype 9 (AAV9), the AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV-RhlO, AAV-Rh74, AAV-2i8 or any other known AAVs. In some embodiments, the VP1 and the wildtype VP1 derived from AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74, or AAV2i8 may have at least 75 %, 80 %, 85 %, 90 %, 95 %, or more sequence identity. In some embodiments case, the VP1 has one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the wildtype VP1 derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, of AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74 or AAV-2i8.

[0052] In some embodiments, the VP2 may be derived from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV2 variants, AAV serotype 3 (AAV3, including serotypes 3A and 3B), AAV serotype 4 (AAV4), the AAV serotype 5 (AAV5), the AAV serotype 6(AAV6), the AAV serotype 7 (AAV7), the AAV serotype 8 (AAV8), the AAV serotype 9 (AAV9), the AAV serotype 10 (AAV10), AAV serotype 11 (AAV 11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV-RhlO, AAV-Rh74, AAV-2i8 or any other known AAVs. In some embodiments, the VP2 and the wildtype VP2 derived from AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAVS, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74, or AAV2i8 may have at least 75 %, 80 %, 85 %, 90 %, 95 %, or more sequence identity. In some embodiments case, the VP2 has one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the wildtype VP2 derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, of AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74 or AAV-2i8.

[0053] The VP3 may be derived from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV2 variants, AAV serotype 3 (AAV3, including serotypes 3A and 3B), AAV serotype 4 (AAV4), the AAV serotype 5 (AAV5), the AAV serotype. 6 (AAV6), the AAV serotype 7 (AAV7), the AAV serotype 8 (AAV8), the AAV serotype 9 (AAV9), the AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV-RhlO, AAV-Rh74, AAV-2i8, AAV-DJ, AAVHSC, LK03, KPI, NP59 or any other known AAVs In some embodiments, the VP3 and the wildtype VP3 derived from AAV1, AAV2, AAV2 variants, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV- Rh74, AAV2i8, AAV-DJ, AAVHSC, LK03, KPI, or NP59 may have at least 75 %, 80 %, 85 %, 90 %, 95 %, or more sequence identity. In some embodiments case, the VP3 has one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the wildtype VP3 derived from AAV1, AAV2, AAV2 variants, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, of AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74, AAV-2i8, AAV-DJ, AAVHSC, LK03, KPI or NP59

[0054] In some embodiments, the cap protein comprises VP1, VP2, VP3, or any combinations thereof derived from AAV of the same serotype; for example, the cap protein may comprise VP1, VP2, VP3, or any combinations thereof derived from AAV2, AAV2 variants, AAV5, or AAV8 In some embodiments, the cap comprises VP1, VP2, VP3, or any combinations thereof derived from different serotypes of AAV; for example, the cap protein may comprise any one or more of VP1, VP2, VP3, or any combination thereof of AAV1, AAV2, AAV2 variants, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, AAV13, AAV-RhlO, AAV-Rh74, AAV-2i8, AAV-DJ, AAVHSC, LKO3, KPI, or NP59.

[0055] In some embodiments, the cap protein may be cloned into pUC57, pFastBacl, modified pUC57, or modified pFastBacl. In some embodiments, the cap protein may be cloned into pUC57. In some embodiments, the cap protein may be cloned into pFastBacl. In some embodiments, the cap protein may be cloned into modified pUC57. In some embodiments, the cap protein may be cloned into modified pFastBacl .

[0056] In some embodiments, the polynucleotide sequence encoding the cap protein is operably linked to a first promoter The first promoter may be any suitable promoter known in the art that can drive the expression of the cap protein. In some embodiments, the first promoter may be a tissue-specific promoter, a constitutive promoter, or a regulatable promoter. In some embodiments, the first promoter can be selected from different sources, for example, the first promoter can be a viral promoter, a plant promoter, or a mammalian promoter.

[0057] The first promoter can include, but are not limited to, a human cytomegalovirus (CMV) immediate-early enhancer or promoter, a SV40 early enhancer or promoter, a JC polyomavirus promoter, a myelin basic Protein (MBP) or a glial fibrillary acidic protein (GFAP) promoter, a herpes simplex virus (HSV-1) latency-related promoter (LAP), a Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, a neuron specific promoter (NSE), a platelet-derived growth factor (PDGF) promoter, hSYN, a melanin aggregation hormone (MCH) promoter, CBA, a matrix metal protein promoter (MMP), a chicken P-actin promoter, CAG, MNDU3, PGK and an EFla promoter.

[0058] In some embodiments, the first promoter is a promoter suitable for expression in insect cells In some embodiments, the promoter suitable for expression in insect cells include, but are not limited to a polh promoter, a plO promoter, a basic promoter, an inducible promoter, an El promoter, or a AE1 promoter. In some embodiments, the first promoter is a polh promoter. In some embodiments, the first promoter is a pl 0 promoter.

[0059] In some embodiments, the 3’ end of a DNA sequence, such as, for example, the cap protein sequence, further comprises a polyadenylation sequence or “poly A sequence”. In some embodiments, the polyadenylation sequences or “poly A sequences” may range from about 1 to about 500 base pairs (bp). In some embodiments, the polyadenylation sequence or “poly A sequence” may be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, 200, or 500 nucleotides.

[0060] In some embodiments, the rep protein can be a replication protein necessary for any rAAV vector to replicate and be packaged into rAAV viral particles. In some embodiments, the rep protein comprises rep78, rep68, rep52 or rep40. In some embodiments, the rep protein maynot comprise all of rep78, rep68, rep52, or rep40, as long as it can allow the rAAV vector to replicate or be packaged into rAAV virus particles. In some embodiment, the rep protein comprises any three of rep78, rep 68, rep52 or rep 40. In some embodiment, the rep protein comprises any two of rep78, rep 68, rep52 or rep 40. In some embodiment, the rep protein comprises any one of rep78, rep 68, rep52 or rep 40. In some embodiment, the rep protein comprises rep78 or rep52. In some embodiment, the rep protein comprises rep78 or rep 40. In some embodiment, the rep protein comprises rep68 or rep52. In some embodiment, the rep protein comprises rep68 or rep40.

[0061] rep78, rep68, rep52, or rep40 may be derived from any AAV serotype In some embodiments, the rep78 may be derived from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3, including serotypes 3A and 3B), AAV serotype 4 (AAV4), the AAV serotype 5 (AAV5), the AAV serotype 6 (AAV6), the AAV serotype 7 (AAV7), the AAV serotype 8 (AAV8), the AAV serotype 9 (AAV9), the AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV-RhlO, AAV-Rh74, AAV-2i8 or any other known AAVs. In some embodiments, the rep78 and the wildtype rep78 derived from AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74, or AAV2i8 may have at least 75 %, 80 %, 85 %, 90 %, 95 %, or more sequence identity. In some embodiments case, the rep78 has one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the wildtype rep78 derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV 13, AAV-RhlO, AAV-Rh74 or AAV-218

[0062] In some embodiments, the rep68 may be derived from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3, including serotypes 3A and 3B), AAV serotype 4 (AAV4), the AAV serotype 5 (AAV5), the AAV serotype 6 (AAV6), the AAV serotype 7 (AAV7), the AAV serotype 8 (AAV8), the AAV serotype 9 (AAV9), the AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV-RhlO, AAV-Rh74, AAV-218 or any other known AAVs. In some embodiments, the rep68 and the wildtype rep68 derived from AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74, or AAV218 may have at least 75 %, 80 %, 85 %, 90 %, 95 %, or more sequence identity. In some embodiments case, the rep68 has one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the wildtype rep68 derived from AAV1, AAV2, AAV3 (including AAV3Aand 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV 13, AAV-RhlO, AAV-Rh74 or AAV-2i8

[0063] In some embodiments, the rep52 may be derived from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3, including serotypes 3A and 3B), AAV serotype 4 (AAV4), the AAV serotype 5 (AAV5), the AAV serotype 6 (AAV6), the AAV serotype 7 (AAV7), the AAV serotype 8 (AAV8), the AAV serotype 9 (AAV9), the AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV-RhlO, AAV-Rh74, AAV-2i8, or any other known AAVs. In some embodiments, the rep52 and the wildtype rep52 derived from AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74, or AAV2i8 may have at least 75 %, 80 %, 85 %, 90 %, 95 %, or more sequence identity. In some embodiments case, the rep52 has one or more amino acid substitutions, deletions, additions, or any combination thereof compared to the wildtype rep52 derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV 13, AAV-RhlO, AAV-Rh74 or AAV-2i8

[0064] In some embodiments, the rep protein comprises rep78, rep68, rep52, or rep40, or any combinations thereof derived from AAV of the same serotype; for example, the rep protein may comprise rep78, rep68, rep52, rep40, or any combinations thereof derived from AAV2. In some embodiments, the rep protein comprises rep78, rep68, rep52, rep40, or any combinations thereof derived from different serotypes of AAVs; for example, the rep protein may comprise any one or more of rep78, rep68, rep52, rep40, or any combination thereof of AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74, or AAV-2i8.

[0065] In some embodiments, the sequence encoding the rep protein is operably linked to a second promoter. The second promoter may be any suitable promoter known in the art that can drive the expression of the rep protein In some embodiments, the second promoter may be a tissue-specific promoter, a constitutive promoter, or a regulatable promoter. In some embodiments, the second promoter can be selected from different sources, for example, the second promoter can be a viral promoter, a plant promoter, or a mammalian promoter

[0066] In some embodiments, the rep protein may be cloned into pUC57, pFastBacl, modified pUC57, or modified pFastBacl. In some embodiments, the rep protein may be cloned into pUC57. In some embodiments, the rep protein may be cloned into pFastBacl. In some embodiments, the rep protein may be cloned into modified pUC57. In some embodiments, the rep protein may be cloned into modified pFastBacl.

[0067] The second promoter can include, but are not limited to, a human cytomegalovirus (CMV) immediate-early enhancer or promoter, a SV40 early enhancer or promoter, a JC polyomavirus promoter, a myelin basic protein (MBP) or a glial fibrillary acidic protein (GFAP) promoter, a herpes simplex virus (HSV-1) latency-related promoter (LAP), a Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, a neuron specific promoter (NSE), a platelet- derived growth factor (PDGF) promoter, hSYN, a melanin aggregation hormone (MCH) promoter, CBA, a matrix metal protein promoter (MPP), a chicken [Lactin promoter, CAG, MNDU3, PGK and an EFla promoter.

[0068] In some embodiments, the second promoter is a promoter suitable for expression in insect cells. In some embodiments, the promoter suitable for expression in insect cells include, but are not limited to a polh promoter, a plO promoter, a basic promoter, an inducible promoter, an El promoter, or a AE1 promoter. In some embodiments, the second promoter is a polh promoter. In some embodiments, the second promoter is a plO promoter.

[0069] In some embodiments, the cap protein and rep protein are derived from AAV of the same serotype; for example, the cap protein and rep protein may be derived from AAV1, AAV2, AAV2 variants, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74, AAV-2i8, or any other known AAVs

[0070] In some embodiments, the cap protein and the rep protein are derived from different serotypes of AAV; for example, the cap protein and the rep protein may be derived from AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-RhlO, AAV-Rh74, AAV-2i8, or any other known AAVs For example, in some embodiments, the cap protein may be derived from AAV5, and the rep protein is derived from the AAV2. In some embodiments, the cap protein may be derived from AAV8 variants, and the rep protein is derived from the AAV2. In some embodiments, the cap protein may be derived from AAV3 variants, and the rep protein is derived from the AAV2

[0071] In some embodiments, the first promoter and the second promoter may be the same promoter. For example, the first promoter and the second promoter may be selected from the group consisting of a polh promoter, a plO promoter, a basic promoter, an inducible promoter, an El promoters, and a AE1 promoter. For example, in some embodiments, the first promoter and the second promoter are both polh promoters. In some embodiments, the first promoter and the second promoter are both plO promoters.

[0072] In some embodiments, the first promoter and the second promoter may comprise different promoters. For example, the first promoter and the second promoter may be from thegroup consisting of a polh promoter, a plO promoter, a basic promoter, an inducible promoter, an El promoters, and a AE1 promoter. For example, in some embodiments, the first promoter is the polh promoter and the second promoter is the plO promoter. In some embodiments, the first promoter is the plO promoter and the second promoter is the polh promoter.

[0073] In some embodiments, the first or second promoter may be cloned into pUC57, pFastBacl, modified pUC57, or modified pFastBacl. In some embodiments, the first or second promoter may be cloned into pUC57. In some embodiments, the first or second promoter may be cloned into pFastBacl . In some embodiments, the first or second promoter may be cloned into modified pUC57 In some embodiments, the first or second promoter may be cloned into modified pFastBacl.

[0074] In some embodiments, the cap protein, rep protein, first promoter, and second promoter may be cloned into pUC57, pFastBacl, modified pUC57, or modified pFastBacl. In some embodiments, the cap protein, rep protein, first promoter, and second promoter may be cloned into pUC57. In some embodiments, the cap protein, rep protein, first promoter, and second promoter may be cloned into pFastBacl. In some embodiments, the cap protein, rep protein, first promoter, and second promoter may be cloned into modified pUC57. In some embodiments, the cap protein, rep protein, first promoter, and second promoter may be cloned into modified pFastBacl

[0075] In some embodiments, the polynucleotide sequence further comprises a sequence encoding a linker, such as, for example a cleavable linker. In some embodiments, the cleavable linker is a sequence comprising a 2A peptide. In some embodiments, the 2A peptide may be selected from the 2A peptides derived from Aphthorvirus or Cardiovirus, such as foot-and- mouth disease virus (FMDV), Equine rhinitis A virus (ERAV), Thosea asigna virus (TaV) or porcine teschovirus (PTV-1). In some embodiments, the sequence encoding the linker further comprises a promoter sequence. In some embodiments, the promoter is an FMDV promoter.Recombinant AAV virus particles

[0076] In another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) particle prepared by introducing or transfecting the composition of the present disclosure into a host cell. In some embodiments, the host cell is an insect cell, a human cell or an animal cell. In some embodiments, the insect cell is Drosophila S2 cells or Sf9 cells. In some embodiments, the animal cell is fibroblasts, Chinese hamster ovary (CHO) cells, COS cells, murine myeloma (NS0) cells, Baby Hamster Kidney (BHK) cells. In some embodiments, the human cell is human embryonic kidney 293 (HEK293) cells, human fibrosarcoma (HT-1080) cells, differentiated hepatocyte-derived carcinoma (Huh-7) cells, HeLa cells, or PER C6 cells. Insome embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is HEK293 cell or its derivatives such as 293T cells.

[0077] In some embodiments, the preparation includes, but is not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection. In some embodiments, the composition is transfected into the 293T cells with a helper plasmid. In some embodiments, the 293T cells are used to produce the rAAV virus particles.

[0078] In some embodiments, the composition of the present disclosure may be delivered into the insect cell by any method known in the art. In some embodiments, the method includes, but is not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and / or infection. In some embodiments, the composition is infected into the insect cell In some embodiments, the composition is stably transfected into the insect cell.

[0079] If necessary, in some cases, the rAAV virus particles can be isolated and purified from the insect cells according to conventional methods known to those skilled in the art. For example, the rAAV can be purified using centrifugation, HPLC, hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, ultrafiltration, gel electrophoresis, affinity chromatography, other purification techniques, or any combinations thereof.Anion Exchange Chromatography (AEX) Purification

[0080] AAV vectors may be produced in adherent or suspension cell culture of various cell lines using transient transfection or virus co-infection methods. Viral particles produced may include full, partial and empty species, which are secreted out of cells into the culture medium or contained inside cells.

[0081] In some embodiments, stable AAV producer cells are generated by transfection and selection of human-derived cells, like HeLa or HEK293 cells, with an rAAV transfer vector containing the ITR cassette and a packaging construct containing Rep genes and Cap genes, sometimes with the help of auxiliary viruses such as adenoviruses (AdV) that provide the helper function Alternatively, a helper virus-free method can use a duo or triple transfection protocol employing two or three plasmids including a constructed helper plasmid. The later system is widely used in research and drug development. Furthermore, development of baculovirus expression vectors may provide another method to produce rAAV viruses in insect SfP cells. All the above technologies may produce rAAV viruses for use in laboratories and clinical trials.

[0082] Since some of the viral particles may reside inside the host cells, a cell lysis step may be required at harvest to release viral particles into the supernatant. For example, cell lysis reagents such as Triton X-100, Tween 20, or other detergents may be utilized.

[0083] After the cell lysis step, the released AAVs may need to be purified. Some purification step may include clarification, concentration, and diafiltration, filtration, chromatography purification by using affinity chromatography and ion exchange chromatography. Other steps may include ultracentrifugation and gradient ultracentrifugation. In some processes, concentration and diafiltration into suitable excipient buffer composition and sterile filtration may complete the purification processes.

[0084] A major challenge in current AAV production is the reduction of empty rAAV capsids, which pose as an impurity that hampers safety and efficacy of the final rAAV drug. The crude rAAV particles may contain about 20 to about 80% empty capsids that need to be removed from the product stream. An initial affinity capture step to capture all rAAV particles from the feed stream followed by a subsequent separation step to separate full rAAV particles from empty rAAV particles are commonly used. Since the full capsids have higher charge density than the empty capsids due to the negatively charged genome that the full capsids carry, ion exchange chromatography, e.g., anion exchange chromatography (AEX), has been employed for such purification step.

[0085] Below is a general protocol to purify AAV capsids using an AEX column and a salt gradient elution. As used herein, a volume of solution applied to a column is generally expressed in terms of a “column volume” (CV) One CV is equivalent to the volume of the column Specifically, after column sanitization with a high salt (NaCl) basic (NaOH) solution (at least 5 CV), column neutralization (at least 5 CV), and column equilibrium (at least 5 CV) with the buffer solution to be used for the purification (e g., a tris buffer solution, or a bis-tris propane buffer solution, a carbonate buffer, or other appropriate buffer solution), the crude AAV product to be purified is loaded onto the AEX column (the total volume may vary, depending on the characteristics of the crude AAV product, among other things). The AEX column is then washed (at least 5 CV) to remove non-binding substance to the AEX column Then the elution is conducted by using a mixture of two salt solutions: Salt Solution A has a low concentration of salt (less than 20 mM); Salt Solution B has a high concentration of salt (at least 300 mM) The elution starts with Salt Solution A only. Then Salt Solution B is gradually added to Salt Solution A such that the salt concentration of the mixture increases in the solution used to elute the AEX column over time. The salt can be KC1, NaCl, KBr, NaBr, etc. The gradient can be run with 0% of Salt Solution B at the start till 50% of Salt Solution B, 75% Salt Solution B, or even 100% Salt Solution B in the end. The gradient can be stepwise and continuous in terms of the changing salt concentration. The total volume of the elution can be from about 20 CV to about 150 CV, depending on the separation efficiency. To optimize this elution condition, a technician can test many variables, including but not limited to: characteristics of the AAV product to be purified,characteristics of the solution of the AAV product to be loaded onto the AEX column, characteristics of the nucleic acid encapsulated in the AAV capsids, the affinities of the AAV capsids (empty or full) and other impurities to the AEX column, the differences in affinities of the empty and full AAV capsids, etc. Throughout the elution, the pH in the column can be maintained substantially the same. The pH of the elution solution can range from pH 6.0 to pH 10 0, depending on various factors, but kept substantially constant during the elution The technician can test various pH values of the column to find the better pH range to run the elution. After most of the AAV capsids have been eluted the column is washed by various solutions to flush the column, sanitize the column, neutralize the column, respectively

[0086] In one example for using the salt gradient process, the eluent solution gradually increases the concentration of NaCl from about 0 mM to about 200 mM over 80 CVs with the pH kept around 9.0. The separation of the full capsids and the empty capsids is based on the charges inside and on the surface of capsid. Full AAV capsids have more charges than the empty AAV capsid, thereby requires higher salt concentration to wash down the AEX column when compared with empty AAV capsids. The eluate with more full capsid has higher salt concentration. A typical chromatogram of salt gradient is illustrated in FIG. 1. According to the chromatogram, the separation of the full AAV capsids from the empty AAV capsids are not best resolved in the sense that there is a significant overlap of the two peaks corresponding to the full AAV capsids and the empty AAV capsids (FIG. 1). After using salt (NaCl) gradient in the elution during the anion-exchange (AEX) purification process to remove residual impurity and empty capsids, the percentage of empty capsids in the final drug substance may range from about 15% to about 30% when tested. FIG. 2 shows a sample analytical ultracentrifugation (AUC) diagram characterizing rAAV particles distribution in the final drug product obtained by the salt gradient process. As shown in FIG. 2, the final drug product comprises about 24% empty AAV capsids, additional partial AAV capsids and AAV aggregates.

[0087] Accordingly, there is still a need for an efficient and simple method that can provide for separation of full rAAVs capsids from process-related impurities and empty capsidsNot to be limited by any theories, one possible reason that the salt gradient can separate the full AAV capsid from the empty AAV capsid is that the presence of a DNA molecule in the full AAV capsid may create a difference in the isoelectric point (PI) of about 0.3, 0.4 or 0.5 unit when compared with that for an empty AAV capsid due the charges on the DNA molecule. The more negative charges on the full AAV capsid may cause stronger retention on the AEX column for the full AAV capsid than for the empty AAV capsid, thereby leading to a later elution for the full AAV capsid from the AEX column. In some embodiments, the theoretical isoelectric points (pl) of a full AAV is about 6.3 (calculated based on secondary amino acid sequence).pH Gradient Elution on AEX

[0088] The mechanism of the pH gradient is different from that of the salt gradient, although both modes utilize charge-charge interaction between capsids and ligand For example, at different pH, the electric charges on the same DNA molecule can be different. At different pH, the electric charges on the same protein can be different due to the presence of dissociable protons on some amino acid residues. Further, at different pH, the conformations of the same protein may be different due to presence or absence of the salt bridge between amino acid residues, among other things Finally, the interactions between the capsid proteins and the DNA molecule encapsulated in the capsid may also change along with the change in pH, further complicated the prediction for which pH gradient condition would be better for the separation of the full and empty AAV capsids. Accordingly, at different pH, the binding affinities of the full AAV capsids may be different from those of the empty AAC capsids. On the one hand, because the changes in affinities to the AEX column, it is rather difficult to predict the separation efficiency for a give AAV capsids batch using the same pH gradient. On the other hand, this possibilities of changing the affinities of the full and empty AAV capsids towards the AEX column under different pH also allow the finetuning the conditions of the pH gradient for each crude AAV capsid batch in order to achieve a better separation between the empty and full AAV capsid

[0089] After some testing, the inventors developed and discovered a new method which uses pH gradient solutions instead of salt gradient solutions to separate full AAV capsids from empty and partial AAV capsids Surprisingly, the new method based on the pH gradient can effectively separate the full AAV capsids from empty AAV capsids. In addition, the new method can reduce the percentage of empty capsids over the total capsids in the final collected AEX fractions to no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 7%, no more than 5%, or no more than 3% in the final drug substance.

[0090] Below is a general protocol to purify AAV capsids using an AEX column and a pH gradient elution Specifically, after column sanitization with a high salt (NaCl, sodium acetate, ammonium acetate, or sodium sulfate) basic (NaOH) solution (at least 5 CV), column neutralization (at least 5 CV), and column equilibrium (at least 5 CV) with the buffered solution (e.g., a Tris buffer, a bis-tris propane buffer, a carbonate buffer, or other appropriate buffer), the crude AAV product to be purified is then loaded onto the AEX column in a solution (the total volume may vary, depending on the characteristics of the crude AAV product, the affinity column used, among other things). The AEX column is then washed with the starting buffer (at least 5 CV) to remove non-binding substance with respect to the AEX column and other impurities that do not bind tightly to the AEX column. Then the elution is carried out by using amixture of two buffers having different pH values: Buffer A has one pH value (high pH); Buffer B has a different pH value (low pH), thereby creating a pH gradient when a mixture of Buffer A and Buffer B is used to elute the AEX column with increasing percentage of Buffer B. The elution can start with Buffer A only or mixture of Buffer A and Buffer B. Then Buffer B is gradually added to Buffer A such that pH decreases in the solution eluting the AEX column over time. T The gradient can be run with 0% of Buffer B at the start till 50% of Buffer B, 75% Buffer B, or even 100% Buffer B in the end. Total volume can be from about 3 CV to about 250 CV, or from about 5 CV to about 150 CV. To optimize this elution condition, a technician can test many variables, including but not limited to: characteristics of the AAV product to be purified, characteristics of the solution of the AAV product to be loaded onto the AEX column, characteristics of the nucleic acid encapsulated in the AAV capsids, the affinities of the AAV capsids (empty or full) and other impurities to the AEX column, the differences in affinities of the empty and full AAV capsids, at what pH the empty AAV capsids can be wash down the AEX column and separated from the bound full AAV capsids, etc. Throughout the elution, the salt content in the column can be maintained substantially the same. The salt content (from the buffer species) of the elution solution can range from about 10 mM to about 40 mM, depending on various factors, but the salt content of the elution buffer can be kept substantially constant during the elution

[0091] The pH of the elution solutions can range from pH 2.7 to pH 10.0, depending on various factors, but the salt content of the elution buffer can be kept substantially constant during the elution. For example, there can be a pH differential between Buffer A and Buffer B: about 1 pH unit, about 1.5 pH units, about 2 pH units, 2.5 pH units, 3.0 pH units, 3.5 pH units, 4 0 pH units, 4.5 pH units, 5.0 pH units, 5.5 pH units, 6.0 pH units, 6.5 units, or 7 units. Buffer A can have a pH value: about 10.0, about 9.5, about 9.0, about 8.5, about 8.0, about 7.5, about 7.0, about 6.5, or about 6.0. Some possible pH choices for Buffer A and Buffer B are listed in Table 2.

[0092] Table 2. Example pH values for Buffer A and Buffer B

[0093] The technician can test various pH values of the column to find the better pH range to run the elution and separate the full AAV capsids from the empty AAV capsids. After most of the AAV capsids have been eluted, the column is washed by various solutions to flush the column, sanitize the column, neutralize the column, respectively.

[0094] In one example of the pH gradient process, the eluent solution gradually decreases the pH from about 9.0 to about 5.0 over about 100 CVs at constant low salt concentration (ranging from 20 mM to about 30 mM). The separation of empty and full capsid using pH gradient can be achieved because the net charge of AAV capsid (both empty and full capsid) changes as pHchanges. When pH of buffer is higher than pl (isoelectronic point) of AAV, AAV carries net negative charge and while pH is lower than pl, AAV carries net positive charge. The theoretical pl of AAV is around pH 6. The separation of empty capsid from full capsid may in part utilize the net charge or charge distribution difference between these two forms of AAV capsid across the pH gradient. A typical chromatogram of pH gradient is shown in FIG 3. Compared to FIG. 1, FIG. 3 shows that the separation between the full AAV capsids and the empty AAV capsids is much better resolved when the pH gradient is used than when the salt gradient is used. According to FIG. 3, there is little overlap between the two peaks for the full and empty AAV capsids, respectively

[0095] FIG. 4 shows an example of analytical ultracentrifugation (AUC) measuring the concentration of empty capsids and full capsids in a final drug substance using the pH gradient in elution. As shown in FIG. 4, the percentage of empty AAV capsids is about 0%. When contrasting FIG. 4 with FIG. 2, the benefits of using the pH gradient over using the salt gradient is revealing.

[0096] Using pH gradient as elution method is not a typical operation in preparative ion exchange chromatography. It requires more robust scale up testing to meet the standard of manufacturing production. Because there are many variables in the manufacturing process for an AEX separation based on a pH gradient, identifying factors important when testing and choosing the AEX separation conditions is important.

[0097] The pH gradient process can adopt an elution profile using a pH gradient from about pH 10 to about pH 2.7, from about pH 9.5 to about pH 2.7, from about pH 9 to about pH 2.7; from about pH 8.5 to about pH 2.7; from about pH 8 to about pH 2.7; from about pH 7.5 to about pH 2.7; about pH 10 to about pH 3; from about pH 9.5 to about pH 3; from about pH 9 to about pH 3; from about pH 8.5 to about pH 3; from about pH 8 to about pH 3; from about pH 7.5 to about pH 3; about pH 10 to about pH 3.5; from about pH 9.5 to about pH 3.5; from about pH 9 to about pH 3.5; from about pH 8.5 to about pH 3.5; from about pH 8 to about pH 3.5; from about pH 7 5 to about pH 3 5; about pH 10 to about pH 4; from about pH 9 5 to about pH 4; from about pH 9 to about pH 4; from about pH 8.5 to about pH 4; from about pH 8 to about pH 4; from about pH 7.5 to about pH 4; pH 10 to about pH 4.5; from about pH 9.5 to about pH 4.5; from about pH 9 to about pH 4.5; from about pH 8.5 to about pH 4.5; from about pH 8 to about pH 4.5; from about pH 7.5 to about pH 4.5; pH 10 to about pH 5; from about pH 9.5 to about pH 5; from about pH 9 to about pH 5; from about pH 8.5 to about pH 5; from about pH 8 to about pH 5; from about pH 7.5 to about pH 5; pH 10 to about pH 5.5; from about pH 9.5 to about pH 5.5; from about pH 9 to about pH 5.5; from about pH 8.5 to about pH 5.5; from about pH 8 to about pH 5.5; or from about pH 7.5 to about pH 5.5; pH 10 to about pH 6; from about pH 9.5 toabout pH 6; from about pH 9 to about pH 6; from about pH 8.5 to about pH 6; from about pH 8 to about pH 6; or from about pH 7.5 to about pH 6.

[0098] Under any of the above pH gradient, the gradient length is at least 3, at least 5, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least245, or at least 250 column volumes. In some embodiments, under any of the above pH gradient, the gradient length is about 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, or 250 column volumes, or more than 250 column volumes. As used herein, the term “gradient length” is the total volume of the elution solutions pass through the AEX column using a gradient, such as a pH gradient or a salt gradient.

[0099] Detection of the capsids can be done by checking the absorbance at wavelength from about 230 to about 280 In some embodiments, the detection is by checking the absorbance at wavelength at about 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, or 280nm.

[0100] The anion exchange chromatography comprises a plurality of quaternary ammonium ligands or diethylaminoethyl group on (i) porous or non-porous particles, (ii) monolith, (iii) membrane, or (iv) hydroxyapatite.

[0101] This pH gradient process can be applied to the preparation of different AAV, including both natural and engineers AAV. In some embodiments, the AAV is AAV1, AAV2, AAV2 7m8, AAV3, AAV4, AAV5, AAV 6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAVRh74, AAV218, Anc80L85, or MyoAAV.

[0102] In some embodiments, steps (b) and (c) of the method are conducted at a temperature from about 2 °C to about 25 °C. In some embodiment, steps (b) and (c) of the method are conducted at a temperature from 16 °C to about 22 °C.

[0103] In some embodiments, the pH gradient comprises an aqueous mobile phase comprising a first buffer and a second buffer. In some embodiments, the first buffer is ammonium hydroxide / ammonium chloride buffer, ammonium hydroxide / ammonium acetate buffer, borate buffer, bicarbonate / carbonate buffer, phosphate buffer, HEPES (4-(2 -hydroxyethyl)- 1 - piperazineethane Sulfonic acid) buffer, HEPPSO (4-(2-hydroxylehtyl)piperazine-l-(2- hydroxypropane sulfonic acid)) buffer, AMPD (2-amino-2-methyl-l,2-propanediol) buffer,-Tl-CABS (4-cyclohexylamino-l-butaneSulfoinc acid) buffer, CAPS (N-cyclohexylo-3- aminopropane sulfonic acid) buffer, CAPSO (3 -cyclohexylamino-2-hydroxyl-l -propane sulfonic acid) buffer and carbonate buffer tris(hydroxymethyl)aminomethane (Tris) buffer, or a bis-tris propane (BTP) buffer. In some embodiments, the first buffer comprises bis-tris propane from about 15 mM to about 25 mM, magnesium chloride from about 1 mM to about 3 mM, and pluronic from about 0.0005% to about 0.002% (v / v). In some embodiments, the first buffer comprises bis-tris propane at about 20 mM, magnesium chloride at about 2 mM, and pluronic at about 0.001% (v / v). The first buffer is at about pH 9. In some embodiments, the first buffer is at a pH lower than pH 10 In some embodiments, the first buffer is not ammonium bicarbonate / ammonium hydroxide buffer at pH 10 or higher.

[0104] In some embodiments, the second buffer is acetate buffer, citrate buffer, succinate buffer, propionate buffer, or dimethyl glutarate buffer. In some embodiments, the second buffer comprises acetate from about 15 mM to about 25 mM, magnesium chloride from about 1 mM to about 3 mM, and pluronic from about 0.0005% to about 0.002% (v / v). In some embodiments, the second buffer comprises acetate at about 20 mM, magnesium chloride at about 2 mM, and pluronic at about 0.001% (v / v). In some embodiments, the second buffer is at about pH 4.5. In some embodiments, the second buffer is at about pH 5.0. In some embodiments, the second buffer is at about pH 5.5. In some embodiments, the second buffer is from about pH 4 5 to about pH 5.5. In some embodiments, the second buffer is at pH above 2 7.. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 0% second buffer to about 100% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 10% second buffer to about 100% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 20% second buffer to about 100% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 30% second buffer to about 100% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 40% second buffer to about 100% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 50% second buffer to about 100% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 60% second buffer to about 100% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 70% second buffer to about 100% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 0% second buffer to about 90% second buffer. In some embodiments, during the application of the pH gradient, the aqueousmobile phase changes from about 10% second buffer to about 90% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 20% second buffer to about 90% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 30% second buffer to about 90% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 40% second buffer to about 90% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 50% second buffer to about 90% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 60% second buffer to about 90% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 0% second buffer to about 80% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 10% second buffer to about 80% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 20% second buffer to about 80% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 30% second buffer to about 80% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 40% second buffer to about 80% second buffer In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 50% second buffer to about 80% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 60% second buffer to about 80% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 0% second buffer to about 70% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 10% second buffer to about 70% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 20% second buffer to about 70% second buffer In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 30% second buffer to about 70% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 40% second buffer to about 70% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 0% second buffer to about 60% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 10% second buffer to about 60% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 20% second buffer to about 60%second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 30% second buffer to about 60% second buffer. In some embodiments, during the application of the pH gradient, the aqueous mobile phase changes from about 40% second buffer to about 60% second buffer. In some embodiments, the acetate in the second buffer is sodium acetate, potassium acetate, or ammonium acetate. In some embodiments, the acetate is sodium acetate.

[0105] In some embodiments, the gradient length for the pH gradient is from about 3 to about 250 column volumes, and may be more than 250 column volumes. In some embodiments, the gradient length for the pH gradient is about 3, about 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 200, about 210, about 220, about 230, about 240, and 250 column volumes.

[0106] In some embodiments, the gradient length for the pH gradient is at least from about 3 or at least from about 250 column volumes. In some embodiments, the gradient length for the pH gradient is at least from about 3, at least from about 5, at least from about 10, at least from about 15, at least from about 20, at least from about 30, at least from about 40, at least from about 50, at least from about 60, at least from about 70, at least from about 80, at least from about 90, at least from about 100, at least from about 120, at least from about 140, at least from about 160, at least from about 180, at least from about 200, at least from about 210, at least from about 220, at least from about 230, at least from about 240, and at least from about 250 column volumes.

[0107] In some embodiments, after the pH gradient process, an average percentage of empty capsids in total capsids in the collected fractions is no more than 10%. In some embodiments, after the pH gradient process, an average percentage of empty capsids in total capsids in the collected fractions is no more than 8%. In some embodiments, after the pH gradient process, an average percentage of empty capsids in total capsids in the collected fractions is no more than 5%. In some embodiments, after the pH gradient process, an average percentage of empty capsids in total capsids in the collected fractions is no more than 3% In some embodiments, after the pH gradient process, an average percentage of empty capsids in total capsids in the collected fractions is no more than 2.5%, no more than 2.0%, no more than 1.5%, or no more than 1.0%.

[0108] Some embodiments of the present disclosure are further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the implementation of the embodiments of the present disclosure described herein, and can therefore be considered to constitute a useful tool for implementingthese embodiments. However, based on the present disclosure, those skilled in the art will understand that without departing from the spirit and scope of the present disclosure, many changes can be made in the specific embodiments disclosed herein, and the same or similar results can still be obtained.EXAMPLES

[0109] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

[0110] Example 1 — pH Gradient Purification Process

[0111] pH gradient process starts with loading the mixture that contains both empty and full capsids to the anion exchange chromatography. The loading mixture is adjusted to high pH and low conductivity (for example, pH about 9.5 or lower, and conductivity from about 3.5 ms / cm to about 6.0 ms / cm for binding of the empty and full capsids, or any values between these two end points when adjusted for the AAV capsid batch to be purified). After loading, a wash buffer (can be the same as the first buffer used in elution gradient, but can be a different buffer) is applied to the AEX column to wash off loosely bound impurity. After the wash step, a pH gradient elution starts by decreasing the first buffer (high pH buffer) from 40% to 0 % and increasing the second buffer (low pH buffer ) from 60% to 100 % over about 100 column volume (CV) or more (can be adjusted depending on the AAV capsid batch). During this pH gradient elution, the full capsid peak is eluted before the empty capsid peak (shown in FIG. 3). The second buffer is low pH buffer (below pH 6, adjusted based on the column used and the AAV capsid batch). Elution peak was collected into fractions and the selected fractions were then pooled to become elution sample. The fractions can be monitored by UV spectroscopy or other means to measure the protein contents in the elution fractions in time or after the elution is complete. FIG. 4 shows an analytical ultracentrifugation (AUG) measuring the concentration of empty capsids and full capsids, among other things, in the combined, collected fractions.

[0112] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the disclosure be limited by the specific examples provided within the specification. While the disclosure has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. Furthermore, it shall be understood that all aspects of the disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is therefore contemplated that the disclosure shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of purifying full adeno-associated virus (AAV) capsids from an AAV fraction comprising empty AAV capsids and full AAV capsids on a preparative scale, comprising:(a) loading an AAV fraction comprising empty AAV capsids and full AAV capsids onto a preparative chromatography;(b) applying an aqueous mobile phase to the preparative chromatography, wherein the aqueous mobile phase comprises a pH gradient from about pH 10 to about pH 2.7 and a gradient length of at least 3 column volumes; and(c) collecting fractions comprising the full AAV capsids.

2. The method of claim 1, wherein the preparative chromatography comprises a plurality of quaternary ammonium ligands or diethylaminoethyl groups on (i) porous or non-porous particles, (ii) monolith, (iii) membrane, or (iv) hydroxyapatite.

3. The method of claim 1 or claim 2, wherein the AAV is natural AAVs or any engineered AAVs.

4. The method of claim 3, wherein the AAV is AAV1, AAV2, AAV2 7m8, AAV3, AAV4, AAVS, AAV 6, AAV7, AAV8, AAV9, AAV10, AAVrhlO, AAVRh74, AAV2i8, Anc80L85, or MyoAAV.

5. The method of any one of claims 1 to 4, wherein the aqueous mobile phase comprises a first buffer and a second buffer.6 The method of claim 5, wherein the first buffer is ammonium hydroxide / ammonium chloride buffer, ammonium hydroxide / ammonium acetate buffer, borate buffer, bicarbonate / carbonate buffer, phosphate buffer, HEPES (4-(2-hy droxy ethyl)- 1 -piperazineethane sulfonic acid) buffer, HEPPSO (4-(2-hydroxylehtyl)piperazine-l-(2-hydroxypropane sulfonic acid)) buffer, AMPD (2-amino-2-methyl-l,2-propanediol) buffer, CABS (4-cyclohexylamino-l-butaneSulfoinc acid) buffer, CAPS (N-cyclohexylo-3-aminopropane sulfonic acid) buffer, CAPSO (3- cyclohexylamino-2-hydroxyl-l-propane sulfonic acid) buffer and carbonate buffer tri s(hydroxymethyl)aminom ethane (Tris) buffer, or a bis-tris propane (BTP)) buffer.

7. The method of claim 5, wherein the first buffer comprises bis-tris propane from about 15 mM to about 25 mM, magnesium chloride from about 1 mM to about 3 mM, and pluronic from about 0.0005% to about 0.002% (v / v).

8. The method of claim 5, wherein the first buffer comprises bis-tris propane at about 20 mM, magnesium chloride at about 2 mM, and pluronic at about 0.001% (v / v).9 The method of any one of claims 1 to 8, wherein (b) and (c) are conducted at a temperature from about 2 °C to about 25 °C.

10. The method of any one of claims 1 to 8, wherein (b) and (c) are conducted at a temperature from about 16 °C to about 22 °C.

11. The method of any one of claims 5 to 10, wherein the first buffer is at about pH 9.012 The method of any one of claims 5 to 11, wherein the second buffer comprises acetate buffer, citrate buffer, succinate buffer, propionate buffer, or dimethyl glutarate buffer.

13. The method of any one of claims 5 to 11, wherein the second buffer comprises acetate at about 20 mM, magnesium chloride at about 2 mM, and pluronic at about 0.001% (v / v)14. The method of any one of claims 5 to 13, wherein the second buffer is at about pH 5.0.

15. The method of any one of claims 1 to 14, wherein the pH gradient is from about pH 9.0 to about pH 5.0.

16. The method of any one of claims 5 to 15, wherein, during the application of the pH gradient, the aqueous mobile phase changes from about 0% second buffer to about 100% second buffer.

17. The method of any one of claims 12 to 16, wherein the acetate in the second buffer is sodium acetate, potassium acetate, or ammonium acetate.

18. The method of claim 17, wherein the acetate is sodium acetate.

19. The method of any one of claims 1 to 18, wherein the gradient length is at least 5 column volumes.

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