Process for concentrating adeno-associated viruses

Abstract

The present invention provides a process for concentrating adeno-associated virus particles using anion exchange chromatography and zonal ultracentrifugation.

Description

[Technical Field]

[0001] The present invention relates to a process for concentrating adeno-associated virus (AAV) particles using anion exchange chromatography and zonal ultracentrifugation. [Background technology]

[0002] Adeno-associated viruses (AAVs) are small, nonpathogenic satellite viruses that are thought to require a helper adenovirus for replication. AAVs are structurally similar to adenoviruses but have a smaller, icosahedral nucleocapsid. AAVs are nonenveloped viruses with a single-stranded DNA genome with at least one inverted terminal repeat (ITR) at each end. For example, the AAV2 serotype can have a single-stranded DNA genome of approximately 4.7 kilobases (kb) with two 145-nucleotide ITRs at each end. Because this virus does not encode a polymerase, it relies on cellular polymerases for genome replication. The ITRs flank two viral genes—rep (replication) and cap (capsid)—which encode nonstructural and structural proteins, respectively. The rep gene encodes four regulatory proteins, called Rep78, Rep68, Rep52, and Rep40, through the use of two promoters and alternative splicing. These proteins are involved in AAV genome replication and packaging. The cap gene generates three capsid proteins, VP1 (virion protein 1), VP2, and VP3, through alternative splicing and translation initiation. The molecular weights of VP1, VP2, and VP3 for AAV2 are 87, 72, and 62 kDa, respectively. These capsid proteins assemble into a roughly spherical protein shell of 60 subunits. The simplicity and nonpathogenic nature of AAV make recombinant AAV (rAAV) a useful gene therapy vector. AAV gene therapy vectors can infect both replicating and nonreplicating cells and deliver transgenes without integrating into the host cell genome. rAAV vectors are often preferred due to their high titer, ability to infect a wide range of cells, mild immune responses, and overall safety. rAAV gene therapy vectors have been found to be highly useful for many diseases, including diabetes and other pancreatic disorders.

[0003] The production of rAAV particles for gene therapy also produces various impurities, and therefore, there remains a need for a process to effectively purify rAAV particles from impurities. Summary of the Invention

[0004] In at least one embodiment, the present invention discloses a method for purifying rAAV particles for gene therapy or therapeutically effective rAAV particles, thereby solving one or more problems in the prior art. Prior to purification, the therapeutically effective rAAV particles are in a composition that also contains AAV-produced impurities. The AAV-produced impurities include a first portion having a net charge different from that of the therapeutically effective rAAV particles and a second portion having a density different from that of the therapeutically effective rAAV particles. In at least one embodiment, the method includes removing the first portion from the composition by anion exchange chromatography (AEX) and removing the second portion from the composition by zonal ultracentrifugation (ZUC). After the AEX and ZUC steps, the composition is substantially devoid of AAV-produced impurities. In a refinement, the first or second portion of the AAV-produced impurities are therapeutically ineffective rAAV particles.

[0005] In at least one embodiment, a method for purifying rAAV particles for gene therapy or therapeutically effective rAAV particles is disclosed. Prior to purification, the therapeutically effective rAAV particles are in a composition that also contains therapeutically ineffective rAAV particles. In at least one embodiment, the method includes removing at least a portion of the therapeutically ineffective rAAV particles from the composition by AEX. After the removal step, in at least one embodiment, the method further includes treating the composition with ZUC. After the AEX and ZUC steps, the composition is substantially devoid of therapeutically ineffective rAAV particles. [Brief explanation of the drawings]

[0006] [Figure 1A] 1 shows particle distribution profiles from rAAV preparations separated by analytical ultracentrifugation. [Figure 1B] 1 shows particle distribution profiles from rAAV preparations separated by analytical ultracentrifugation. [Figure 2A]1 shows the effect of rAAV preparations containing light and heavy capsids on transgene expression in rAAV-infected cells. [Figure 2B] 1 shows the effect of rAAV preparations containing light and heavy capsids on transgene expression in rAAV-infected cells. [Figure 3A] 1 shows the effect of rAAV preparations containing light and heavy capsids on transgene expression in rAAV-infected cells. [Figure 3B] 1 shows the effect of rAAV preparations containing light and heavy capsids on transgene expression in rAAV-infected cells. [Figure 4] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 5A] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 5B] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 6A] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 6B] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 7] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 8A] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 8B] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 9A] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 9B] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 10] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 11A]1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 11B] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 11C] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 11D] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 12A] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 12B] 1 is an image showing labeled light and heavy capsids infecting HepG2 cells. [Figure 13] 1 is a flow chart of steps in a purification method according to various embodiments. [Figure 14] 1 is a flowchart illustrating steps of AEX processing in accordance with various embodiments. [Figure 15] 10 is a flowchart illustrating steps of a tangential flow filtration process after AEX processing according to various embodiments. [Figure 16] 1 is a flowchart illustrating steps of ZUC processing in accordance with various embodiments. [Figure 17] 10 is a flowchart illustrating steps of a tangential flow filtration process after AEX processing according to various embodiments. [Figure 18] 1 is a zeta potential analysis showing the difference in net charge versus pH between heavy capsids, ZUC light capsids, and AEX light capsids. [Figure 19] 1 shows the particle distribution profile from an rAAV preparation after anion exchange chromatography. The rAAV preparation was separated by analytical ultracentrifugation. [Figure 20] Cryo-electron microscopy images of light and heavy capsids from an rAAV preparation after anion exchange chromatography. Arrows indicate dense particles (i.e., heavy capsids) and "non-dense" particles (i.e., light capsids). [Figure 21]1 is a graph showing vector genome titer and density of different fractions of an rAAV preparation undergoing zonal ultracentrifugation. [Figure 22] 1 shows particle distribution profiles from rAAV preparations after zonal ultracentrifugation. The rAAV preparations were separated by analytical ultracentrifugation. [Figure 23] Figure 23A is a gel containing fractions of an rAAV preparation separated by zonal ultracentrifugation. Figure 23B is an alkaline agarose gel containing DNA isolated from the ultracentrifugation fractions. [Figure 24] Cryo-electron microscopy images of light and heavy capsids from rAAV preparations after zonal ultracentrifugation. Arrows indicate dense particles (i.e., heavy capsids) and "non-dense" particles (i.e., light capsids). [Figure 25A] Figure 25 shows an analysis of an rAAV preparation undergoing anion exchange chromatography and zonal ultracentrifugation. Figure 25A shows the absorbance spectrum of the rAAV preparation during anion exchange chromatography. Figure 25B shows the capsid and vector genome titers for different fractions of the rAAV preparation during zonal ultracentrifugation. [Figure 25B] Figure 25 shows an analysis of an rAAV preparation undergoing anion exchange chromatography and zonal ultracentrifugation. Figure 25A shows the absorbance spectrum of the rAAV preparation during anion exchange chromatography. Figure 25B shows the capsid and vector genome titers for different fractions of the rAAV preparation during zonal ultracentrifugation. [Figure 26] 1 shows capsid titers for different fractions of rAAV preparations subjected to zonal ultracentrifugation with and without prior anion exchange chromatography treatment. [Figure 27]Figure 27 shows an analysis of light capsids when subjected to anion exchange chromatography and zonal ultracentrifugation. Figure 27A shows the absorbance spectrum of the rAAV preparation during anion exchange chromatography. The circled peak in Figure 27A was then processed by zonal ultracentrifugation. Figure 27B shows the capsid and vector genome titers for different fractions of the circled peak in Figure 27A during zonal ultracentrifugation. The circled peak in Figure 27B was again processed by anion exchange chromatography. Figure 27C shows the absorbance spectrum of the circled peak in Figure 27B during anion exchange chromatography. [Figure 28] Figure 28 shows the concentration of rAAV associated with the impurity Rep protein after immunochromatographic purification using an affinity resin such as AVB Sepharose, after anion exchange chromatography, and after zonal ultracentrifugation. Figure 28 highlights that anion exchange chromatography removed a sufficient concentration of rAAV associated with Rep protein from the AVB Sepharose-purified composition, which contained therapeutically effective rAAV. Figure 28 further highlights that zonal ultracentrifugation removed additional rAAV associated with Rep protein that was not removed by anion exchange chromatography. [Figure 29] 1 shows the removal of rAAV associated with Rep protein during anion exchange chromatography. After the composition was processed by anion exchange chromatography, the concentration of Rep protein was assessed. Neither the eluate nor the wash contained sufficient concentrations of Rep protein. A sufficient concentration of Rep protein was identified when the anion exchange chromatography column was regenerated to remove impurities remaining after the loading, washing, and elution steps. [Figure 30] This figure shows the removal of rAAV associated with Rep protein during zonal ultracentrifugation. The isolated fraction (i.e., "pool") has a significantly lower concentration of Rep protein compared to the non-isolated fraction (i.e., "post-pool"). Note also that the concentration of encapsulated vector genomes is significantly increased in the pooled fraction compared to the post-pooled fraction. [Figure 31] Figure 31 shows the removal of impurity deamidated capsids after immunochromatographic purification using an affinity resin such as AVB Sepharose, after anion exchange chromatography, and after zonal ultracentrifugation. Figure 31 highlights that anion exchange chromatography removed a significant concentration of deamidated capsids from an AVB Sepharose-purified composition containing therapeutically effective rAAV. Figure 31 further highlights that zonal ultracentrifugation removed additional deamidated capsids that were not removed by anion exchange chromatography. [Figure 32]

[0023] Figure 1 shows the removal of deamidated capsids during anion exchange chromatography. After the composition was processed by anion exchange chromatography, the concentration of deamidated capsids was assessed. The eluate contained a sufficiently reduced concentration of deamidated capsids. A sufficient concentration of deamidated capsids was identified in the eluted wash buffer when the anion exchange chromatography column was regenerated to remove impurities remaining after the loading, washing, and elution steps. [Figure 33] Figure 1 shows the removal of deamidated capsids during zonal ultracentrifugation. The isolated fraction (i.e., "pool") has a significantly lower concentration of deamidated capsids compared to the non-isolated fraction (i.e., "post-pool"). Note also that the concentration of encapsulated vector genomes is significantly increased in the pooled fraction compared to the post-pooled fraction. DETAILED DESCRIPTION OF THE INVENTION

[0007] As desired, detailed embodiments of the present disclosure are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary and that they may be embodied in various and alternative forms.

[0008] Except in the examples, or where otherwise expressly indicated, all numerical quantities herein expressing quantities of ingredients or conditions of reaction and / or use should be understood to be modified by the word "about." For example, a reference to "about X" includes the reference to "X." In one example, the term "about" is understood to be within normal tolerances in the art, e.g., within two standard deviations of the mean. In different examples, "about" refers to a variability of ±0.0001%, ±0.0005%, ±0.001%, ±0.005%, ±0.01%, ±0.05%, ±0.1%, ±0.5%, ±1%, ±5%, or ±10%. In further examples, "about" can be understood to be within ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, or ±2%.

[0009] Unless the context clearly dictates otherwise, all numerical values ​​provided herein are modified by the term about. All ranges are inclusive of the range endpoints. The initial definition of an acronym or other abbreviation applies to all subsequent uses of the same abbreviation herein and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation. And, unless expressly stated to the contrary, measurements of a property are determined by the same techniques as earlier or later references to the same property.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0011] It is also to be understood that this disclosure is not limited to the specific embodiments and methods described below, as specific components and / or conditions may, of course, vary. Further, the terminology used herein is used only to describe particular embodiments and is not intended to be limiting in any way.

[0012] It should also be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a singular element is intended to include plural elements.

[0013] The terms "or" and "and" can be used interchangeably and can be understood to mean "and / or."

[0014] The term "comprising" is synonymous with "with," "including," "having," "containing," or "characterized by." These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.

[0015] The phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause in the body of a claim rather than immediately following a preamble, it limits only the elements recited in that clause and does not exclude other elements from the claim as a whole.

[0016] The phrase "consisting essentially of" limits the scope of a claim to specified materials or steps, in addition to those that do not materially affect the basic and novel characteristics of the claimed subject matter.

[0017] The terms "comprising," "consisting of," and "consisting essentially of" can be used interchangeably. Where one of these three terms is used, the subject matter of this disclosure and claims can include the use of either of the other two terms.

[0018] As used herein, the terms "heterologous gene," "heterologous sequence," "heterologous," "heterologous regulatory sequence," "heterologous transgene," or "transgene" mean that the referenced gene or regulatory sequence does not naturally occur in the AAV vector or particle but has been artificially introduced therein. For example, these terms refer to a nucleic acid that includes both a heterologous gene and a heterologous regulatory sequence operably linked to the heterologous gene that controls expression of that gene in a host cell. It is contemplated that the transgene herein can encode a biomolecule (e.g., a therapeutic biomolecule), such as a protein (e.g., an enzyme), a polypeptide, a peptide, RNA (e.g., tRNA, dsRNA, ribosomal RNA, catalytic RNA, siRNA, miRNA, pre-miRNA, lncRNA, snoRNA, small hairpin RNA, trans-splicing RNA, and antisense RNA), one or more components of a gene or base editing system, such as a CRISPR gene editing system, an antisense oligonucleotide (AON), antisense oligonucleotide (AON)-mediated exon skipping, a poison exon that causes nonsense-mediated decay (NMD), or a dominant-negative mutant.

[0019] The term "vector" is understood to refer to any genetic element, such as a nucleic acid molecule, plasmid, phage, transposon, cosmid, bacmid, miniplasmid (e.g., a plasmid lacking bacterial elements), doggybone DNA (e.g., a minimal closed linear construct), chromosome, virus, virion, etc., which, when associated with the appropriate control elements, is capable of replication and transfer of genetic sequences between cells. An "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), artificial chromosomes, and viruses that incorporate recombinant polynucleotides.

[0020] The term "recombinant" refers to a nucleic acid molecule or protein formed by using recombinant DNA technology. For example, a recombinant nucleic acid molecule can be formed by combining nucleic acid sequences and sequence elements. A recombinant protein can be a protein produced by a cell that has received a recombinant nucleic acid molecule.

[0021] The terms "encode," "encoded," and "encoding" refer to the inherent property of a particular sequence of nucleotides in a nucleic acid molecule, such as a gene, complementary DNA (cDNA), or messenger RNA (mRNA), to serve as a template for the synthesis of other polymers and macromolecules in biological processes. Thus, a gene encodes a protein when transcription and translation of the mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0022] The present invention relates to methods for purifying rAAV particles for gene therapy or therapeutically effective rAAV particles, including rAAV particles that are disclosed in or can be made according to known methods, for example, as disclosed in US9,504,762, WO2019 / 222136, US2019 / 0376081, and WO2019 / 217513, the disclosures of which are incorporated herein by reference in their entireties.

[0023] According to the present invention, production of rAAV particles is an inefficient process that produces various impurities, including therapeutically ineffective rAAV particles. These impurities limit the ability of purification techniques to further separate therapeutically effective rAAV particles from the impurities. To this end, the inventors have overcome the limitations of the current state of the art by developing a method for isolating therapeutically effective rAAV particles from the impurities.

[0024] In various embodiments, methods and processes are provided for purifying therapeutically effective rAAV particles from a composition comprising AAV production impurities, including therapeutically effective rAAV particles and therapeutically ineffective rAAV particles. The composition of various embodiments is the production of rAAV particles. The AAV production impurities may also include impurities that have a different net charge than the therapeutically effective rAAV particles or impurities that have a different density than the AAV particles.

[0025] Various embodiments of the method and process include subjecting a composition to AEX, which removes impurities having a net charge different from that of therapeutically effective rAAV particles from the composition. These impurities include therapeutically ineffective rAAV particles. It has been discovered that the use of ZUC for the purification of therapeutically effective rAAV particles is substantially limited due to the presence of therapeutically ineffective rAAV particles, which are less soluble and more prone to aggregation than therapeutically effective rAAV particles. Without wishing to be bound by theory, these properties may overload the ability of ZUC to isolate therapeutically effective rAAV particles. For example, therapeutically ineffective rAAV particles without AEX treatment may precipitate in ZUC, preventing their separation from therapeutically effective rAAV particles. To this end, AEX removes a sufficient amount of therapeutically ineffective rAAV particles from the composition so that a composition with an increased concentration of therapeutically effective rAAV particles can be efficiently loaded and processed by ZUC. For example, the increased concentration of therapeutically effective rAAV particles is an economically viable amount that can be processed by ZUC each time. For example, AEX treatment can allow titers of at least 0.1 x 10e16 vector genomes (vg) per load to be processed by ZUC up to 10 x 10e17 vg per load. In various embodiments, therapeutically ineffective rAAV particles comprise capsids with associated Rep proteins. In other embodiments, therapeutically ineffective rAAV particles comprise capsids with one or more VP1 proteins with deamidated N-terminal amino acids.

[0026] In various refinements, AEX removes or removes at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99+%, or 100% of impurities having a net charge different from that of the therapeutically active rAAV particles. In other refinements, the percentage of impurities removed by AEX ranges between any two of the percentages provided above. In various refinements, AEX removes or removes at least 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 99+% of therapeutically inactive rAAV particles from the composition. In another refinement, the percentage of therapeutically ineffective rAAV particles removed from the composition by AEX ranges between any two of the percentages provided above.

[0027] In various refinements, AEX allows for subsequent processing of the composition with ZUC or allows the original composition to have a greater amount of therapeutically effective rAAV particles processed by ZUC.

[0028] In various refinements, the AEX is at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.7, at least 3.8, at least 3.9, at least 4, at least 4.1, at least 4.2, at least 4.3, at least 4.4, at least At least a Log of at least 4.5, at least 4.6, at least 4.7, at least 4.8, at least 4.9, at least 5, at least 5.1, at least 5.2, at least 5.3, at least 5.4, at least 5.5, at least 5.6, at least 5.7, at least 5.8, at least 5.9, at least 6, at least 6.1, at least 6.2, at least 6.3, at least 6.4, at least 6.5, at least 6.6, at least 6.7, at least 6.8, at least 6.9, or at least 7 10 The concentration of contaminating viruses in the composition is reduced to a value.

[0029] In various refinements, the AEX step involves processing the composition through a membrane filter or column containing a strongly basic anion exchange resin. Examples of strongly basic anion exchange resins include quaternized polyethyleneimines, with Type I resins having trimethylammonium groups such as trimethylammoniumethyl (TMAE) and Type II resins having dimethylethanolamine groups such as diethylaminoethyl (DEAE). Examples of filters or columns containing strongly basic anion exchange resins include Mustang Q (Pall), Sartobind Q (Sartorius), POROS 50 HQ (Thermofisher), POROS 50 XQ (Thermofisher), Fractogel TMAE (EMD Millipore), Fractogel DEAE (EMD Millipore), Eshmuno Q (EMD Millipore), CIMmultus-QA (BIA separation), Nuvia Q (Bio-Rad), Q Sepharose XL (Cytiva), Q Sepharose HP (Cytiva), Capto Q Impres (Cytiva), Source 15Q (Cytiva), Source 30Q (Cytiva), Mono Q (Cytiva), TSKgel Q-STAT (TOSOH bioscience), TSKgel SuperQ-5PW(20) (TOSOH Biosciences), and Toyopearl. Examples of weakly basic anion exchange resins include SuperQ 650M (Tosoh Biosciences), Toyopearl GigaCap Q 650M (Tosoh Biosciences), and Capto Adhere Impres (Multimodal, Cytiva). Examples of weakly basic anion exchange resins include diethylaminoethyl (DEAE), dimethylaminopropyl, or diethylaminopropyl (ANX). Examples of filters and columns containing weakly basic anion exchange resins include Sartobind STIC PA (Sartorius), DEAE Sepharose FF (Cytiva), Poros 50 D (Thermofisher), POROS50PI (ThermoFisher),Suitable buffers and buffering agents for use in AEX include Fractogel EMD DEAE(M) (EMD Millipore), MacroPrep DEAE Support (Bio-Rad), DEAE Ceramic HyperD20 (Sartorius), Toyopearl NH2-750F (Tosoh Biosciences), or Toyopearl DEAE650 M (Sigma Aldrich). Suitable buffers and buffering agents for use in AEX may include, for example, N-methylpiperazine, piperazine, bis-tris(hydroxymethyl)aminomethane (Tris), bis-trispropane, MES, Hepes, BTP, phosphate buffer, N-methyldiethanolamine, 1,3-diaminopropane, ethanolamine, acetate such as sodium acetate or lithium acetate, or ions provided from various sources such as citrate. In various refinements, the bed height of the column is at least 7 centimeters (cm), 7 cm, 7.1 cm, 7.2 cm, 7.3 cm, 7.4 cm, 7.5 cm, 7.6 cm, 7.7 cm, 7.8 cm, 7.9 cm, 8 cm, 8.1 cm, 8.2 cm, 8.3 cm, 8.4 cm, 8.5 cm, 8.6 cm, 8.7 cm, 8.8 cm, 8.9 cm, 9 cm, 9.1 cm, 9.2 cm, 9.3 cm, 9.4 cm, 9.5 cm, 9.6 cm, 9.7 cm, 9.8 cm, 9.9 cm, 10 cm, 10.1 cm, 10.2 cm, 10.3 cm, 10.4 cm, 10.5 cm, 10.6 cm, 10.7 cm, 10.8 cm, 10.9 cm, 11cm, 11.1cm, 11.2cm, 11.3cm, 11.4cm, 11.5cm, 11.6cm, 11.7cm, 11.8cm, 11.9cm, 12cm, 12.1cm, 12.2cm, 12.3cm, 12.4cm, 12.5cm, 12.6cm, 12.7cm, 12.8cm, 12.9cm, 13cm, 13.1cm, 13.2cm, 13.3cm, 13.4cm, 13.5cm, 13.6cm, 13.7cm, 13.8cm, 13.9cm, 14cm, 14.1cm, 14.2cm, 14.3cm, 14.4cm, 14.5cm, 14.6cm, 14.7cm, 14.8cm, 14.9cm, or 15cm. In other modifications, the column bed height is greater than 15 cm (15.1 cm, 15.2 cm, 15.3 cm, 15.4 cm,15.5cm、15.6cm、15.7cm、15.8cm、15.9cm、16cm、16.1cm、16.2cm、16.3cm、16.4cm、16.5cm、16.6cm、16.7cm、16.8cm、16.9cm、17cm、17.1cm、17.2cm、17.3cm、17.4cm、17.5cm、17.6cm、17.7cm、17.8cm、17.9cm、18cm、18.1cm、18.2cm、18.3cm、18.4cm、18.5cm、18.6cm、18.7cm、18.8cm、18.9cm、19cm、19.1cm、19.2cm、19.3cm、19.4cm、19.5cm、19.6cm、19.7cm、19.8cm、19.9cm、20cm、20.1cm、20.2cm、20.3cm、20.4cm、20.5cm、20.6cm、20.7cm、20.8cm、20.9cm、21cm、21.1cm、21.2cm、21.3cm、21.4cm、21.5cm、21.6cm、21.7cm、21.8cm、21.9cm、22cm、22.1cm、22.2cm、22.3cm、22.4cm、22.5cm、22.6cm、22.7cm、22.8cm、22.9cm、23cm、23.1cm、23.2cm、23.3cm、23.4cm、23.5cm、23.6cm、23.7cm、23.8cm、23.9cm、24cm、24.1cm、24.2cm、24.3cm、24.4cm、24.5cm、24.6cm、24.7cm、24.8cm、24.9cm、25cm、25.1cm、25.2cm、25.3cm、25.4cm、25.5cm、25.6cm、25.7cm、25.8cm、25.9cm、26cm、26.1cm、26.2cm、26.3cm、26.4cm、26.5cm、26.6cm、26.7cm、26.8cm、26.9cm、27cm、27.1cm、27.2cm、27.3cm、27.4cm、27.5cm、27.6cm、27.7cm、27.8cm、27.9cm、28cm、28.1cm、28.2cm、28.3cm、28.4cm、28.5cm、28.6cm、28.7cm、28.8cm、28.9cm、29cm、29.1cm、29.2cm、29.3cm、29.4cm、29.5cm、29.6cm、29.7cm、29.8cm、29.9cm、30 cm). In other refinements, the bed height of the column ranges between any two of the bed heights provided above.

[0030] In various refinements, the AEX operation is carried out at a pH of at least 6, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11. In other refinements, the pH at which the AEX operation is carried out ranges between any two of the pHs provided above.

[0031] In various refinements, the AEX operation is carried out at a temperature of at least 4 degrees Celsius (°C), 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or 36°C. In other refinements, the temperature at which the AEX operation is carried out ranges between any two of the temperatures provided above.

[0032] In various refinements, the composition loaded onto the AEX column for AEX processing may be at least 0.1 x 10e16 vector genomes per liter (vg / L), 0.15 x 10e16 vg / L, 0.5 x 10e16 vg / L, 1 x 10e16 vg / L, 1.5 x 10e16 vg / L, 2 x 10e16 vg / L, 2.5 x 10e16 vg / L, 3 x 10e16 vg / L, 3.5 x 10e16 vg / L, The antibody has a titer of 4x10e16vg / L, 4.5x10e16vg / L, 5x10e16vg / L, 5.5x10e16vg / L, 6x10e16vg / L, 6.5x10e16vg / L, 7x10e16vg / L, 7.5x10e16vg / L, 8x10e16vg / L, 8.5x10e16vg / L, 9x10e16vg / L, 9.5x10e16vg / L, or 10x10e16vg / L. In other refinements, the titer ranges between any two of the titers provided above.

[0033] In various refinements, the composition packed into the AEX column for AEX processing has a solubility of at least 0.0 millisiemens per centimeter (mS / cm), 0.0 mS / cm, 0.001 mS / cm, 0.002 mS / cm, 0.003 mS / cm, 0.004 mS / cm, 0.005 mS / cm, 0.006 mS / cm, 0.007 mS / cm, 0.008 mS / cm, 0.009 mS / cm, 0.01 mS / cm, 0.02 mS / cm, 0.03 mS / cm, 0.04 mS / cm, 0.05 mS / cm, 0.06mS / cm, 0.07mS / cm, 0.08mS / cm, 0.09mS / cm, 0.1mS / cm, 0.1mS / cm, 0.2mS / cm, 0.3mS / cm, 0.4mS / cm, 0.5mS / cm, 0.6mS / cm, 0.7mS / cm, 0.8mS / cm, 0.9mS / cm, 1mS / cm, 1.1mS / cm, 1.2mS / cm, 1.3mS / cm, 1.4mS / cm, 1.5mS / cm, 1.6mS / cm, 1.7mS / cm, 1.8mS / cm, 1.9mS / cm, 2mS / cm, 2.1mS / cm, 2.2mS / cm, 2.3mS / cm, 2.4mS / cm, 2.5mS / cm, 2.6mS / cm, 2.7mS / cm, 2.8mS / cm, 2.9mS / cm, 3mS / cm, 3.1mS / cm, 3.2mS / cm, 3.3mS / cm, 3.4mS / cm, 3.5mS / cm, 3.6mS / cm, 3.7mS / cm, 3.8mS / cm, 3.9mS / cm, 4mS / cm, 4.1mS / cm, 4.2mS / cm, 4.3mS / cm, 4.4mS / cm, 4.5mS / cm, 4.6m In another refinement, the composition loaded into the AEX column for AEX processing has a conductivity of less than 1 mS / cm.In another refinement, the conductivity of the composition packed in the AEX column ranges between any two of the conductivities provided above.

[0034] In various refinements, the composition loaded onto the AEX column for AEX processing has a pH of at least 7, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11. In other refinements, the pH of the composition loaded onto the AEX column ranges between any two of the pHs provided above.

[0035] In various refinements, the column is washed with a buffer after the composition is run through the AEX column. In various refinements, the conductivity of the wash buffer is at least 1 mS / cm, 1 mS / cm, 1.1 mS / cm, 1.2 mS / cm, 1.3 mS / cm, 1.4 mS / cm, 1.5 mS / cm, 1.6 mS / cm, 1.7 mS / cm, 1.8 mS / cm, 1.9 mS / cm, 2 mS / cm, 2.1 mS / cm, 2.2 mS / cm, 2.3 mS / cm, 2.4 mS / cm, 2.5 mS / cm, 2.6 mS / cm, 2.7 mS / cm, 2.8 mS / cm, 2.9 mS / cm, 3 mS / cm, 3.1 mS / cm, 3.2 mS / cm, 3.3 mS / cm, 3.4 mS / cm, 3.5 mS / cm, 3.6 mS / cm, 3.7 mS / cm, 3.8 mS / cm m, 3.9mS / cm, 4mS / cm, 4.1mS / cm, 4.2mS / cm, 4.3mS / cm, 4.4mS / cm, 4.5mS / cm, 4.6mS / cm , 4.7mS / cm, 4.8mS / cm, 4.9mS / cm, 5mS / cm, 5.1mS / cm, 5.2mS / cm, 5.3mS / cm, 5.4mS / cm, 5 In another refinement, the conductivity of the wash buffer is greater than 7 mS / cm. In another refinement, the conductivity of the wash buffer is between any two of the conductivities provided above.

[0036] As the column is washed with various embodiments of wash buffer, the ultraviolet (UV) absorbance of the wash buffer exiting the column is monitored at 260 nanometer (nm) and 280 nm wavelengths, and the ratio of the 260 nm wavelength to the 280 nm wavelength (A 260 :A 280 ) can eliminate human error and variations between different purifications. In various refinements, the A of the wash buffer leaving the column 260 :A 280is at least 0.5, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or 1.5. In another refinement, the A of the wash buffer exiting the column 260 :A 280 The ratio ranges between any two of the ratios provided above.

[0037] In various refinements, the composition is eluted with an elution buffer containing a concentration of a buffering agent after washing the AEX column with a wash buffer.In various refinements, the concentration of the buffer is at least 0.5 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM , 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63mM, 64mM, 65mM, 66mM, 67mM, 68mM, 69 mM, 70mM, 71mM, 72mM, 73mM, 74mM, 75mM, 76mM, 77mM, 78mM, 79mM, 80mM, 81m M, 82mM, 83mM, 84mM, 85mM, 86mM, 87mM, 88mM, 89mM, 90mM, 91mM, 92mM, 93mM, 94mM, 95mM, 96mM, 97mM, 98mM, 99mM, 100mM, 101mM, 102mM, 103mM, 104mM, 1 05mM, 106mM, 107mM, 108mM, 109mM, 110mM, 111mM, 112mM, 113mM, 114mM, 11 5mM, 116mM, 117mM, 118mM, 119mM, 120mM, 121mM, 122mM, 123mM, 124mM, 125 mM, 126mM, 127mM, 128mM, 129mM, 130mM, 131mM, 132mM, 133mM, 134mM, 135mM , 136mM, 137mM, 138mM, 139mM, 140mM, 141mM, 142mM, 143mM, 144mM, 145mM, 146mM, 147mM, 148mM, 149mM, 150mM, 151mM, 152mM, 153mM, 154mM, 155mM, 156mM, 157mM, 158mM, 159mM, 160mM, 161mM, 162mM, 163mM, 164mM, 165mM, 166mM, 167mM, 168mM, 169mM, 170mM, 171mM, 172mM, 173mM, 174mM, or 175mM.In other refinements, the concentration of the buffering agent ranges between any two of the concentrations provided above. Various refinements of the elution buffer also include a concentration of at least 1 mS / cm, 1 mS / cm, 1.1 mS / cm, 1.2 mS / cm, 1.3 mS / cm, 1.4 mS / cm, 1.5 mS / cm, 1.6 mS / cm, 1.7 mS / cm, 1.8 mS / cm, 1.9 mS / cm, 2 mS / cm, 2.1 mS / cm, 2.2 mS / cm, 2.3 mS / cm, 2.4 mS / cm, 2.5 mS / cm, 2.6 mS / cm, 2.7 mS / cm, 2.8 mS / cm, 2.9 mS / cm, 3 mS / cm, 4 mS / cm, 5 mS / cm, 6 mS / cm, 7 mS / cm, 8 mS / cm, 9 mS / cm, 10 mS / cm, 11 mS / cm, 12 mS / cm, 13 mS / cm, 14 mS / cm, 15 mS / cm, 16 mS / cm, 17 mS / cm, 18 mS / cm, 19 mS / cm, 20 mS / cm, 21 mS / cm, 22 mS / cm, 23 mS / cm, 24 mS / cm, 25 mS / cm, 26 mS / cm, 27 mS / cm, 28 mS / cm, 29 mS / cm, 30 mS / cm, 31 mS / cm, 32 mS / cm, 33 mS / cm, 34 mS / cm, 35 mS / cm, 36 mS / cm, 37 mS / cm, 38 mS / cm, 39 mS / cm, , 3.1mS / cm, 3.2mS / cm, 3.3mS / cm, 3.4mS / cm, 3.5mS / cm, 3.6mS / cm, 3.7mS / cm, 3.8mS / cm, 3.9mS / cm, 4mS / cm, 4.1mS / cm, 4.2mS / cm, 4.3mS / cm, 4.4mS / cm, 4.5mS / cm, 4.6mS / cm, 4.7mS / cm, 4.8mS / cm, 4.9mS / cm, 5mS / cm, 5.1mS / cm, 5.2mS / cm, 5.3mS / cm, 5.4mS / cm, 5.5mS / cm, 5.6mS / cm, 5.7mS / cm, 5.8mS / cm, 5.9mS / cm, 6mS / cm, 6.1mS / cm, 6.2mS / cm, 6.3mS / cm, 6.4mS / cm, 6.5mS / cm, 6.6mS / cm, 6.7mS / cm, 6.8mS / cm, 6.9mS / cm, 7mS / cm, 7.1mS / cm, 7.2mS / cm, 7.3mS / cm, 7.4mS / cm, 7.5mS / cm, 7.6mS / cm, 7.7mS / cm, 7. The elution buffer has a conductivity of 8 mS / cm, 7.9 mS / cm, 8 mS / cm, 8.1 mS / cm, 8.2 mS / cm, 8.3 mS / cm, 8.4 mS / cm, 8.5 mS / cm, 8.6 mS / cm, 8.7 mS / cm, 8.8 mS / cm, 8.9 mS / cm, 9 mS / cm, 9.1 mS / cm, 9.2 mS / cm, 9.3 mS / cm, 9.4 mS / cm, 9.5 mS / cm, 9.6 mS / cm, 9.7 mS / cm, 9.8 mS / cm, 9.9 mS / cm, or 10 mS / cm. In another refinement, the conductivity of the elution buffer ranges between any two of the conductivities provided above.

[0038] In various refinements, the AEX operation includes processing the composition through an AEX column, and the wash step, or elution step, is performed at a rate of at least 50 centimeters per hour (cm / hr), 50 cm / hr, 55 cm / hr, 60 cm / hr, 65 cm / hr, 70 cm / hr, 75 cm / hr, 80 cm / hr, 85 cm / hr, 90 cm / hr, 95 cm / hr, 100 cm / hr, 105 cm / hr, 110 cm / hr, 115 cm / hr, 120 cm / hr, 125 cm / hr, 130 cm / hr, 135 cm / hr, 140 cm / hr, 145 cm / hr, 150 cm / hr, 155 cm / hr, 160 cm / hr, 165 cm / hr, 170 cm / hr, 175 cm / hr, 180 cm / hr, 185 cm / hr, 190 cm / hr, 195 cm / hr, 200 cm / hr, 210 cm / hr, 215 cm / hr, 220 cm / hr, 230 cm / hr, 240 cm / hr, 250 cm / hr, 260 cm / hr, 270 cm / hr, 280 cm / hr, 290 cm / hr, 300 cm / hr, 310 cm / hr, 320 cm / hr, 330 cm / hr, 340 cm / hr, 350 cm / hr, 360 cm / hr, 370 cm / hr, 380 cm / hr, 390 cm / hr, 400 cm / hr, 410 cm / hr, 420 cm / hr, 430 cm / hr, 440 cm / hr, 450 cm / hr, 460 cm / hr, 470 cm The AEX operation is performed at a flow rate of 5 cm / hr, 130 cm / hr, 135 cm / hr, 140 cm / hr, 145 cm / hr, 150 cm / hr, 155 cm / hr, 160 cm / hr, 170 cm / hr, 180 cm / hr, 190 cm / hr, 200 cm / hr, 210 cm / hr, 220 cm / hr, 230 cm / hr, 240 cm / hr, 250 cm / hr, 260 cm / hr, 270 cm / hr, 280 cm / hr, 290 cm / hr, or 300 cm / hr. In other refinements, the flow rate at which the AEX operation is performed ranges between any two of the flow rates provided above.

[0039] During the elution step, the eluted composition is detected at a wavelength of 260 nm (A 260 ), collection of the composition begins when the eluted composition reaches a UV absorbance of at least 0.1 absorbance units (AU) / cm, 0.1 AU / cm, 0.15 AU / cm, 0.2 AU / cm, 0.25 AU / cm, 0.3 AU / cm, 0.35 AU / cm, 0.4 AU / cm, 0.45 AU / cm, 0.5 AU / cm, 0.55 AU / cm, 0.6 AU / cm, 0.65 AU / cm, 0.7 AU / cm, 0.75 AU / cm, 0.8 AU / cm, 0.85 AU / cm, 0.9 AU / cm, 0.95 AU / cm, or 1 AU / cm. In other refinements, the absorbance at which collection begins is between any two of the absorbances provided above.

[0040] During the elution step, the eluted composition is adjusted to the maximum A 260 A is a percentage of 260In various refinements, the percentage is at least 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%. In other refinements, the maximum A 260 The percentages range between any two of the percentages provided above.

[0041] In various refinements, the pH of the composition eluted from the AEX column or the eluate is or is adjusted to at least 6, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11. In another refinement, the pH of the composition eluted from the AEX column ranges between any two of the pHs provided above.

[0042] The methods and processes of various embodiments include subjecting the composition to ZUC, which removes impurities from the composition that have a density different from that of the therapeutically active rAAV particles.

[0043] In various refinements, ZUC removes or removes at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99+%, or 100% of impurities having a density different from that of the therapeutically effective rAAV particles. In other refinements, the percentage of impurities removed by ZUC ranges between any two of the percentages provided above. In various refinements, the ZUC reduces the contaminating virus concentration in the composition by at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.3, at least 3.4, at least 3.5, at least 3.6, at least 3.7, at least 3.8, at least 3.9, or at least 4 Log 10 Decreases value.

[0044] In various refinements of the ZUC process, a gradient compound is added to the composition, which is then loaded between the cushion layer and the overlay layer in the rotor for zonal ultracentrifugation. After ZUC is complete, a replacement solution is pumped into the rotor to force the cushion layer, composition, and overlay layer out of the ZUC rotor. Alternatively, the cushion layer, composition, and overlay layer can be pumped out of the ZUC rotor without the use of a replacement solution. In both loading and unloading the ZUC rotor with layers, the ZUC rotor can be rotating or stationary. In the ZUC process, the overlay layer is first pumped into a rotating or stationary ZUC rotor, followed by the composition with the gradient compound and cushion layer. The cushion layer, overlay layer, and replacement solution also contain a gradient compound. Examples of gradient-forming compositions include cesium chloride (CsCl), iodixanol, or sucrose. The cushion layer prevents particles (e.g., therapeutically active rAAV) from pelleting against the rotor walls, and the overlay layer prevents particles from migrating from the gradient formed by the gradient compound. In a refinement, the concentrations of the gradient compound in the cushion layer, composition, and overlay layer are different from one another. In another refinement, the concentration of the gradient compound in the cushion layer is greater than in the composition. In a further refinement, the concentration of the gradient compound in the compound is greater than in the overlay layer.

[0045] In various refinements, the concentration of the gradient compound in the cushion layer, composition, overlay layer, or displacement solution is at least 15%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%. In another refinement, the concentration of the gradient compound in the cushion layer, composition, overlay layer, or displacement solution is within any two of the concentration ranges provided above.

[0046] In various refinements, the weight of the overlay layer pumped into the ZUC rotor is at least 117 grams, 117g, 118g, 119g, 120g, 121g, 122g, 123g, 124g, 125g, 126g, 127g, 128g, 129g, 130g, 131g, 132g, 133g, 134g, 135g, 136g, 137g, 138g, 139g, 140g, 141g, 142g, 143g, 144g, 145g, 146g, 147g, 148g, 149g, 150g, 151g, 152g, 153g, 154g, 155g, 156g, 157g, 158g, 159g, 160g, 161g, 162g, 163g, 164g, 165g, 166g, 167g, 168g, 16 9g, 170g, 171g, 172g, 173g, 174g, 175g, 176g, 177g, 178g, 179g, 180g, 181g, 182g, 183g, 184g, 185g, 186g, 187g, 188g, 189g, 190g, 191g, 192g, 193g, 19 4g, 195g, 196g, 197g, 198g, 199g, 200g, 201g, 202g, 203g, 204g, 205g, 206g, 207g, 208g, 209g, 210g, 211g, 212g, 213g, 214g, 215g, 216g, 217g, 218g, 21 9g, 220g, 221g, 222g, 223g, 224g, 225g, 226g, 227g, 228g, 229g, 230g, 231g, 232g, 233g, 234g, 235g, 236g, 237g, 238g, 239g, 240g, 241g, 242g, 243g, 24 4g, 245g, 246g, 247g, 248g, 249g, 250g, 251g, 252g, 253g, 254g, 255g, 256g, 257g, 258g, 259g, 260g, 261g, 262g, 263g, 264g, 265g, 266g, 267g, 268g, 26 9g, 270g, 271g, 272g, 273g, 274g, 275g, 276g, 277g, 278g, 279g, 280g, 281g, 282g, 283g, 284g, 285g, 286g, 287g, 288g, 289g, 290g, 291g, 292g, 293g, 29 4g, 295g, 296g, 297g, 298g, 299g, 300g, 301g, 302g, 303g, 304g, 305g, 306g,307g, 308g, 309g, 310g, 311g, 312g, 313g, 314g, 315g, 316g, 317g, 318g, 319g, 320g, 321g, 322g, 323g, 324g, 325g, 326g, 327g, 328g, 329g, 330g, 331g, 332g, 333g, 334g, 335g, 336g, 337g, 338g, 339g, 340g, or 341g. In an alternative refinement, the weight of the overlay layer pumped into the ZUC rotor is at least 1100 g, 1110 g, 1120 g, 1130 g, 1140 g, 1150 g, 1160 g, 1170 g, 1180 g, 1190 g, 1200 g, 1210 g, 1220 g, 1230 g, 1240 g, 1250 g, 1260 g, 1270 g, 1280 g, 1290 g, or 1300 g. In another refinement, the weight of the overlay layer pumped into the ZUC rotor ranges between any two of the weights provided above.

[0047] In various refinements, the weight of the cushion layer pumped into the ZUC rotor is at least 534g, 534g, 535g, 536g, 537g, 538g, 539g, 540g, 541g, 542g, 543g, 544g, 545g, 546g, 547g, 548g, 549g, 550g, 551g, 552g, 553g, 554g, 555g, 556g, 557g, 558g, 559g, 560g, , 561g, 562g, 563g, 564g, 565g, 566g, 567g, 568g, 569g, 570g, 571g, 572g, 573g, 574g, 575g, 576g, 577g, 578g, 579g, 580g, 581g, 582g, 583g, 584g, 585g, 586g, 587g, 588g, 589g, 590g, 591g, 592g, 593g, 594g, 595g, 596g, 5 97g, 598g, 599g, 600g, 601g, 602g, 603g, 604g, 605g, 606g, 607g, 608g, 609g, 610g, 611g, 612g, 613g, 614g, 61 5g, 616g, 617g, 618g, 619g, 620g, 621g, 622g, 623g, 624g, 625g, 626g, 627g, 628g, 629g, 630g, 631g, 632g, 633 g, 634 g, 635 g, 636 g, 637 g, 638 g, 639 g, 640 g, 641 g, 642 g, 643 g, 644 g, 645 g, 646 g, 647 g, 648 g, 649 g, 650 g, 651 g, 652 g, 653 g, 654 g, 655 g, 656 g, 657 g, 658 g, 659 g, 660 g, 661 g, 662 g, 663 g, 664 g, 665 g, 666 g, 667 g, or 668 g.In an alternative refinement, the weight of the cushion layer pumped into the ZUC rotor is at least 3600g, 3600g, 3601g, 3602g, 3603g, 3604g, 3605g, 3606g, 3607g, 3608g, 3609g, 3610g, 3611g, 3612g, 3613g, 3614g, 3615g, 3616g, 3617g, 3618g, 3619g, 3620g, 3621g, 3622g, 3623g, 3624g, 3625g, 3626g, 3627g, 3628g, 3629g, 3630g, 3631g, 3632g, 3633g, 3634g, 3635g, 3636g, 3637g, 3638g, 3639g, 3640g, 3641g, 3642g, 3643g, 3644g, 3645g, 3646g, 3647g, 3648g, 3649g, 3650g, 3651g, 3652g, 3653g, 3654g, 3655g, 3656g, 3657g, 3658g, 3659g, 3660g, 3661g, 3662g, 3663g, 3664g, 3665g, 3666g, 3667g, 3668g 19g, 3620g, 3621g, 3622g, 3623g, 3624g, 3625g, 3626g, 3627g, 3628g, 3629g, 3630g, 3631g, 3632g, 3633g, 3634g, 3635g, 3636g, 3637g, 3638g, 3639g, 3640g, 3641g, 3642g, 3643g, 3644g, 3645g, 3646g , 3647g, 3648g, 3649g, 3650g, 3651g, 3652g, 3653g, 3654g, 3655g, 3656g, 3657g, 3658g, 3659g, 366 0g, 3661g, 3662g, 3663g, 3664g, 3665g, 3666g, 3667g, 3668g, 3669g, 3670g, 3671g, 3672g, 3673g, 36 74g, 3675g, 3676g, 3677g, 3678g, 3679g, 3680g, 3681g, 3682g, 3683g, 3684g, 3685g, 3686g, 3687g, 3688g, 3689g, 3690g, 3691g, 3692g, 3693g, 3694g, 3695g, 3696g, 3697g, 3698g, 3699g, or 3700g. In another refinement, the weight of the cushion layer pumped into the ZUC rotor ranges between any two of the weights provided above.

[0048] In various refinements, the composition loaded into the ZUC rotor for ZUC processing is at least 0.1 x 10e16 vg / loading, 0.1 x 10e16 vg / loading, 0.5 x 10e16 vg / loading, 1 x 10e16 vg / loading, 1.5 x 10e16 vg / loading, 2 x 10e16 vg / loading, 2.5 x 10e16 vg / loading, 3 x 10e16 vg / loading, 3.5 x 10e1 6vg / fill, 4×10e16vg / fill, 4.5×10e16vg / fill, 5×10e16vg / fill, 5.5×10e16vg / fill, 6×10e16vg / fill, 6.5×10e16vg / fill, 7×10e16vg / fill, 7.5×10e16vg / fill, 8×10e16vg / fill, 8.5×10e16vg / fill, 9×10e16vg / fill, 9.5×1 0e16vg / fill, 10×10e16vg / fill, 0.1×10e17vg / fill, 0.5×10e17vg / fill, 1×10e17vg / fill, 1.5×10e17vg / fill, 2×10e17vg / fill, 2.5×10e17vg / fill, 3×10e17vg / fill, 3.5×10e17vg / fill, 4×10e17vg / fill, 4.5×10e17vg / fill The formulation has a titer of 5x10e17vg / fill, 5x10e17vg / fill, 5.5x10e17vg / fill, 6x10e17vg / fill, 6.5x10e17vg / fill, 7x10e17vg / fill, 7.5x10e17vg / fill, 8x10e17vg / fill, 8.5x10e17vg / fill, 9x10e17vg / fill, 9.5x10e17vg / fill, or 10x10e17vg / fill. In other refinements, the titer ranges between any two of the titers provided above.

[0049] In various refinements, the density of the composition loaded into the ZUC rotor for ZUC processing is at least 1.347 grams per milliliter (g / mL), 1.3471 g / mL, 1.3472 g / mL, 1.3473 g / mL, 1.3474 g / mL, 1.3475 g / mL, 1.3476 g / mL, 1.3477 g / mL, 1.3478 g / mL, 1.3479 g / mL, 1.348 g / mL, 1.3481 g / mL, 1.3482 g / mL, 1.3483 g / mL, 1.3484 g / mL, 1.3485 g / mL, 1.3486 g / mL, 1.3487 g / mL, 1.3488 g / mL, 1.3489 g / mL, 1.3490 g / mL, 1.3491 g / mL, 1.3492 g / mL, 1.3493 g / mL, 1.3494 g / mL, 1.3495 g / mL, 1.3496 g / mL, 1.3497 g / mL, 1.3498 g / mL, 1.349 ...9 g / mL, 1.3490 g / mL, 1.3491 g / mL, 1.3492 g / mL, 87g / mL, 1.3488g / mL, 1.3489g / mL, 1.349g / mL, 1.3491g / mL, 1.3492g / mL, 1.3493g / mL, 1.3494g / mL, 1.3495g / mL, 1.3496g / mL, 1.3497g / mL, 1.3498g / m L, 1.3499g / mL, 1.35g / mL, 1.3501g / mL, 1.3502g / mL, 1.3503g / mL, 1.3504g / mL, 1.3505g / mL, 1.3506g / mL, 1.3507g / mL, 1.3508g / mL, 1.3509g / mL, 1.35 1g / mL, 1.3511g / mL, 1.3512g / mL, 1.3513g / mL, 1.3514g / mL, 1.3515g / mL, 1.3516g / mL, 1.3517g / mL, 1.3518g / mL, 1.3519g / mL, 1.352g / mL, 1.3521g / m L, 1.3522g / mL, 1.3523g / mL, 1.3524g / mL, 1.3525g / mL, 1.3526g / mL, 1.3527g / mL, 1.3528g / mL, 1.3529g / mL, 1.353g / mL, 1.3531g / mL, 1.3532g / mL, 1.3 533g / mL, 1.3534g / mL, 1.3535g / mL, 1.3536g / mL, 1.3537g / mL, 1.3538g / mL, 1.3539g / mL, 1.354g / mL, 1.3541g / mL, 1.3542g / mL, 1.3543g / mL, 1.3544g / mL, 1.3545g / mL, 1.3546g / mL, 1.3547g / mL, 1.3548g / mL, 1.3549g / mL, 1.355g / mL, 1.3551g / mL, 1.3552g / mL, 1.3553g / mL, 1.3554g / mL, 1.3555g / mL, 1.3556g / mL、1.3557g / mL、1.3558g / mL、1.3559g / mL、1.356g / mL、1.3561g / mL、1.3562g / mL、1.3563g / mL、1.3564g / mL、1.3565g / mL、1.3566g / mL、1.3567g / mL、1.3568g / mL、1.3569g / mL、1.357g / mL、1.3571g / mL、1.3572g / mL、1.3573g / mL、1.3574g / mL、1.3575g / mL、1.3576g / mL、1.3577g / mL、1.3578g / mL、1.3579g / mL、1.358g / mL、1.3581g / mL、1.3582g / mL、1.3583g / mL、1.3584g / mL、1.3585g / mL、1.3586g / mL、1.3587g / mL、1.3588g / mL、1.3589g / mL、1.359g / mL、1.3591g / mL、1.3592g / mL、1.3593g / mL、1.3594g / mL、1.3595g / mL、1.3596g / mL、1.3597g / mL、1.3598g / mL、1.3599g / mL、1.36g / mL、1.3601g / mL、1.3602g / mL、1.3603g / mL、1.3604g / mL、1.3605g / mL、1.3606g / mL、1.3607g / mL、1.3608g / mL、1.3609g / mL、1.361g / mL、1.3611g / mL、1.3612g / mL、1.3613g / mL、1.3614g / mL、1、1.3675g / mL、1.3676g / mL、1.3677g / mL、1.3678g / mL、1.3679g / mL、1.368g / mL、1.3681g / mL、1.3682g / mL、1.3683g / mL、1.3684g / mL、1.3685g / mL、1.3686g / mL、1.3687g / mL、1.3688g / mL、1.3689g / mL、1.369g / mL、1.3691g / mL、1.3692g / mL、1.3693g / mL、1.3694g / mL、1.3695g / mL、1.3696g / mL、1.3697g / mL、1.3698g / mL、1.3699g / mL、1.37g / mL、1.3701g / mL、1.3702g / mL、1.3703g / mL、1.3704g / mL、1.3705g / mL、1.3706g / mL、1.In another refinement, the density of the composition loaded into the ZUC rotor for ZUC processing ranges between any two of the densities provided above.

[0050] In various refinements, the cushion layer, composition, overlay layer, or replacement solution is delivered at a rate of at least 20 milliliters per minute (mL / min), 20 mL / min, 21 mL / min, 22 mL / min, 23 mL / min, 24 mL / min, 25 mL / min, 26 mL / min, 27 mL / min, 28 mL / min, 29 mL / min, 30 mL / min, 31 mL / min, 32 mL / min, 33 mL / min, 34 mL / min, 35 mL / min, 36 mL / min, 37 mL / min, 38 mL / min, 39 mL / min, 40 mL / min, 41 mL / min, 42 mL / min, In an alternative refinement, the cushion layer, composition, overlay layer, or replacement solution is loaded into the ZUC rotor at a flow rate of 200 mL / min or greater. In another refinement, the flow rate at which the cushion layer, composition, overlay layer, or replacement solution is loaded into the ZUC rotor ranges between any two of the flow rates provided above.

[0051] After loading, the packed ZUC rotor is centrifuged at a speed for a period of time to form a gradient and separate particles by density.

[0052] In various refinements, the ZUC rotor is designed to operate at a speed of at least 10,000 revolutions per minute (rpm), 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, 14,000 rpm, 15,000 rpm, 16,000 rpm, 17,000 rpm, 18,000 rpm, 19,000 rpm, 20,000 rpm, 21,000 rpm, 22,000 rpm, 23,000 rpm, 24,000 rpm, 25,000 rpm, 26,000 rpm, 27,000 rpm, 28,000 rpm , 29,000 rpm, 30,000 rpm, 31,000 rpm, 32,000 rpm, 33,000 rpm, 34,000 rpm, 35,000 rpm, 36,000 rpm, 37,000 rpm, 38,000 rpm, 39,000 rpm, 40,000 rpm, 41,000 rpm, 42,000 rpm, 43,000 rpm, 44,000 rpm, 45,000 rpm, 46,000 rpm, 47,000 rpm, 48,000 rpm, 49,000 rpm, or 50,000 rpm. In other refinements, the speed at which the ZUC rotor is centrifuged ranges between any two of the speeds provided above.

[0053] In an alternative refinement, the ZUC rotor is centrifuged at a force (G) of at least 50,000 g, 50,000 G, 55,000 G, 60,000 G, 65,000 G, 70,000 G, 75,000 G, 80,000 G, 85,000 G, 90,000 G, 95,000 G, 100,000 G, 105,000 G, 110,000 G, 115,000 G, 120,000 G, or 125,000 G. In another alternative refinement, the speed at which the ZUC rotor is centrifuged ranges between any two of the speeds provided above.

[0054] In various refinements, the ZUC rotor is centrifuged for at least 13 hours (hr), 13 hr, 13.5 hr, 14 hr, 14.5 hr, 15 hr, 15.5 hr, 16 hr, 16.5 hr, 17 hr, 17.5 hr, 18 hr, 18.5 hr, 19 hr, 19.5 hr, 20 hr, 20.5 hr, 21 hr, 21.5 hr, 22 hr, 22.5 hr, 23 hr, 23.5 hr, 24 hr, 24.5 hr, or 25 hr. In other refinements, the time the packed ZUC rotor is centrifuged ranges between any two of the times provided above.

[0055] In various refinements, the packed ZUC rotor is heated to at least 10°C, 10°C, 10.5°C, 11°C, 11.5°C, 12°C, 12.5°C, 13°C, 13.5°C, 14°C, 14.5°C, 15°C, 15.5°C, 16°C, 16.5°C, 17°C, 17.5°C, 18°C, 18.5°C, 19°C, 19.5°C, 20°C, 20.5°C, 21°C, 21.5°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75° The packed ZUC rotor is centrifuged at a temperature of 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, 26°C, 26.5°C, 27°C, 27.5°C, 28°C, 28.5°C, 29°C, 29.5°C, 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, 33.5°C, 34°C, 34.5°C, 35°C, 35.5°C, or 36°C. In another refinement, the temperature at which the packed ZUC rotor is centrifuged ranges between any two of the temperatures identified above.

[0056] After the particles are separated by ultracentrifugation, the packed rotor can be stationary or centrifuged at a speed (e.g., 1000 rpm, 1500 rpm, 2000 rpm, 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm, or 5000 rpm) to recover the ZUC-treated composition by pumping a displacement solution into the rotor to force the cushion layer, composition, and overlay layer out of the ZUC rotor. In various refinements, the packed rotor is centrifuged for at least 0 minutes (min), at least 1 minute, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes, 160 minutes, 170 minutes, 180 minutes, 190 minutes, 200 minutes, 210 minutes, 220 minutes, 230 minutes, 240 minutes, 250 minutes, 260 minutes, 270 minutes, 280 minutes, 290 minutes, 300 minutes, 310 minutes, 320 minutes, 330 minutes, 340 minutes, 350 minutes, or 360 minutes. In other refinements, the time the rotor is centrifuged ranges between any two of the times provided above.

[0057] In various embodiments, AEX and ZUC remove or eliminate at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99+%, or 100% of AAV-produced impurities. In different embodiments, the percentage of AAV-produced impurities removed by AEX and ZUC ranges between any two of the above percentages. In an improvement, the composition is substantially devoid of AAV-produced impurities after AEX and ZUC treatment. As noted above, when rAAV is produced, there is a mixture of heavy capsids containing the complete transgene of interest, partial capsids containing a portion of the transgene of interest, and light capsids. As emphasized below, empty and light capsids have no therapeutic benefit and increase the patient's exposure to foreign proteins, nucleic acids, etc., thereby increasing the likelihood of an adverse immune response in the patient. Therefore, it is preferable to remove empty and light capsids from AAV as much as possible. The combined use of AEX and ZUC can obtain heavy and partial capsids with 99+% purity, with ZUC treatment performed after AEX. In this regard, if AEX is not used as the first step, empty and light capsids overload the capacity of ZUC, resulting in precipitation during ZUC treatment. On the other hand, using AEX without ZUC does not completely remove empty and light capsids. Therefore, the present invention relates to a method for purifying AAV heavy and partial capsids that are at least 85% pure (i.e., free of light and empty capsids). In further refinements, the heavy and partial capsids are at least 90% pure, or the heavy and partial capsids are 99+% pure, or the composition has no detectable light or empty capsids.

[0058] Various embodiment methods and processes include processing the composition through tangential flow filtration (TFF) between the AEX and ZUC treatments, where the TFF step includes ultrafiltration and diafiltration steps, and the AEX elution buffer is removed from the composition and replaced with a loading buffer containing gradient-forming compounds for the ZUC treatment.

[0059] In various refinements, the AEX-treated composition loaded for TFF has a viscosity of at least 0.1 x 10e17 vg / m² (m 2 ), 0.1×10e17vg / m 2 , 0.5×10e17vg / m 2 , 1×10e17vg / m 2 , 1.5×10e17vg / m 2 , 2×10e17vg / m 2 , 2.5×10e17vg / m 2 , 3×10e17vg / m 2 , 3.5×10e17vg / m 2 , 4×10e17vg / m 2 , 4.5×10e17vg / m 2 , 5×10e17vg / m 2 , 5.5×10e17vg / m 2 , 6×10e17vg / m 2 , 6.5×10e17vg / m 2 , 7×10e17vg / m 2 , 7.5×10e17vg / m 2 , 8×10e17vg / m 2 , 8.5×10e17vg / m 2 , 9×10e17vg / m 2 , 9.5×10e17vg / m 2 , or 10 x 10e17vg / m 2 In another refinement, the titer ranges between any two of the titers provided above.

[0060] In various refinements, the TFF is at least 2 pounds per square inch (psi) (0.137895 bar), 2 psi (0.137895 bar), 3 psi (0.206843 bar), 4 psi (0.27579 bar), 5 psi (0.344738 bar), 6 psi (0.413685 bar), 7 psi (0.482633 bar), 8 psi (0.551581 bar), 9 psi (0.620528 bar), 10 psi (0.689476 bar), 11 psi (0.758423 bar), 12 psi (0 .827371bar), 13psi(0.896318bar), 14psi(0.965266bar), 15psi(1.03421bar), 16psi(1.10316bar), 17psi(1.17211bar), 18psi(1.24106bar) ), 19psi (1.31bar), 20psi (1.37895bar), 21psi (1.4479bar), 22psi (1.51685bar), 23psi (1.58579bar), 24psi (1.65474bar), 25psi (1.72369ba r), 26psi (1.79264bar), 27psi (1.86158bar), 28psi (1.93053bar), 29psi (1.99948bar), 30psi (2.06843bar), 31psi (2.13737bar), 32psi (2.2 0632bar), 33psi(2.27527bar), 34psi(2.34422bar), 35psi(2.41317bar), 36psi(2.48211bar), 37psi(2.55106bar), 38psi(2.62001bar), 39ps The AEX-treated composition is filtered at a transmembrane pressure (TMP) of 1 (2.68896 bar), 40 psi (2.7579 bar), 41 psi (2.82685 bar), 42 psi (2.8958 bar), 43 psi (2.96475 bar), 44 psi (3.03369 bar), 45 psi (3.10264 bar), 46 psi (3.17159 bar), 47 psi (3.24054 bar), 48 psi (3.30948 bar), 49 psi (3.37843 bar), or 50 psi (3.44738 bar).In another refinement, the TMP of the TFF for the AEX-treated composition ranges between any two of the TMPs provided above.

[0061] In various refinements, the TFF is at least 1 L / min / m 2 , 1L / min / m 2 ,2L / min / m 2 ,3L / min / m 2 ,4L / min / m 2 ,5L / min / m 2 ,6L / min / m 2 ,7L / min / m 2 ,8L / min / m 2 ,9L / min / m 2 , 10L / min / m 2 , 11L / min / m 2 , 12L / min / m 2 , 13L / min / m 2 , 14L / min / m 2 , or 15 L / min / m 2 In another refinement, the crossflow of the TFF for the AEX-treated composition ranges between any two of the crossflows provided above.

[0062] In various improvements, TFF is at least 1×10e13vg / mL、1×10e13vg / mL、1.1×10e13vg / mL、1.2×10e13vg / mL、1.3×10e13vg / mL、1.4×10e13vg / mL、1.5×10e13vg / mL、1.6 ×10e13vg / mL、1.7×10e13vg / mL、1.8×10e13vg / mL、1.9×10e13vg / mL、2×10e 13vg / mL、2.1×10e13vg / mL、2.2×10e13vg / mL、2.3×10e13vg / mL、2.4×10e13v g / mL, 2.5×10e13vg / mL, 2.6×10e13vg / mL, 2.7×10e13vg / mL, 2.8×10e13vg / mL, 2.9×10e13vg / mL, 3×10e13vg / mL, 3.1×10e13vg / mL, 3.2×10e13vg / mL, 3.3×10e13vg / mL, 3.4×10e13vg / mL, 3.5×10e13vg / mL, 3.6×10e13vg / mL, 3.7×10e13vg / mL, 3.8×10e13vg / mL, 3.9×10e13vg / mL, 4×10e13vg / mL, 4.1×10e1 3vg / mL、4.2×10e13vg / mL、4.3×10e13vg / mL、4.4×10e13vg / mL、4.5×10e13v g / mL、4.6×10e13vg / mL、4.7×10e13vg / mL、4.8×10e13vg / mL、4.9×10e13vg / mL、5×10e13vg / mL、5.1×10e13vg / mL、5.2×10e13vg / mL、5.3×10e13vg / mL、5.4×10e13vg / mL、5.5×10e13vg / mL、5.6×10e13vg / mL、5.7×10e13vg / mL、5.8× 10e13vg / mL、5.9×10e13vg / mL、6×10e13vg / mL、6.1×10e13vg / mL、6.2×10e1 3vg / mL、6.3×10e13vg / mL、6.4×10e13vg / mL、6.5×10e13vg / mL、6.6×10e13v g / mL、6.7×10e13vg / mL、6.8×10e13vg / mL、6.9×10e13vg / mL、7×10e13vg / mL、7.1×10e13vg / mL、7.2×10e13vg / mL、7.3×10e13vg / mL、7.4×10e13vg / mL、7.The AEX treated composition is filtered to a retentate concentration of 5×10e13 vg / mL, 7.6×10e13 vg / mL, 7.7×10e13 vg / mL, 7.8×10e13 vg / mL, 7.9×10e13 vg / mL, 8×10e13 vg / mL, 8.1×10e13 vg / mL, 8.2×10e13 vg / mL, 8.3×10e13 vg / mL, 8.4×10e13 vg / mL, 8.5×10e13 vg / mL, 8.6×10e13 vg / mL, 8.7×10e13 vg / mL, 8.8×10e13 vg / mL, 8.9×10e13 vg / mL, or 9×10e13 vg / mL. In another refinement, the retentate concentration ranges between any two of the concentrations provided above.

[0063] In various refinements, the TFF diafilters the AEX-treated composition with 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 diavolumes of diafiltration against TFF buffer.

[0064] The method and process of various embodiments include processing the composition through TFF after ZUC treatment. TFF includes ultrafiltration and diafiltration steps. This TFF step includes ultrafiltration and diafiltration steps, in which the ZUC buffer containing the gradient-forming compound is removed from the composition and replaced with an AEX elution buffer, which is then removed from the composition and replaced with a formulation buffer containing a pharmaceutically acceptable carrier, to prepare a pharmaceutical composition.

[0065] In various refinements, the ZUC-treated composition loaded for TFF has a viscosity of at least 0.1 x 10e17 vg / m² (m 2 ), 0.1×10e17vg / m 2 , 0.5×10e17vg / m 2 , 1×10e17vg / m 2 , 1.5×10e17vg / m 2 , 2×10e17vg / m 2, 2.5×10e17vg / m 2 , 3×10e17vg / m 2 , 3.5×10e17vg / m 2 , 4×10e17vg / m 2 , 4.5×10e17vg / m 2 , 5×10e17vg / m 2 , 5.5×10e17vg / m 2 , 6×10e17vg / m 2 , 6.5×10e17vg / m 2 , 7×10e17vg / m 2 , 7.5×10e17vg / m 2 , 8×10e17vg / m 2 , 8.5×10e17vg / m 2 , 9×10e17vg / m 2 , 9.5×10e17vg / m 2 , or 10 x 10e17vg / m 2 In another refinement, the titer ranges between any two of the titers provided above.

[0066] In various refinements, the TFF is at least 2 psi (0.137895 bar), 2 psi (0.137895 bar), 3 psi (0.206843 bar), 4 psi (0.27579 bar), 5 psi (0.344738 bar), 6 psi (0.413685 bar), 7 psi (0.482633 bar), 8 psi (0.551581 bar), 9 psi (0.620528 bar), 10 psi (0.689476 bar), 11 psi (0.758423 bar), 12 psi (0.827371 bar), 13 psi (0.827371 bar), 14 psi (0.827371 bar), 15 psi (0.827371 bar), 16 psi (0.827371 bar), 17 psi (0.827371 bar), 18 psi (0.827371 bar), 19 psi (0.951581 bar), 20 psi (0.951581 bar), 21 psi (0.951581 bar), 22 psi (0.951581 bar), 23 psi (0.951581 bar), 24 psi (0.951581 bar), 25 psi (0.951581 bar), 26 psi (0.951581 bar), 27 psi (0.951581 bar), 28 psi (0.951581 bar), 29 psi (0.951581 bar), 30 psi (0.951581 bar), 31 psi (0.951581 bar), 32 psi (0.951581 r), 13psi (0.896318bar), 14psi (0.965266bar), 15psi (1.03421bar), 16psi (1.10316bar), 17psi (1.17211bar), 18psi (1.24106bar), 19psi (1.31bar), 20psi(1.37895bar), 21psi(1.4479bar), 22psi(1.51685bar), 23psi(1.58579bar), 24psi(1.65474bar), 25psi(1.72369bar), 2 6psi (1.79264bar), 27psi (1.86158bar), 28psi (1.93053bar), 29psi (1.99948bar), 30psi (2.06843bar), 31psi (2.13737bar), 32psi (2.206 32bar), 33psi(2.27527bar), 34psi(2.34422bar), 35psi(2.41317bar), 36psi(2.48211bar), 37psi(2.55106bar), 38psi(2.62001bar), 39p The ZUC treated composition is filtered at a TMP of 40 psi, 41 psi, 42 psi, 43 psi, 44 psi, 45 psi, 46 psi, 47 psi, 48 psi, 49 psi, or 50 psi.In another refinement, the TMP of the TFF for the ZUC-treated composition ranges between any two TMPs provided above.

[0067] In various refinements, the TFF is at least 1 L / min / m 2 , 1L / min / m 2 ,2L / min / m 2 ,3L / min / m 2 ,4L / min / m 2 ,5L / min / m 2 ,6L / min / m 2 ,7L / min / m 2 ,8L / min / m 2 ,9L / min / m 2 , 10L / min / m 2 , 11L / min / m 2 , 12L / min / m 2 , 13L / min / m 2 , 14L / min / m 2 , or 15 L / min / m 2 In another refinement, the crossflow of the TFF for the ZUC-treated composition ranges between any two of the crossflows provided above.

[0068] In various improvements, TFF is at least 1×10e13vg / mL、1×10e13vg / mL、1.1×10e13vg / mL、1.2×10e13vg / mL、1.3×10e13vg / mL、1.4×10e13vg / mL、1.5×10e13vg / mL、1.6 ×10e13vg / mL、1.7×10e13vg / mL、1.8×10e13vg / mL、1.9×10e13vg / mL、2×10e 13vg / mL、2.1×10e13vg / mL、2.2×10e13vg / mL、2.3×10e13vg / mL、2.4×10e13v g / mL, 2.5×10e13vg / mL, 2.6×10e13vg / mL, 2.7×10e13vg / mL, 2.8×10e13vg / mL, 2.9×10e13vg / mL, 3×10e13vg / mL, 3.1×10e13vg / mL, 3.2×10e13vg / mL, 3.3×10e13vg / mL, 3.4×10e13vg / mL, 3.5×10e13vg / mL, 3.6×10e13vg / mL, 3.7×10e13vg / mL, 3.8×10e13vg / mL, 3.9×10e13vg / mL, 4×10e13vg / mL, 4.1×10e1 3vg / mL、4.2×10e13vg / mL、4.3×10e13vg / mL、4.4×10e13vg / mL、4.5×10e13v g / mL、4.6×10e13vg / mL、4.7×10e13vg / mL、4.8×10e13vg / mL、4.9×10e13vg / mL、5×10e13vg / mL、5.1×10e13vg / mL、5.2×10e13vg / mL、5.3×10e13vg / mL、5.4×10e13vg / mL、5.5×10e13vg / mL、5.6×10e13vg / mL、5.7×10e13vg / mL、5.8× 10e13vg / mL、5.9×10e13vg / mL、6×10e13vg / mL、6.1×10e13vg / mL、6.2×10e1 3vg / mL、6.3×10e13vg / mL、6.4×10e13vg / mL、6.5×10e13vg / mL、6.6×10e13v g / mL、6.7×10e13vg / mL、6.8×10e13vg / mL、6.9×10e13vg / mL、7×10e13vg / mL、7.1×10e13vg / mL、7.2×10e13vg / mL、7.3×10e13vg / mL、7.4×10e13vg / mL、7.5×10e13vg / mL, 7.6×10e13vg / mL, 7.7×10e13vg / mL, 7.8×10e13vg / mL, 7.9×10e13vg / mL, 8×10e13vg / mL, 8.1×10e13vg / mL, 8.2× 10e13vg / mL, 8.3×10e13vg / mL, 8.4×10e13vg / mL, 8.5×10e13vg / mL, 8.6×10e13vg / mL, 8.7×10e13vg / mL, 8.8×10e13vg / mL, 8.9×1 The ZUC-treated composition is filtered to a retentate concentration of 0x10e13 vg / mL, 9x10e13 vg / mL, 1x10e14 vg / mL, 1.1x10e14 vg / mL, 1.2x10e14 vg / mL, 1.3x10e14 vg / mL, 1.4x10e14 vg / mL, 1.5x10e14 vg / mL, 1.6x10e14 vg / mL, 1.7x10e14 vg / mL, 1.8x10e14 vg / mL, 1.9x10e14 vg / mL, or 2x10e14 vg / mL. In other refinements, the retentate concentration ranges between any two of the concentrations provided above.

[0069] In various refinements, the TFF diafilters the ZUC-treated composition with 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 diavolumes of diafiltration against TFF buffer.

[0070] definition "Anion exchange chromatography" or "AEX" refers to the process of separating analytes from a mixture by flowing the mixture through an anion exchange material, which represents a fixed matrix carrying covalently bound positively charged substituents. Anion exchange materials are typically provided as anion exchange chromatography columns. Anion exchange materials have the ability to exchange their non-covalently bound counterions for similarly charged binding partners or ions from the surrounding solution (e.g., the mixture). Depending on the chemical nature of the charged groups / substituents, anion exchange materials can be further classified as strong or weak ion exchange materials depending on the strength of the covalently bound charged substituents. Strong anion exchange materials have quaternary ammonium groups, and weak anion exchange materials have diethylaminoethyl groups as the charged substituents. Anion exchange chromatography involves the steps of equilibrating a column with a buffer, flowing a composition through the column, washing the column, and eluting the composition from the column.

[0071] "Zonal ultracentrifugation" or "ZUC" refers to the process of centrifuging a composition using a zonal rotor. Examples of zonal rotors and zonal ultracentrifugation systems are disclosed in U.S. Patent Nos. 6,051,189, 7,862,494, 7,837,609, 9,862,936, and 9,956,564, all of which are incorporated herein by reference in their entireties. One example of zonal ultracentrifugation is isopycnic density gradient sedimentation, which relies on differences in the buoyancy properties of constituent particles dispersed in a dense solution as the basis for component separation.

[0072] "Tangential flow filtration" or "TFF" refers to an ultrafiltration process in which a solution containing capsids to be concentrated flows tangentially along the surface of an ultrafiltration membrane. The membrane has a pore size with a specific cutoff value that prevents capsids from flowing through the membrane as a permeate. Thus, the capsids are part of the retentate. Tangential flow filtration also includes diafiltration, in which the original solution is removed as a permeate and replaced with another solution. For example, tangential flow filtration replaces the elution buffer from a composition after anion exchange chromatography with a loading buffer for zonal ultracentrifugation. In another example, tangential flow filtration replaces the elution buffer from a composition after zonal ultracentrifugation with a formulation buffer containing a pharmaceutically acceptable carrier to prepare a pharmaceutical composition.

[0073] "Pharmaceutical product" refers to a product suitable for pharmaceutical use in a subject animal, including humans and mammals. For example, the pharmaceutical product is a rAAV virion.

[0074] A "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a subject animal, including humans and mammals. A pharmaceutical composition includes a pharmaceutically effective amount of a pharmaceutical agent, such as an AAV virion, and also includes a pharmaceutically acceptable carrier. Pharmaceutical compositions encompass compositions containing an active ingredient, inactive ingredients that make up the carrier, and any product resulting directly or indirectly from the combination, complexation, or aggregation of any two or more components, or from the dissociation of one or more components, or from other types of reactions or interactions of one or more components. Thus, pharmaceutical compositions encompass any composition made by mixing the virions provided herein with a pharmaceutically acceptable carrier.

[0075] A "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical excipients, vehicles, diluents, stabilizers, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers, such as, but not limited to, phosphate buffered saline solution, 5% dextrose in water, and emulsions such as oil / water or water / oil emulsions, as well as various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). The pharmaceutical carrier used can depend on the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intrathecal, intramuscular, intravenous, or intraperitoneal injection, or topical, transdermal, or transmucosal administration). "Pharmaceutically acceptable salts" are salts that can be incorporated into oxalate-degrading enzyme compositions for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0076] "Pharmaceutically acceptable" or "pharmacologically acceptable" means a material that is biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0077] "Subject" encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other ape and monkey species; livestock such as cows, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice, and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or sex.

[0078] A "contaminating virus" refers to a virus that contaminates a composition during the manufacturing process. The contaminating virus compromises the safety of the pharmaceutical product for administration to a subject. Examples of contaminating viruses include baculoviruses such as Autographa californica nuclear polyhedrosis virus (AcNPV), encephalomyocarditis virus (EMC), porcine parvovirus (PPV), reovirus (Reo-3), simian vacuolar virus 40 (SV-40), vesicular stomatitis virus (VSV), or retroviruses such as murine leukemia virus (X-MuLV).

[0079] Adeno-associated virus Therapeutically effective rAAV particles include those disclosed in US9,504,762, WO2019 / 222136, and US2019 / 0376081, the disclosures of which are incorporated by reference in their entireties.

[0080] "AAV" is a common abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus with a genome enclosed by a capsid. Currently, there are 13 characterized AAV serotypes. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, New York. However, since it is well known that the various serotypes are very closely related, both structurally and functionally, even at the genetic level, it is fully expected that these same principles will be applicable to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174 in Parvoviruses and Human Disease, J.R.P.Tattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974).) For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes, and all possess three related capsid proteins. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome and the presence of similar self-annealing segments at the ends corresponding to the ITRs. Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0081] As used herein, "AAV viral particle" refers to an infectious viral particle composed of at least one AAV capsid protein and an encapsidated AAV genome. "Recombinant AAV" or "rAAV," "rAAV virion," or "rAAV viral particle," or "rAAV vector particle," or "AAV virus" refers to a viral particle composed of at least one capsid or Cap protein and an encapsidated rAAV vector genome as described herein. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector." Thus, since such a vector is contained within the rAAV vector particle, the production of an AAV vector particle necessarily includes the production of an rAAV vector. In different embodiments, the rAAV viral particles include those of EP 2,698,163, EP 2,859,016, EP 3,044,231, EP 3,352,787, EP 3,491,008, EP 3,794,016, EP 3,794,112, US 9,393,323, US 9,447,168, US 9,504,762, US 9,764,045, US 10,124,041, US 10,463,718, US 10,512,675, US 10,709,796, US 10,792,336, US 2017 / 0087219, US 2019 / 0376081, US 2020 US2020 / 0024579, US2020 / 0061161, US2020 / 0069819, US2020 / 0362368, WO2015 / 038625, WO2017 / 053677, WO2018 / 022608, WO2019 / 217513, WO2019 / 222132, WO2019 / 222136, WO2020 / 232044, WO2021 / 097157, WO2021 / 183895, and WO2021 / 202943, the disclosures of which are incorporated herein by reference in their entireties.

[0082] "Capsid" refers to the structure in which the rAAV vector genome is packaged. The capsid contains either the VP1 protein or the VP3 protein, but more typically contains all three VP1, VP2, and VP3 proteins, as found in native AAV. The sequence of the capsid proteins determines the serotype of the rAAV virion. rAAV virions are classified as AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, is AAV-rh.10 (AAVrh10), AAV-DJ (AAVDJ), AAV-DJ8 (AAVDJ8), AAV-1, AAV-2, AAV-2G9, AAV-3, AAV3a, AAV3b, AAV3-3, AAV 4, AAV4-4, AAV-5, AAV-6, AAV6.1, AAV6.2, AAV6.1.2, AAV-7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.2 4, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV-10, AAV-11, AAV-12, AAV16.3, AAV24.1, AAV27.3, AAV 42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV4 2-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV 43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, A AV1-8 / 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 / r h.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、AAV5、AAV2 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、AAV rh.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 、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、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.5 6、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.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、A AVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAV rh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVr h.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh.53、AAVrh.54、AAVrh.56、AAVrh.5 7、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh8R、AAVrh8R A586R mutant、AAVrh8R R533A mutation、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhEr1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVh Er1.7、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 SM10-2, AAV Shuffle100-1, AAV Shuffle100-3, AAV Shuffle100-7, AAV Shuffle10-2, AAV Shuffle10-6, AAV Shuffle10-8, AAV Shuffle100-2, AAV SM10-1, AAV SM10-8, AAV SM100-3, AAV SM100-10, 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, authentic AAV (ttAAV), UPENN AAV10, or Japanese AAV10 serotype, AAV_po.6, AAV_po., AAV_po.5, AAV_LK03, AAV_ra.1, AAV_bat_YNM, AAV_bat_Brazil, AAV_mo.1, AAV_Chicken_DA-1, or AAV_Mouse_NY1, Bba21, Bba26, Bba27, Bba29, Bba30, Bba31, Bba32, Bba33, Bba34, Bba35, Bba36, Bba37, Bba38, Bba41, Bba42, Bba43, Bba44, Bce14, Bce15, Bce16, Bce17, Bce18, Bce20, Bce35, Bce36, Bce39, Bce40, Bce41, Bce42, Bce43, Bce44, Bce45, Bce46, Bey20, Bey22, Bey23, Bma42, Bma43, Bpo1, Bpo2 , Bpo3, Bpo4, Bpo6, Bpo8, Bpo13, Bpo18, Bpo20, Bpo23, Bpo24, Bpo27, Bpo28, Bpo29, Bpo33, Bpo35, Bpo36, Bpo37, Brh26, Brh27, Brh28, Brh29, Brh30, Brh31, Brh32, Brh33, Bfm17, Bfm18, Bfm20, Bfm21, Bfm24, Bfm25, Bfm27, Bfm32, Bfm33, Bfm34, Bfm35, AAV-rh10, AAV-rh39, AAV-rh43, AAVanc80L65, or a variant of any of these (e.g., These capsid proteins include those derived from several AAV serotypes, including non-naturally occurring mixed serotypes (see U.S. Patent No. 8,318,480 for disclosure of non-naturally occurring mixed serotypes). Exemplary capsids are also provided in International Application Publication Nos. WO 2018 / 022608 and WO 2019 / 222136, which are incorporated herein in their entireties. The capsid proteins may also be variants of native VP1, VP2, and VP3, including mutated, chimeric, or shuffled proteins. The capsid proteins may be of rh.10 or other subtypes within various clades of AAV, various clades and subtypes being disclosed, for example, in U.S. Patent No. 7,906,111. In various embodiments, the capsid of the AAV viral particle is selected from the group consisting of AAV-1 (Genbank Accession No. AAD27757.1), AAV-2 (NCBI Reference SEQ ID NO: YP_680426.1), AAV-3 (NCBI Reference SEQ ID NO: NP_043941.1), AAV-3B (Genbank Accession No. AAB95452.1), AAV-4 (NCBI Reference SEQ ID NO: NP_044927.1), AAV-5 (NCBI Reference SEQ ID NO: YP_068409.1), AAV-6 (Genbank Accession No. AAB95450.1), AAV-7 (NCBI Reference SEQ ID NO: YP_077178.1), AAV-8 (NCBI Reference SEQ ID NO: YP_077179.1), AAV-9 (Genbank Accession No. A and / or AAV-10 (Genbank Accession No. AAT46337.1), AAV-11 (Genbank Accession No. AAT46339.1), AAV-12 (Genbank Accession No. ABI16639.1), AAV-13 (Genbank Accession No. ABZ10812.1), or the amino acid sequences disclosed in WO2018 / 022608 and WO2019 / 222136.The construction and use of AAV proteins of different serotypes are described in Chao et al., Mol. Ther. 2:619-623, 2000; Davidson et al., PNAS 97:3428-3432, 2000; Xiao et al., J. Virol. 72:2224-2232, 1998; Halbert et al., J. Virol. 74:1524-1532, 2000; Halbert et al., J. Virol. 75:6615-6624, 2001; and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, 2001.

[0083] "AAV vector," "rAAV vector," "vector genome," and "rAAV vector genome" refer to either single-stranded or double-stranded nucleic acid having an AAV 5' inverted terminal repeat (ITR) sequence and an AAV 3' ITR flanking a protein-coding sequence (preferably a functional therapeutic protein-coding sequence, e.g., FVIII, FIX, and PAH) operably linked to transcriptional regulatory elements heterologous to the AAV viral genome, i.e., one or more promoters and / or enhancers, and optionally, a polyadenylation sequence, and / or one or more introns inserted between the exons of the protein-coding sequence. The term "gene of interest" (GOI) can also refer to the rAAV vector genome. A single-stranded rAAV vector refers to a nucleic acid present in the genome of an AAV viral particle and can be either the sense or antisense strand of a nucleic acid sequence disclosed herein. Sizes of such single-stranded nucleic acids are provided in bases. A double-stranded rAAV vector refers to the nucleic acid present in the DNA of a plasmid, e.g., pUC19, or in the genome of a double-stranded virus, e.g., baculovirus, used to express or transfer the rAAV vector nucleic acid. The size of such double-stranded nucleic acids is provided in base pairs (bp). The term "ITR," as used herein, refers to the art-recognized regions found at the 5' and 3' ends of the rAAV genome that function in cis as an origin of DNA replication and as a packaging signal for the viral genome. The AAV ITRs, together with the Rep coding region, provide efficient removal and rescue from endosomes and integration of the nucleotide sequence intervening between the two adjacent ITRs into the host cell genome. The sequences of specific AAV-related ITRs are disclosed in Yan et al., J. Virol. 79(1):364-379 (2005). ITRs are also found in "flip" or "flop" configurations in which the sequence between the AA' inverted repeats (forming the arms of the hairpin) is present in the reverse complement (Wilmott, Patrick, et al. Human gene therapy methods 30.6 (2019): 206-213).The construction and use of AAV vector genomes of different serotypes are described in Chao et al., Mol. Ther. 2:619-623, 2000; Davidson et al., PNAS 97:3428-3432, 2000; Xiao et al., J. Virol. 72:2224-2232, 1998; Halbert et al., J. Virol. 74:1524-1532, 2000; Halbert et al., J. Virol. 75:6615-6624, 2001; and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, 2001. Due to the wide range of constructs available and extensive characterization, the exemplary AAV vector genomes disclosed below are derived from serotype 2.

[0084] The terms "therapeutically effective AAV," "therapeutically effective AAV particle," "therapeutic AAV," "therapeutically effective rAAV," "therapeutically effective rAAV particle," "therapeutically effective rAAV," and "therapeutically effective rAAV" refer to a recombinant AAV capable of infecting cells such that the infected cells express (e.g., by transcription and / or translation) an element of interest (e.g., a nucleotide sequence, a protein, etc.). To this extent, therapeutically effective rAAV particles can include AAV particles having capsids or vector genomes (vg) with different properties. For example, therapeutically effective rAAV particles can have capsids with different post-translational modifications. In other examples, therapeutically effective AAV particles can contain vg with different sizes / lengths, plus or minus strand sequences, different flip / flop ITR configurations (flip / flop, flop / flip, flip / flip, flop / flop, etc.), different numbers of ITRs (1, 2, 3, etc.), or truncations. For example, overlapping homologous recombination occurs in rAAV-infected cells between nucleic acids with 5' and 3' truncations, such that a "complete" nucleic acid encoding a large protein is generated, thereby reconstituting a functional, full-length gene. In another example, complementary nucleic acid sequences with 5' and 3' truncations interact with each other to form a "complete" nucleic acid during second strand synthesis. The "complete" nucleic acid encodes a large protein, thereby reconstituting a functional, full-length gene. Therapeutically effective rAAV particles are also referred to as heavy capsids, complete capsids, or partially complete capsids.

[0085] The term "therapeutically effective amount" refers to an amount of a therapeutic agent that, when administered to a subject suffering from or susceptible to a disease, disorder, or condition, is sufficient to treat, diagnose, prevent, or delay the onset of symptoms of the disease, disorder, or condition. It will be understood by those skilled in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose. The term "therapeutically effective" refers to any element or composition of a therapeutic agent that acts sufficiently such that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, a therapeutically effective amount of the therapeutic agent is sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of the disease, disorder, and / or condition. For example, as described above, a therapeutically effective rAAV can infect cells such that the infected cells express (e.g., by transcription and / or translation) an element of interest (e.g., a nucleotide sequence, a protein, etc.). A therapeutically effective rAAV has a vector genome that is used by a cell infected with the therapeutically effective rAAV to produce a therapeutically effective nucleotide sequence that is used by the infected cell to produce an element of interest (e.g., a nucleotide sequence, a protein, etc.) by various methods, such as replication, transcription, or translation. Note also that a "therapeutic agent" includes a therapeutically effective rAAV or a therapeutic rAAV virus.

[0086] For example, a "therapeutic rAAV virus," which refers to an rAAV virion, rAAV virus particle, rAAV vector particle, or rAAV virus containing a heterologous polynucleotide encoding a therapeutic protein, can be used to replace or supplement a protein in vivo. A "therapeutic protein" is a polypeptide having biological activity that replaces or compensates for the loss or reduction in activity of a corresponding endogenous protein. For example, functional phenylalanine hydroxylase (PAH) is a therapeutic protein for phenylketonuria (PKU). Thus, for example, a recombinant rAAV PAH virus can be used in a medicament for treating a subject suffering from PKU. The medicament may be administered intravenously (IV), and administration of the medicament results in expression of the PAH protein in the subject's bloodstream sufficient to alter neurotransmitter metabolite or neurotransmitter levels in the subject. Optionally, the medicament may also include prophylactic and / or therapeutic corticosteroids for the prevention and / or treatment of any liver toxicity associated with administration of the rAAV PAH virus. Medicaments comprising prophylactic or therapeutic corticosteroid treatment can include at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more mg / day of corticosteroid. Medicaments comprising prophylactic or therapeutic corticosteroids can be administered for a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10, or more weeks. PKU therapy can also optionally include tyrosine supplementation.

[0087] "Therapeutically ineffective AAV particles," "therapeutically ineffective AAV," "therapeutically ineffective rAAV particles," or "therapeutically ineffective rAAV" refer to AAV particles that are unable to infect cells, or AAV particles in which cells infected with therapeutically ineffective rAAV particles are unable to express (e.g., by transcription and / or translation) elements of interest (e.g., nucleotide sequences, proteins, etc.). Therapeutically ineffective rAAV particles may contribute to reduced efficacy per unit dose of capsid and may increase the risk of an immune response due to the necessary increase in foreign protein introduced into a patient for an effective amount of heavy / full / partially complete capsid. Therapeutically ineffective rAAV particles can include AAV particles with capsids or vg having different properties, referred to as empty capsids or light capsids. For example, empty capsids have no vg or have an unquantifiable or undetectable concentration of vg. In another example, the light capsid may have a vg with an incomplete expression cassette that does not express a gene of interest. In one example, the vector genome of the light capsid has one or more sizes that are insufficient for cells infected with the capsid to produce a therapeutically effective nucleotide sequence. In another example, the light capsid has a vector genome with one or more sizes that reduce expression of an element by cells infected with the capsid compared to expression of the element by cells infected under the same conditions but lacking infection by the capsid, and a therapeutically effective rAAV encoding the element. In different examples, the size of the vector genome of a light capsid is 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% or less, 43% or less, 42% or less, 41% or less, 40% or less, 39% or less, 38% or less, 37% or less, 36% or less, 35% or less, 34% or less, 33% or less, 32% or less, 31% or less, 30% or less than the size of the vector genome of a therapeutically effective rAAV. % or less, 29% or less, 28% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.Empty or light capsids may also have different capsid properties that may impair the infectivity of the capsid. In another example, therapeutically ineffective rAAV particles include rAAV particles having Rep proteins associated with them. rAAV associated with Rep proteins may include, for example, large Rep proteins (e.g., Rep78 or Rep68 proteins), small Rep proteins (e.g., Rep52 or Rep40 proteins), or a combination thereof. rAAV associated with Rep proteins may also include Rep proteins that are removed from the capsid during different steps, such as the washing or renaturation step in AEX processing or the isolation of post-pooling fractions during ZUC processing. Alternatively, rAAV associated with Rep proteins may include large Rep proteins, small Rep proteins, or a combination thereof, that bind to the capsid. Such bonds include different binding mechanisms, such as covalent bonds, ionic bonds, hydrogen / electrostatic bonds, or van der Waals forces. In a different example, when a portion of the vector genome is not encapsulated within the capsid, the Rep protein can bind to different portions of the capsid or rAAV particle containing the vector genome. These capsids can lack the vector genome or can have a partial / complete vector genome but cannot infect cells. In another example, therapeutically ineffective rAAV particles include rAAV particles with deamidated capsids. For example, deamidated capsids include capsids with deamidated VP1, VP2, or VP3 proteins. For example, the conserved NG (Asp-Gly) residue in the N-terminal region of VP1 is vulnerable to deamidation.Different deamidated capsids and their effects on infectivity, transgene expression, or efficacy are described in Giles, April R., et al., "Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function," Molecular Therapy 26.12 (2018): 2848-2862, and Frederick, Amy, et al., "Engineered Capsids for Efficient Gene Delivery to the Retina and Cornea," Human Gene Therapy 31.13-14 (2020): 756-774. Without being bound by any particular theory, heavy / full or partially full capsids differ from light or empty capsids in their charge and / or density.

[0088] "AAV production impurities" refer to impurities that may impair the efficacy of a therapeutically effective rAAV. AAV production impurities arise during rAAV preparation and include therapeutically ineffective rAAV, aggregates of rAAV particles, exogenous high molecular weight DNA, small nucleotides, proteins, buffer components, etc.

[0089] The transgene incorporated into the AAV capsid can be, but is not limited to, any heterologous gene of therapeutic interest. The transgene is a nucleic acid sequence heterologous to the vector sequences flanking the transgene that encodes a polypeptide, protein, or other product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows transcription, translation, and / or expression of the transgene in a host cell.

[0090] The composition of the transgene sequence depends on the use to which the resulting vector will be put. For example, one type of transgene sequence contains a reporter sequence that generates a detectable signal upon expression. Such reporter sequences include, but are not limited to, DNA sequences encoding b-lactamase, b-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane-bound proteins including, for example, CD2, CD4, CD8, influenza hemagglutinin protein, and others known in the art for which high-affinity antibodies exist or can be generated by conventional means, as well as fusion proteins, particularly those containing membrane-bound proteins appropriately fused to an antigen tag domain derived from hemagglutinin or Myc.

[0091] These coding sequences, when associated with the regulatory elements that drive their expression, provide signals that are detectable by conventional means, including enzymatic, radioactive, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays (including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry). For example, if the marker sequence is the LacZ gene, the presence of a vector bearing a signal is detected by assaying for beta-galactosidase activity. If the transgene is green fluorescent protein or luciferase, a vector bearing a signal can be measured visually by color or light production in a luminometer.

[0092] However, transgenes are typically non-marker sequences that encode products useful in biology and medicine, such as proteins, peptides, RNA, enzymes, dominant-negative mutants, or catalytic RNAs. Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNA, siRNA, small hairpin RNA, trans-splicing RNA, and antisense RNA. An example of a useful RNA sequence is one that inhibits or eliminates the expression of a target nucleic acid sequence in a treated animal. Typically, suitable target sequences include tumor targets and viral diseases. For examples of such targets, see the tumor targets and viruses described below in the section related to immunogens.

[0093] Transgenes can be used to correct or ameliorate genetic defects. Genetic defects can include those in which a normal gene is expressed at lower than normal levels or in which a functional gene product is not expressed. A preferred type of transgene sequence encodes a therapeutic protein or polypeptide to be expressed in a host cell. A vector can also contain multiple transgenes, for example, to correct or ameliorate genetic defects caused by a multisubunit protein. In some situations, different transgenes can be used to encode each subunit of a protein or to encode different peptides or proteins. This is desirable when the DNA encoding the protein subunits is large (e.g., immunoglobulins, platelet-derived growth factor, or dystrophin protein). To produce a multisubunit protein, cells are infected with a recombinant virus containing each of the different subunits. Alternatively, different subunits of a protein can be encoded by the same transgene. In this case, a single transgene contains DNA encoding each subunit, separated by an internal ribozyme entry site (IRES). This is desirable when the size of the DNA encoding each subunit is small, e.g., when the total size of the DNA encoding the subunits and the IRES is less than 5 kilobases (Kb). It should also be noted that longer genomes (i.e., >5 Kb) may be feasible through partial genome recombination in target cells. As an alternative to an IRES, the DNA may be separated by a sequence encoding a 2A peptide that self-cleaves in a post-translational event. See, e.g., Donnelly et al., J. Gen. Virol., 78(Pt1):13-21 (January 1997); Furler et al., Gene Ther., 8(11):864-873 (June 2001); Klump et al., Gene Ther., 8(10):811-817 (May 2001). This 2A peptide is significantly smaller than an IRES and is well suited for use when space is a limiting factor.More often, when the transgene is large, consists of multiple subunits, or two transgenes are delivered simultaneously, rAAVs carrying the desired transgenes or subunits are co-administered to allow them to concatenate in vivo to form a single vector genome. In such embodiments, for co-expression in host cells, a first AAV may carry an expression cassette expressing a single transgene, and a second AAV may carry an expression cassette expressing a different transgene. However, the selected transgene may encode any biologically active product or other product (e.g., a product desired for research).

[0094] A suitable transgene can be readily selected by one of skill in the art. The selection of the transgene is not to be construed as a limitation of the present invention. The transgene may be a heterologous protein, which may be a therapeutic protein. Exemplary therapeutic proteins include, but are not limited to, blood factors, such as b-globin, hemoglobin, tissue plasminogen activator, and clotting factors; colony-stimulating factors (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, and the like; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factors (FGFs, e.g., basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), bone morphogenetic proteins (BGF), and the like. MP), epidermal growth factor (EGF), growth differentiation factor 9 (GDF-9), hepatoma-derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophic factors, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), etc.; soluble receptors, such as soluble TNF-a receptor, soluble VEGF receptor, soluble interleukin receptor (e.g., soluble IL-1 receptor and soluble type II IL-1 receptor), soluble g / d T cell receptors, ligand-binding fragments of soluble receptors, etc.; enzymes, such as α-glucosidase, imiglucarase, β-glucocerebrosidase, and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as 1P-10, interferon-gamma-induced monokine (Mig), Groa / IL-8, RANTES, MIP-1a, MIR-1b, MCP-1, PF-4, etc.; angiogenic agents, such as vascular endothelial growth factors (VEGFs, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), glioma-derived growth factor, angiogenin, angiogenin-2, etc.; anti-angiogenic agents, such as soluble VEGF receptors; protein vaccines;Neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyroid-stimulating hormone, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyroid-stimulating hormone-releasing hormone These include estrogen, vasoactive intestinal peptide, sleep peptide, etc.; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle-stimulating hormone (FSH); human alpha-1 antitrypsin; leukemia inhibitory factor (LIF); tissue factor, luteinizing hormone; macrophage-activating factor; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); tissue inhibitor of metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotropin; fibrin; hirudin; IF-1 receptor antagonists, etc. Some other non-limiting examples of proteins of interest include ciliary neurotrophic factor (CNTF); brain-derived neurotrophic factor (BDNF); neurotrophin 3 and 4 / 5 (NT-3 and 4 / 5); glial cell line-derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); hemophilia-related coagulation proteins, e.g., factor VIII, factor IX, factor X; hereditary angioedema-associated proteins, e.g., C1 inhibitor; dystrophin, mini-dystrophin, or micro-dystrophin; lysosomal acidic lipase (LYL) phenylalanine hydroxylase (PAH); glycogen storage disease-related enzymes, e.g., glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporters (e.g., GFUT2), aldolase A, β-enolase, and glycogen synthase; lysosomal enzymes (e.g., beta-N-acetylhexosaminidase A);and any variants thereof. Other transgenes include transgenes encoding cardiac myosin binding protein C, β-myosin heavy chain, cardiac troponin T, cardiac troponin I, myosin ventricular essential light chain 1, myosin ventricular regulatory light chain 2, cardiac alpha actin (ACTC), α-tropomyosin, titin, four and a half LIM protein 1, and other transgenes disclosed in U.S. Patent No. WO 2014 / 170470. AAV vectors also contain conventional control elements or sequences operably linked to the transgene in a manner that allows its transcription, translation, and / or expression in cells transfected with a plasmid vector or infected with a virus. As used herein, "operably linked" sequences include both expression control sequences contiguous with a gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. Suitable genes include those described in Anguela et al. "Entering the Modern Era of Gene Therapy", Annual Rev. of Med. Vol. 70, pages 272-288 (2019), and Dunbar et al., "Gene Comes of Age", Science, Vol. 359, Issue 6372, ed. 4672 (2018).

[0095] Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (e.g., Kozak consensus sequences); sequences that increase protein stability; and, if necessary, sequences that increase secretion of the encoded product. Many expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and may be used.

[0096] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the b-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 promoter [Invitrogen]. Inducible promoters allow for the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or specific physiological states (e.g., acute phase, specific differentiation states of cells, or only in replicating cells). Inducible promoters and induction systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by one of ordinary skill in the art.Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, and the T7 polymerase promoter system [WO98 / 10088]; the ecdysone insect promoter [No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)]; the tetracycline-repressible system [Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)]; and the tetracycline-inducible system [Gossen et al., Science, 268:1766-1769 (1995); Harvey et al. al, Curr. Opin. Chem. Biol., 2:512-518 (1998)], the RU486 inducible system [Wang et al, Nat. Biotech., 15:239-243 (1997), and Wang et al, Gene Ther., 4:432-441 (1997)], and the rapamycin inducible system [Magari et al, J. Clin. Invest., 100:2865-2872 (1997)]. Other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, a specific differentiation state of cells, or only in replicating cells.

[0097] Optionally, the native promoter of the transgene can be used. When it is desired that the expression of the transgene mimics the native expression, the native promoter can be preferred. When the expression of the transgene must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus, the native promoter can be used. In further embodiments, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic the native expression.

[0098] Transgene can also comprise a gene operably linked to a tissue-specific promoter.For example, if expression in skeletal muscle is desired, a promoter active in muscle must be used.These include promoters derived from genes encoding skeletal b-actin, myosin light chain 2A, dystrophin, and muscle creatine kinase, as well as synthetic muscle promoters that have higher activity than naturally occurring promoters (see Li et al., Nat.Biotech., 17:241-245(1999)). Examples of promoters that are tissue-specific include, inter alia, liver (albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), lymphocyte (CD2, Hansal et al. al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell receptor chain), neurons, e.g., the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), the neurofilament light chain gene (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene (Piccioli et al., Neuron, 15:373-84 (1995)).

[0099] Recombinant AAV can be used to produce a protein of interest in vitro, for example, in cell culture. For example, AAV can be used in a method for producing a protein of interest in vitro, which method includes providing a recombinant AAV containing a nucleotide sequence encoding a heterologous protein and contacting the recombinant AAV with cells in cell culture, thereby causing the recombinant AAV to express the protein of interest in the cells. The size of the nucleotide sequence encoding the protein of interest can vary. For example, the nucleotide sequence can be at least about 0.1 kilobases (kb), at least about 0.2 kb, at least about 0.3 kb, at least about 0.4 kb, at least about 0.5 kb, at least about 0.6 kb, at least about 0.7 kb, at least about 0.8 kb, at least about 0.9 kb, at least about 1 kb, at least about 1.1 kb, at least about 1.2 kb, at least about 1.3 kb, at least about 1.4 kb, at least about 1.5 kb, at least about 1.6 kb, at least about 1.7 kb, or at least about 1. The nucleotide sequence can be 8 kb, at least about 2.0 kb, at least about 2.2 kb, at least about 2.4 kb, at least about 2.6 kb, at least about 2.8 kb, at least about 3.0 kb, at least about 3.2 kb, at least about 3.4 kb, at least about 3.5 kb in length, at least about 4.0 kb in length, at least about 5.0 kb in length, at least about 6.0 kb in length, at least about 7.0 kb in length, at least about 8.0 kb in length, at least about 9.0 kb in length, or at least about 10.0 kb in length. In some embodiments, the nucleotide sequence is at least about 1.4 kb in length.

[0100] Recombinant AAVs can also be used to produce proteins of interest in vivo in animals, such as mammals. Some embodiments provide methods for producing proteins of interest in vivo, including providing a recombinant AAV containing a nucleotide sequence encoding the protein of interest and administering the recombinant AAV to a subject, whereby the recombinant AAV expresses the protein of interest in the subject. In some embodiments, the subject can be a non-human mammal, such as a monkey, dog, cat, mouse, or cow. The size of the nucleotide sequence encoding the protein of interest can vary. For example, the nucleotide sequence may be at least about 0.1 kb, at least about 0.2 kb, at least about 0.3 kb, at least about 0.4 kb, at least about 0.5 kb, at least about 0.6 kb, at least about 0.7 kb, at least about 0.8 kb, at least about 0.9 kb, at least about 1 kb, at least about 1.1 kb, at least about 1.2 kb, at least about 1.3 kb, at least about 1.4 kb, at least about 1.5 kb, at least about 1.6 kb, at least about 1.7 kb, at least about 1.8 kb, It can be at least about 2.0 kb, at least about 2.2 kb, at least about 2.4 kb, at least about 2.6 kb, at least about 2.8 kb, at least about 3.0 kb, at least about 3.2 kb, at least about 3.4 kb, at least about 3.5 kb in length, at least about 4.0 kb in length, at least about 5.0 kb in length, at least about 6.0 kb in length, at least about 7.0 kb in length, at least about 8.0 kb in length, at least about 9.0 kb in length, or at least about 10.0 kb in length. In some embodiments, the nucleotide is at least about 1.4 kb in length.

[0101] Of particular interest is the use of recombinant AAVs to express one or more therapeutic proteins to treat various diseases or disorders. Non-limiting examples of diseases include cancers such as carcinoma, sarcoma, leukemia, and lymphoma; and autoimmune diseases such as multiple sclerosis. Non-limiting examples of carcinomas include esophageal cancer, hepatocellular carcinoma, basal cell carcinoma, squamous cell carcinoma (various tissues), bladder cancer including transitional cell carcinoma, bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma. Non-limiting examples of sarcomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Non-limiting examples of solid tumors include glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Non-limiting examples of leukemia include chronic myeloproliferative syndrome, acute myeloid leukemia, chronic lymphocytic leukemia, including B-cell CLL, T-cell CLL, prolymphocytic leukemia, and hairy cell leukemia, and acute lymphoblastic leukemia. Examples of lymphomas include, but are not limited to, B-cell lymphomas, such as Burkitt's lymphoma and Hodgkin's lymphoma.

[0102] Other non-limiting examples of diseases that can be treated using the rAAVs and methods disclosed herein include sickle cell anemia, cystic fibrosis, lysosomal acid lipase (LAL) deficiency, Tay-Sachs disease, phenylketonuria, mucopolysaccharidoses, glycogen storage diseases (GSDs, e.g., GSD types I, II, III, IV, V, VI, VII, VIII, IX, XI, XII, XIII, and XIV), galactosemia, muscular dystrophies (e.g., Duchenne muscular dystrophy), cardiomyopathies (e.g., hypertrophic cardiomyopathy, dilated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, etc.), and genetic diseases including hemophilia A (classical hemophilia), hemophilia B (Christmas disease), Wilson's disease, Fabry disease, Gaucher disease, hereditary angioedema (HAE), and alpha-1 antitrypsin deficiency. Additionally, the rAAVs and methods disclosed herein can be used to treat other disorders that can be treated by local expression of a transgene in the liver or expression of a secreted protein from the liver or hepatocytes.

[0103] The amount of heterologous protein expressed in a subject (e.g., in the serum of a subject) can vary. For example, in some embodiments, the protein is expressed at a concentration of at least about 9 milligrams (mg) / mL, at least about 10 mg / mL, at least about 11 mg / mL, at least about 12 mg / mL, at least about 13 mg / mL, at least about 14 mg / mL, at least about 15 mg / mL, at least about 16 mg / mL, at least about 17 mg / mL, at least about 18 mg / mL, at least about 19 mg / mL, at least about 20 mg / mL, at least about 21 mg / mL, at least about 22 mg / mL, at least about 23 mg / mL, at least about 24 mg / mL, at least about 25 mg / mL, at least about 26 mg / mL, at least about 27 mg / mL, at least about 28 mg / mL, at least about 29 mg / mL, at least about 30 mg / mL, at least about 31 mg / mL, at least about 32 mg / mL, at least about 33 mg / mL, at least about 34 mg / mL, at least about 35 mg / mL, at least about 36 mg / mL, at least about 37 mg / mL, at least about 38 mg / mL, at least about 39 mg / mL, at least about 40 mg / mL, at least about 41 mg / mL, at least about 42 mg / mL, at least about 43 mg / mL, at least about 44 mg / mL, at least about 45 mg / mL, at least about 46 mg / mL, at least about 47 mg / mL, at least about 48 mg / mL, at least about 49 mg / mL, at least about 50 mg / mL, at least about 51 mg / mL, at least about 52 mg / mL, at least about 53 mg / mL, at least about 54 mg / mL, at least about 5 The antibody may be expressed in the serum of a subject in an amount of at least about 9 mg / mL, at least about 30 mg / mL, at least about 31 mg / mL, at least about 32 mg / mL, at least about 33 mg / mL, at least about 34 mg / mL, at least about 35 mg / mL, at least about 36 mg / mL, at least about 37 mg / mL, at least about 38 mg / mL, at least about 39 mg / mL, at least about 40 mg / mL, at least about 41 mg / mL, at least about 42 mg / mL, at least about 43 mg / mL, at least about 44 mg / mL, at least about 45 mg / mL, at least about 46 mg / mL, at least about 47 mg / mL, at least about 48 mg / mL, at least about 49 mg / mL, or at least about 50 mg / mL. The protein of interest may be expressed in the serum of a subject in an amount of about 9 pg / mL, about 10 pg / mL, about 50 pg / mL, about 100 pg / mL, about 200 pg / mL, about 300 pg / mL, about 400 pg / mL, about 500 pg / mL, about 600 pg / mL, about 700 pg / mL, about 800 pg / mL, about 900 pg / mL, about 1000 pg / mL, about 1500 pg / mL, about 2000 pg / mL, about 2500 pg / mL, or a range between any two of those values.Those skilled in the art will understand that the expression level required for a protein of interest to be therapeutically effective may vary depending on factors such as, but not limited to, the particular protein of interest and the subject being treated, and that an effective amount of protein can be readily determined by one of skill in the art using conventional methods known in the art without undue experimentation.

[0104] Methods for generating adeno-associated viruses Any method known in the art can be used to prepare the novel rAAV viral particles of the present disclosure. In some embodiments, the novel rAAV viral particles are produced in mammalian cells (e.g., HEK293). In some embodiments, the novel rAAV viral particles are produced in insect cells (e.g., Sf9). In some embodiments, the AAV viral particles are prepared by providing a host cell with an AAV genome vector containing a transgene along with a Rep gene and a Cap gene. In some embodiments, the AAV genome vector contains a transgene, an AAV Rep gene, and an AAV Cap gene. In some embodiments, the rAAV viral particles are prepared by providing a host cell with two or more vectors. For example, in some embodiments, an AAV genome vector containing a transgene is introduced (e.g., transfected or transduced) into a cell harboring a vector (e.g., a plasmid or baculovirus) containing an AAV Rep gene and an AAV Cap gene. In some embodiments, cells transfected or transduced with an AAV genome vector comprising a transgene, a vector (e.g., a plasmid or baculovirus) comprising the AAV Rep gene, and a vector (e.g., a plasmid or baculovirus) comprising the AAV Cap gene.

[0105] Methods for producing AAV viral particles are described, for example, in U.S. Pat. Nos. 6,204,059, 6,756,283, 6,258,595, 6,261,551, 6,270,996, 6,281,010, 6,365,394, 6,475,769, 6,482,634, 6,485,966, 6,943,019, 6,953,690, 7,022,519, 7,238,526, 7,291,498, and 7,491,508, 7,064,764, 7,194,191, 7,566,118, 7,666,120, 7,766,130, 7,776,140, ​​7,786,150, 7,866,160, 7,876,170, 7,876,180, 7,976,190, 7,976,190, 7,976,190, 7,976,150, 7,976,160, 7,976,170, 7,976,180, 7,976,19 ... S8137948, or International Publication No. 1996 / 039530, WO1998 / 010088, WO1999 / 014354, WO1999 / 015685, WO1999 / 047691, WO2000 / 055342, WO2000 / 075353, WO2001 / 023597, WO2015 / 191508, WO2019 / 217513, WO2018 / 022608, WO2019 / 222136, WO2020 / 232044, WO2019 / 222132, Methods In Molecular Biology, ed. Richard, Humana Press, NJ (1995), O'Reilly et al., Baculovirus Expression Vectors, A Laboratory Manual, Oxford University Press (1994), Samulski et al., J. Vir. 63:3822-8 (1989), Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88:4646-50 (1991), Ruffing et al., J. Vir. 66:6922-30 (1992), Kimbauer et al., Vir., 219:37-44 (1996), Zhao et al., Vir. 272:382-93 (2000), the contents of each of which are incorporated herein by reference in their entirety.

[0106] For example, cells such as insect cells, yeast cells, and mammalian cells (e.g., human cells or non-human mammalian cells) can produce rAAV. For example, cells can produce rAAV if they are provided with AAV helper functions, AAV non-helper functions, and nucleotide sequences that the cells use to produce an AAV vector genome. In various embodiments, the AAV helper functions, AAV non-helper functions, and nucleotide sequences that the cells use to produce rAAV are provided by vectors delivered to the cells, for example, via transfection with a transfection reagent, via transduction / infection with other recombinant viruses, by integrating the nucleotide sequences into the genome of the cells, or by other methods. Examples of such cells include mammalian cell lines such as HEK293, HeLa, CHO, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT1080, A549, Cos-7, ARPE-19, and MRC-5 cells. In other examples, the insect cell line used can be derived from Spodoptera frugiperda such as Sf9, SF21, SF900+, Drosophila cell lines, mosquito cell lines such as Aedes albopictus-derived cell lines, silkworm cell lines such as Bombyx mori cell lines, Trichopiusia ni cell lines such as High Five cells, or Lepidoptera cell lines such as Ascalapha odorata cell lines. Preferred insect cells are cells derived from insect species susceptible to baculovirus infection, including High Five, Sf9, Sf-RVN, Se301, SeIZD2109, SeUCR1, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38.

[0107] As previously described, the term "vector" is understood to refer to any genetic element, such as a plasmid, phage, transposon, cosmid, bacmid, miniplasmid (e.g., a plasmid lacking bacterial elements), doggybone DNA (e.g., a minimal closed linear construct), chromosome, virus, or virion (e.g., baculovirus), which, when associated with appropriate control elements, is capable of replicating and transferring genetic sequences between cells. As used herein, "insect cell-compatible vector" or "vector" refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector may be used as long as it is insect cell-compatible. While the vector may be integrated into the insect cell genome, the presence of the vector in the insect cell need not be permanent; transient episomal vectors are also included. The vector may be introduced by any known means, such as chemical treatment, electroporation, or infection of the cells. Baculovirus vectors and methods for their use are described in the above-cited references regarding molecular engineering of insect cells.

[0108] The vector from which the cell produces the rAAV vector genome further comprises a promoter and a restriction site downstream of the promoter, located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR, to allow insertion of a polynucleotide encoding one or more proteins of interest. The vector may also contain post-transcriptional regulatory elements downstream of the restriction site and upstream of the 3' AAV ITR. The viral construct may further comprise a polynucleotide inserted into the restriction site and operably linked to the promoter, the polynucleotide comprising the coding region for the protein of interest.

[0109] The term "AAV helper" refers to AAV-derived coding sequences that can be expressed to provide AAV gene products that in turn function in trans for productive AAV replication. AAV helper functions therefore include both major AAV open reading frames (ORFs), rep and cap. Rep expression products have been shown to have many functions, including, inter alia: recognition, binding, and nicking of AAV origins of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. Capsid (Cap) expression products supply necessary packaging functions. AAV helper functions are used herein to complement AAV functions in trans that are missing from the AAV vector genome.

[0110] In various embodiments, vectors providing AAV helper functions include nucleotide sequences encoding capsid proteins or Rep proteins. Any AAV serotype (AAV1 (NCBI Reference SEQ ID NO: / Genbank Accession No. NC_002077.1), AAV2 (NCBI Reference SEQ ID NO: / Genbank Accession No. NC_001401.2), AAV3 (NCBI Reference SEQ ID NO: / Genbank Accession No. NC_001729.1), AAV3B (NCBI Reference SEQ ID NO: / Genbank Accession No. AF028705.1), AAV4 (NCBI Reference SEQ ID NO: / Genbank Accession No. NC_001829.1), AAV5 (NCBI Reference SEQ ID NO: / Genbank Accession No. NC_006152.1), AAV6 (NCBI Reference SEQ ID NO: / Genbank Accession No. AF028704.1), AAV7 (NCBI Reference SEQ ID NO: / Genbank Accession No. AF028704.1), AAV8 (NCBI Reference SEQ ID NO: / Genbank Accession No. AF028704.1), AAV9 (NCBI Reference SEQ ID NO: / Genbank Accession No. AF028704.1), AAV10 (NCBI Reference SEQ ID NO: / Genbank Accession No. AF028704.1), AAV11 (NCBI Reference SEQ ID NO: / Genbank Accession No. AF028704.1), AAV12 (NCBI Reference SEQ ID NO: / Genbank Accession No. AF028704.1), AAV13 (NCBI Reference SEQ ID NO: / Genbank Accession No. AF028704.1), AAV14 (NCBI Reference SEQ ID NO: / Genbank Accession No. AF028704.1), AAV15 (NCBI Reference SEQ ID NO: / Genbank .1), AAV7 (NCBI Reference SEQ ID NO. / Genbank Accession No. NC_006260.1), AAV8 (NCBI Reference SEQ ID NO. / Genbank Accession No. NC_006261.1), AAV9 (NCBI Reference SEQ ID NO. / Genbank Accession No. AX753250.1), AAV10 (NCBI Reference SEQ ID NO. / Genbank Accession No. AY631965.1), AAV11 (NCBI Reference SEQ ID NO. / Genbank Accession No. AY631966.1), AAV12 (NCBI Reference SEQ ID NO. / Genbank Accession No. DQ813647.1), AAV13 (NCBI Reference SEQ ID NO. / Genbank Accession No. EU285562.1), is AAV-rh.10 (AAVrh10), AAV-DJ (AAVDJ), AAV-DJ8 (AAVDJ8), AAV-1, AAV-2, AAV-2G9, AA V-3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV-5, AAV-6, AAV6.1, AAV6.2, AAV6.1.2, AA V-7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AA V9.61, AAV9.68, AAV9.84, AAV9.9, AAV-10, AAV-11, AAV-12, 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、AAV223.2 23.4、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、AA V3.1 / 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 / rh.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、AAVF3、AAVF5、AAVH2、AAVrh.72、AA Vhu.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. hu.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、AAVhu.24、AA Vhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、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. u.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. hu.14 / 9、AAVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、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.37、AAVrh.37、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. h.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh.8R、AAVrh.8R. A586R mutation、AAVrh8R R533A mutation、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhEr1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、AAVhEr1.36、AAVhEr r2.29、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-L K04、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 SM10-2、AAVシャッフル100-1、AAVシャッフル100-3、AAVシャッフル100-7、AAVシャッフル10-6、AAVシャッフル10-8、AAV SM10-1、AAV SM10-8、AAV SM100-3 SM100-10、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 AAV (ttAAV), UPENN AAV10, or Japanese AAV10 serotype, AAV_po.6, AAV_po., AAV_po.5, AAV_LK03, AAV_ra.1, AAV_bat_YNM, AAV_bat_Brazil, AAV_mo.1, AAV_Chicken_DA-1, or AAV_Mouse_NY1, Bba21, Bba26, Bba27, Bba29, Bba30, Bba31, Bba32, Bba33, Bba34, Bba35, Bba36, Bba37, Bba38, Bba41, Bba42, Bba43, Bba44, Bce14, Bce15, Bce16, Bce17, Bce18, Bce20, Bce35, Bce36, Bce39, Bce40, Bce41, Bce42, Bce43, Bce44, Bce45, Bce46, Bey20, Bey22, Bey23, Bma42, Bma43, Bpo1, Bpo2, Bpo3, Bpo4, Bpo6, Bpo8, Bpo13, Bpo AAV-rh10, AAV-rh39, AAV-rh43, AAVanc80L65, or any variant thereof) may be used herein to generate a recombinant AAV. Exemplary capsids are also provided in International Application Nos. 2018 / 022608 and WO2019 / 222136, which are incorporated herein in their entireties. Each NCBI reference sequence or Genbank accession number provided above is also incorporated herein by reference. In some embodiments, the AAV cap gene encodes a capsid from serotype 1, serotype 2, serotype 3, serotype 3B, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13, or a variant thereof.

[0111] For production, cells with AAV helper functions produce sufficient recombinant capsid proteins to form capsids. This includes at least the VP1 and VP3 proteins, but more typically includes all three VP1, VP2, and VP3 proteins, as found in naturally occurring AAV. The sequence of the capsid proteins determines the serotype of the AAV virions produced by the host cells. Capsids useful in the present invention include those derived from several AAV serotypes, including 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or mixed serotypes (see, e.g., U.S. Patent No. 8,318,480 for a disclosure of non-naturally occurring mixed serotypes). Capsid proteins may also be variants of naturally occurring VP1, VP2, and VP3, including mutated, chimeric, or shuffled proteins. The capsid protein may be of rh.10 or other subtypes within the various clades of AAV, the various clades and subtypes being disclosed, for example, in U.S. Patent No. 7,906,111. Due to the wide range of construct availability and extensive characterization, the exemplary AAV vectors disclosed below are derived from serotype 2. The construction and use of AAV vectors and AAV proteins of different serotypes are described in Chao et al., Mol. Ther. 2:619-623, 2000; Davidson et al., PNAS 97:3428-3432, 2000; Xiao et al., J. Virol. 72:2224-2232, 1998; Halbert et al., J. Virol. 74:1524-1532, 2000; Halbert et al., J. Virol. 75:6615-6624, 2001; and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, 2001.

[0112] In various embodiments, the nucleotide sequence encoding the VP protein can be operably linked to a suitable expression control sequence. In various embodiments, the nucleotide sequence encoding the Rep protein can be operably linked to a suitable expression control sequence, such as a eukaryotic promoter. For example, the nucleotide sequence can be operably linked to a eukaryotic promoter. In another example, the nucleotide sequence can be operably linked to a baculovirus promoter, such as the polyhedrin (Polh) promoter, ΔIE1 promoter, p5 promoter, p10 promoter, p40 promoter, metallothionein promoter, 39K promoter, p6.9 promoter, and orf46 promoter.

[0113] For production, cells with AAV helper functions produce Rep proteins to facilitate rAAV production. It has been found that infectious particles can be generated when at least one large Rep protein (Rep78 or Rep68) and at least one small Rep protein (Rep52 and Rep40) are expressed in cells. In certain embodiments, all four of Rep78, Rep68, Rep52, and Rep40 are expressed. Alternatively, Rep78 and Rep52, Rep78 and Rep40, Rep68 and Rep52, or Rep68 and Rep40 are expressed. The following example demonstrates the use of a Rep78 / Rep52 combination. The Rep proteins can be derived from AAV-2 or other serotypes. In various embodiments, the nucleotide sequence encoding the Rep protein can be operably linked to suitable expression control sequences. In various embodiments, the nucleotide sequence encoding the Rep protein can be operably linked to suitable expression control sequences, such as a eukaryotic promoter. For example, the nucleotide sequence can be operably linked to a eukaryotic promoter. In another example, the nucleotide sequence can be operably linked to a baculovirus promoter, such as the polyhedrin (Polh) promoter, ΔIE1 promoter, p5 promoter, p10 promoter, p40 promoter, metallothionein promoter, 39K promoter, p6.9 promoter, and orf46 promoter.

[0114] Cells with AAV helper functions can also produce assembly activating proteins (AAPs), which aid in capsid assembly. In various embodiments, the nucleotide sequence encoding the AAP can be operably linked to a suitable expression control sequence. For example, the nucleotide sequence can be operably linked to a eukaryotic promoter. In another example, the nucleotide sequence can be operably linked to a baculovirus promoter, such as the polyhedrin (Polh) promoter, ΔIE1 promoter, p5 promoter, p10 promoter, p40 promoter, metallothionein promoter, 39K promoter, p6.9 promoter, and orf46 promoter.

[0115] The term "non-AAV helper functions" refers to non-AAV-derived viral and / or cellular functions on which AAV depends for replication. Thus, this term refers to proteins and RNAs required for AAV replication, including those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (excluding herpes simplex virus type 1), and vaccinia virus.

[0116] The term "non-AAV helper function vector" generally refers to a nucleic acid molecule containing a nucleotide sequence that provides accessory functions. The accessory function vector can be transfected into a suitable host cell, and the vector can then support AAV virion production in the host cell. Explicitly excluded from this term are naturally occurring infectious virus particles, such as adenovirus, herpesvirus, or vaccinia virus particles. Thus, accessory function vectors can be in the form of plasmids, phages, transposons, or cosmids. In particular, it has been demonstrated that a full complement of adenovirus genes is not required for accessory helper function. For example, adenovirus mutants incapable of DNA replication and late gene synthesis have been shown to be permissive for AAV replication. Ito et al. (1970) J. Gen. Virol. 9:243; Ishibashi et al. (1971) Virology 45:317. Similarly, mutants in the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing accessory functions. Carter et al., (1983) Virology 126:505. However, adenoviruses deleted in the E1 region or deleted in the E4 region cannot support AAV replication. Thus, the E1A and E4 regions are likely required, directly or indirectly, for AAV replication. Laughlin et al., (1982) J. Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983) Virology 126:505. Other characterized Ad mutants include:E1B (Laughlin et al. (1982), supra, Janik et al. (1981), supra, Ostrove et al., (1980) Virology 104:502); E2A (Handa et al., (1975) J. Gen. Virol. 29:239, Strauss et al., (1976) J. Virol. 17:140, Myers et al., (1980) J. Virol. 35:665, Jay et al., (1981) Proc. Natl. Acad. Sci. USA78: 2927, Myers et al., (1981) J. Biol. Chem. 256: 567), E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P.Tijssen ed., 1990), E3 (Carter et al. (1983), supra), and E4 (Carter et al. (1983), supra; Carter (1995)). Studies of accessory functions provided by adenoviruses with mutations in the E1B coding region have yielded conflicting results, although Samulski et al. (1988) J. Virol. 62:206-210 recently reported that E1B55k, but not E1B19k, is required for AAV virion production. In addition, WO 97 / 17458 and Matshushita et al. (1998) Gene Therapy 5:938-945 describe accessory function vectors encoding various Ad genes. A particularly preferred accessory function vector comprises an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus E2A 72 kD coding region, an adenovirus E1A coding region, and an adenovirus E1B region lacking an intact E1B55k coding region. Such vectors are described in WO 01 / 83797.

[0117] Host cells commonly used for the production of rAAV viral particles include, but are not limited to, HEK293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines described in U.S. Patent No. 6,156,303, U.S. Patent No. 5,387,484, U.S. Patent No. 5,741,683, U.S. Patent No. 5,691,176, and U.S. Patent No. 5,688,676, U.S. Patent Application Publication No. 2002 / 0081721, and International Patent Publication Nos. 2000 / 047757, WO2000 / 024916, and WO1996 / 017947, the contents of each of which are incorporated by reference in their entirety. In some embodiments, the HEK293 cells may be HEK-293T cells. Other examples of mammalian cells that can be used to produce AAV viral particles include A549, WEH1, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, W138, Saos, C2C12, L cells, HT1080, HepG2, and primary mammalian fibroblasts, hepatocytes, and myoblasts. In some embodiments, host cells used to produce AAV viral particles are derived from mammalian species, including, but not limited to, humans, monkeys, mice, rats, rabbits, and hamsters. In some embodiments, host cells used to produce AAV viral particles are derived from cell types, including, but not limited to, fibroblasts, hepatocytes, tumor cells, cell line-transformed cells, and the like. The use of insect cells for the expression of heterologous proteins is well documented, as are methods for introducing nucleic acids, e.g., vectors, e.g., insect cell-compatible vectors, into such cells and methods for maintaining such cells in culture.(For example, METHODS IN MOLECULAR BIOLOGY, ed. Richard, Humana Press, NJ (1995), O'Reilly et al., BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994), Samulski et al. al., J. Vir. (1989) vol. 63, pp. 3822-3828, Kajigaya et al., Proc. Nat'l. Acad. Sci. USA (1991) vol. 88, pp. 4646-4650, Ruffing et al., J. Vir. (1992) vol. 66, pp. 6922-6930, Kirnbauer et al., Vir. (1996) vol. 219, pp. 37-44, Zhao et al. (See, e.g., W. et al., Vir. (2000) vol. 272, pp. 382-393, and U.S. Patent No. 6,204,059.) Examples of insect cell lines that can be used include Spodoptera frugiperda, e.g., Sf9, Sf21, Sf900+, Drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus-derived cell lines, silkworm cell lines, e.g., Bombyx mori cell lines, Trichoplusia ni cell lines, e.g., High Five cell lines, or Lepidoptera cell lines, e.g., Ascalapha odorata cell lines. Exemplary insect cells are cells from insect species susceptible to baculovirus infection, including High Five, Sf9, Sf-RVN, Se301, SeIZD2109, SeUCR1, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5, and Ao38.

[0118] In some embodiments, the novel rAAV viral particles are produced in insect cells. Growth conditions for insect cells in culture and production of heterologous products in insect cells in culture are well known in the art; see U.S. Patent No. 6,204,059, the contents of which are incorporated herein by reference in their entirety.

[0119] In various embodiments, insect cells containing vectors for rAAV production are provided. Recombinant baculoviruses (rBVs) containing nucleotide sequences for rAAV production can be used to deliver these nucleotide sequences into insect cells for rAAV production. Baculoviruses, such as rBVs, are enveloped DNA viruses of arthropods, two of which are well-known expression vectors for producing recombinant proteins in cell culture. Baculoviruses have circular, double-stranded genomes (80-200 kbp) that can be engineered to enable delivery of large genome contents to specific cells. Viruses used as vectors are typically Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) or Bombyx mori nuclear polyhedrosis virus (Bm-NPV) (Katou, Yasuhiro, et al., Virology 404.2 (2010):204-214). Baculoviruses are commonly used to infect insect cells for recombinant protein expression.In particular, the expression of heterologous genes in insects has been described, for example, in U.S. Pat. No. 4,745,051; Friesen, P.D., and L.K. Miller., Current Topics in Microbiology and Immunology 131 (1986): 31-49; EP 127839; EP 155476; Vlak, Just M., et al., Journal of General Virology 69.4 (1988): 765-776; Miller, Lois K., Annual Reviews in Microbiology 42.1 (1988): 177-199; Carbonell, Luis F., et al., Gene 73.2 (1988): 409-418; Maeda, Susumu, et al., Nature 315.6020 (1985): 592-594; Lebacq-Verheyden, Anne-Marie, et al., Molecular and Cellular biology 8.8 (1988): 3129-3135, Smith, Gale E., et al., Proceedings of the National Academy of Sciences 82.24 (1985): 8404-8408, Miyajima, Atsushi, et al., Gene 58.2-3 (1987): 273-281, and Martin, Brian M., et al., DNA 7.2 (1988): 99-106.Numerous baculovirus strains and variants that can be used for protein production, as well as corresponding permissive insect host cells, are described in Luckow, Verne A., and Max D. Summers., Bio / technology 6.1 (1988): 47-55; Miller et al. (1986) Genetic Engineering, Principles and Methods, Vol. 8 (eds. J. Setlow and A. Hollaender), Plenum Press, NY, pp. 277-298, 1986); Maeda, Susumu, et al., Nature 315.6020 (1985): 592-594; and McKenna, Kevin A., Huazhu Hong, and Robert R. Granados., Journal of Invertebrate Pathology 71.1 (1998): 82-90.

[0120] To generate recombinant baculovirus (rBV), a donor vector and bacmid or a transcription vector and linear baculovirus DNA are used. Bacmids are propagated as large plasmids in bacteria such as Escherichia coli. When transfected into insect cells, bacmids generate baculovirus. Traditional baculovirus generation, such as Invitrogen's Bac-to-Bac system, generates recombinant baculovirus by site-specific transposition in E. coli. High molecular weight bacmid DNA is then isolated and transfected into Sf9 or Sf21 cells, from which recombinant baculovirus is isolated and amplified.

[0121] Insect cells can be separately transfected with bacmids carrying the nucleotide sequence for the rAAV vector genome or carrying nucleotide sequences that provide AAV helper functions to generate rBVs, which are then used to coinfect naive insect cells to generate rAAV.

[0122] In various embodiments, the transfected cells are cultured for about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, about 144 hours, about 168 hours, about 192 hours, about 216 hours, about 240 hours, or a time between any of these two time points after transfection.

[0123] As mentioned above, increased concentrations of therapeutically effective rAAV particles are economically feasible amounts that can be processed by ZUC each time. In one example, the process of various embodiments can enable the production of higher concentrations of rAAV, allowing larger cell culture volumes to be used for rAAV production. For example, host cells capable of producing rAAV can be cultured in volumes of at least 5 milliliters (mL), at least 10 mL, at least 20 mL, at least 50 mL, at least 100 mL, at least 500 mL, at least 1 liter (L), at least 10 L, at least 50 L, at least 100 L, at least 250 L, at least 500 L, at least 1000 L, at least 1500 L, at least 2000 L, or at least 2500 L. Culturing can also be carried out in roller tubes, shake flasks, or bioreactors.

[0124] Clarification: A number of methods can be used to remove rAAV virions from cultured host cells. In one example, the cells can be lysed and the virus purified. Alternatively, the virus can be expressed in the supernatant. rAAV virions can be purified by centrifugation, filtration, tangential flow filtration, chromatography, or a combination thereof.

[0125] In one example of such a method, insect cells are resuspended in lysis buffer (20 mM Tris-Cl pH=8, 150 mM NaCl, 0.5% deoxychloate) and lysed using glass beads. The lysate is treated with benzonase (Sigma, St. Louis, Mo.), centrifuged at 4000 g, and the supernatant is chromatographed on a Streamline HE column (Pharmacia), Phenyl Sepharose, and POROS HE (Potter et al., Methods Enzymol 346:413-30, 2002).

[0126] Encapsidation / infectivity: To assess encapsidation of vector genomes, purified AAV virions can be treated with nucleases to degrade any non-encapsidated DNA. Encapsidated DNA is protected from nucleases and is therefore detectable after nuclease treatment. The following example shows vector genomes that survive benzonase treatment, as determined by Southern blotting.

[0127] To evaluate virion performance, purified rAAV virions are used to infect HEK293 cells in culture or injected into mouse skeletal muscle, and their infectivity is assessed by scoring cells expressing GFP. If the payload gene is not optically accessible, other detection techniques, such as Western blotting, immunoassays, PCR, reverse transcription-PCR, or functional assays, can be used to assess infectivity. Example 3 of U.S. Patent Publication No. 2015 / 0071883 uses immunoassays and clotting assays to evaluate transduction of Rag2 mice with factor VIII-expressing rAAV. Therefore, measures of infectivity may vary depending on the payload gene and model system.

[0128] Generally, a virion contains 10 7The virions are infectious such that when HEK293 cells are incubated in the presence of a virion solution containing (10^7, 1E07) viral genomes, the foreign gene is expressed in detectable amounts by the cells. Alternatively, the virions are infectious such that when HEK293 cells are incubated in the presence of a virion solution containing 10^6 viral genomes, the foreign gene is expressed in detectable amounts by the cells.

[0129] Formulations: Various formulations of rAAV are known in the art. Purified rAAV can be diluted or dialyzed in saline, optionally containing buffers, carriers, and / or stabilizers. Known AAV formulations include those using polaxamer, PEG, sugars, polyhydric alcohols, or polyvalent ion salts. See, e.g., U.S. Patent Nos. 8,852,607 and 7,704,721. An exemplary formulation is 1.38 mg / ml sodium phosphate monobasic monohydrate, 1.42 mg / ml sodium phosphate dibasic (dry), 8.18 mg / ml sodium chloride, 20 mg / ml mannitol, and 2.0 mg / ml poloxamer 188 (Pluronic F-68), pH 7.4.

[0130] In other examples, the rAAV pharmaceutical formulations of the present invention comprise one or more pharmaceutically acceptable excipients to provide the formulation with advantageous properties for storage and / or administration to a subject for treatment. In certain embodiments, the pharmaceutical formulations of the present invention can be stored at temperatures below 65°C for at least two weeks, preferably at least four weeks, more preferably at least six weeks, and even more preferably at least about eight weeks without a detectable change in stability. In this context, the term "stable" means that the rAAV present in the formulation essentially retains its physical stability, chemical stability, and / or biological activity during storage. In certain embodiments of the present invention, the recombinant AAV virus present in the pharmaceutical formulation retains at least about 80% of its biological activity in human patients, more preferably at least about 85%, 90%, 95%, 98%, or 99% of its biological activity in human patients, during storage at -65°C for a specified period of time.

[0131] In various examples, formulations comprising rAAV further comprise one or more buffering agents. Other buffering agents are disclosed in Sek, D. "Breaking old habits: moving away from commonly used buffers in pharmaceuticals," European Pharmaceutical Review 3 (2012). For example, formulations of the invention comprise dibasic sodium phosphate at a concentration of about 0.1 mg / ml to about 3 mg / ml, about 0.5 mg / ml to about 2.5 mg / ml, about 1 mg / ml to about 2 mg / ml, or about 1.4 mg / ml to about 1.6 mg / ml. In particularly preferred embodiments, rAAV formulations of the invention comprise dibasic (dry) sodium phosphate at about 1.42 mg / ml. Another buffering agent that may find use in the rAAV formulations of the invention is monobasic sodium phosphate monohydrate, which in some embodiments finds use at a concentration of about 0.1 mg / ml to about 3 mg / ml, about 0.5 mg / ml to about 2.5 mg / ml, about 1 mg / ml to about 2 mg / ml, or about 1.3 mg / ml to about 1.5 mg / ml. In a particularly preferred embodiment, the rAAV formulations of the invention comprise about 1.38 mg / ml of monobasic sodium phosphate monohydrate. In an even more particularly preferred embodiment of the invention, the rAAV formulations of the invention comprise about 1.42 mg / ml of dibasic sodium phosphate and about 1.38 mg / ml of monobasic sodium phosphate monohydrate.

[0132] In another aspect, the rAAV formulations of the invention may include one or more isotonicity agents, such as sodium chloride, preferably at a concentration of about 1 mg / ml to about 20 mg / ml, e.g., about 1 mg / ml to about 10 mg / ml, about 5 mg / ml to about 15 mg / ml, or about 8 mg / ml to about 20 mg / ml. In a particularly preferred embodiment, the formulations of the invention contain about 8.18 mg / ml of sodium chloride. Other buffers and isotonicity agents known in the art are suitable and routinely available for use in the formulations of the present disclosure.

[0133] In another aspect, the rAAV formulations of the invention may contain one or more bulking agents. Exemplary bulking agents include, but are not limited to, mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24). In certain preferred embodiments, the formulations of the invention contain mannitol, which may be present in an amount of about 5 mg / ml to about 40 mg / ml, or about 10 mg / ml to about 30 mg / ml, or about 15 mg / ml to about 25 mg / ml. In particularly preferred embodiments, mannitol is present at a concentration of about 20 mg / ml.

[0134] In yet another embodiment, the rAAV formulations of the invention may include one or more surfactants, which may be non-ionic surfactants.

[0135] Other aspects and advantages of the present disclosure will be understood in light of the following illustrative examples.

[0136] Exemplary Embodiments of the Invention Example 1 - Effect of light capsids on infectivity and transgene expression As previously described, the production of therapeutically effective rAAV particles is not a completely efficient process. AAV production results in a mixture of therapeutically effective rAAV particles, therapeutically ineffective rAAV particles, and production impurities (e.g., low-molecular-weight DNA and small nucleotides, exogenous high-molecular-weight DNA, buffer components, etc.). Therapeutically effective rAAV particles can infect cells such that the infected cells express (e.g., by transcription and / or translation) elements of interest (e.g., nucleotide sequences, proteins, etc.). To this extent, therapeutically effective rAAV particles can include AAV particles with capsids or vg with different properties. For example, therapeutically effective rAAV particles can have capsids with different post-translational modifications. In other examples, therapeutically effective rAAV particles can have vg with different sizes / lengths, plus or minus strand sequences, different flip / flop inverted terminal repeat (ITR) configurations, different numbers of ITRs, or truncations. Therapeutically effective rAAV particles are also referred to as "heavy," "full," or "partial" capsids. Therapeutically ineffective rAAV particles are unable to infect cells, or cells infected with therapeutically ineffective rAAV particles are unable to express (e.g., by transcription and / or translation) elements of interest (e.g., nucleotide sequences, proteins, etc.). Therapeutically ineffective rAAV particles may contribute to reduced efficacy per unit dose of capsid and may increase the risk of an immune response due to the increased number of foreign proteins required to be introduced into a patient for an effective amount of heavy / full capsid. Therapeutically ineffective rAAV particles may include AAV particles with capsids or vg having different properties, referred to as empty or light capsids. For example, empty capsids have no vg or have an unquantifiable vg concentration. Empty or light capsids may also have different capsid properties. Without being bound to any particular theory, heavy / full / partially full capsids differ from light or empty capsids in their charge and / or density.

[0137] Figures 1A and 1B show exemplary particle profiles of AAV preparations using analytical ultracentrifugation. As shown in Figure 1B, it can be seen that some low molecular weight impurities may be present, but the major impurities are approximately 4% empty capsids and approximately 6-7% aggregates. Therapeutically effective capsids from the preparations comprise approximately 62% full / heavy capsids and approximately 25% partially full / partial capsids.

[0138] Figures 2A, 2B, 3A, and 3B show the effect of the presence of light particles on transgene expression of a gene of interest. HepG2 cells were infected with therapeutically effective rAAV particles (i.e., heavy capsids) carrying a transgene for gene of interest #1 (GOI1) and a known concentration of therapeutically ineffective rAAV particles (i.e., light capsids). As shown in Figures 2A, 2B, 3A, and 3B, increasing the concentration of light capsids reduced transgene expression in HepG2 cells. Figure 3B highlights that when light capsids account for approximately 50% of the capsids infecting the cells, the relative potency decreased by approximately 48%. Thus, the presence of light particles reduces the transgene expression and potency of heavy particles in HepG2 cells.

[0139] Figures 4, 5A, 5B, 6A, 6B, 7, 8A, 8B, 9A, 9B, 10, 11A, 11B, 11C, 11D, 12A, and 12B show that light capsids can bind to HepG2 cells, enter HepG2 cells, and enter the nuclei of HepG2 cells. In this study, heavy capsids were labeled with either a green-fluorescent cyanine 3 (Cy3) dye or a red-fluorescent cyanine 5 (Cy5) dye. Light capsids were also labeled with Cy3 or Cy5. Specifically, Cy3 / Cy5 NHS esters were used to label the primary amines on the heavy / light capsids to obtain stable conjugation.

[0140] Labeled particles were added to HepG2 cells. Specifically, prior to AAV transduction, HepG2 cells were seeded on a cell imaging plate. The cells were incubated with labeled AAV at 4°C for 1 hour to prevent internalization by binding to cell surface receptors and inhibiting endocytosis. The cells were then incubated at 37°C for 4–8 hours to allow heavy and light capsids to transduce HepG2 cells. The cells were then washed with CMEM and PBS, fixed with 4% PFA for 10 minutes, washed three more times with PBS, and mounted with antifade buffer for confocal microscopy.

[0141] FIG. 4 shows a control without cyanine dye, which shows no strong signal, while FIGS. 5A and 5B show Cy3- and Cy5-labeled light particles, respectively, bound to HepG2 cell surface receptors.

[0142] 6A and 6B show that Cy3-labeled (left panel) and Cy5-labeled (right panel) light particles are detected in the nuclei of HepG2 cells after 8 hours of incubation at 37°C.

[0143] FIG. 7 shows Cy5-labeled heavy particles bound to HepG2 cell surface receptors.

[0144] 8A, 8B, 9A, and 9B show that both Cy3-labeled light particles and Cy5-labeled heavy particles bind to HepG2 cell surface receptors.

[0145] FIG. 10 shows that Cy3-labeled light particles and Cy5-labeled heavy particles are detected in the nuclei of HepG2 cells after 8 hours of incubation at 37°C.

[0146] Figures 11A, 11B, 11C, and 11D show that both Cy3-labeled light particles and Cy5-labeled heavy particles bind to HepG2 cell surface receptors after 1 hour at 4°C. Figures 11A, 11B, and 11C show that after 1 hour at 4°C, red indicates Cy5-labeled heavy particles and green indicates C3-labeled light particles. Figure 11D shows binding of heavy and light particles in the nuclei of HepG2 cells after 8 hours at 37°C.

[0147] As noted above, Figure 11D shows the binding of heavy and light particles in the nuclei of HepG2 cells after 8 hours at 37°C. Figure 11D also shows that Cy3-labeled light particles and Cy5-labeled heavy particles are detected in the nuclei of HepG2 cells after 8 hours of incubation at 37°C. As shown in Figures 12A and 12B, the highlighted areas (see boxes and arrows) show colocalization of Cy3 and Cy5 dyes, which may indicate that heavy and light particles use the same cellular machinery. This colocalization may explain the reduced efficacy caused by the presence of light capsids and further substantiate the desire to obtain AAV preparations that are free or substantially free of light capsids, i.e., that contain more than 99%, preferably more than 99.5%, of light capsids.

[0148] Considering the adverse effects of light and empty capsids, their removal is important for enhancing the efficacy of AAV gene therapy agents.

[0149] Example 2 - Purification of AAV capsids encoding gene of interest #1, gene of interest #2, and gene of interest #3 The following examples disclose the production of rAAV carrying either GOI1, gene of interest #2 (GOI2), or gene of interest #3 (GOI3). GOI1 has a polynucleotide size of less than 5.5 Kb. GOI2 has a polynucleotide size of less than 6 Kb. GOI3 has a polynucleotide size of less than 5.5 Kb and is different from GOI1. rAAV carrying GOI1 and GOI2 were pseudotyped with AAV5 capsids. rAAV carrying GOI3 was pseudotyped with AAV9 capsids.

[0150] All downstream columns and TFF operations are carried out at ambient temperature, with the intermediate pool held at chilled temperatures if extended hold times are required.

[0151] As starting material, cell-free culture medium containing AAV capsids carrying GOI1, GOI2, or GO3 ("harvested pool material") was used.

[0152] As shown in Figure 13, processing 10 of the collected pooled material involves purifying 100 capsids from the collected pooled material and processing the capsids through AEX200, TFF300, ZUC400, and TFF500 to prepare a final preparation of capsid 600. Processing 11 of the collected pooled material containing rAAV with GOI1 pseudotyped with an AAV5 capsid involves purifying 110 capsids from the collected pooled material and processing the capsids through AEX210, TFF310, ZUC410, and TFF510 to prepare a final preparation of capsid 610. Processing 12 of the collected pooled material containing rAAV with GOI2 pseudotyped with AAV5 capsids involves purifying 120 capsids from the collected pooled material and processing the capsids through AEX220, TFF320, ZUC420, and TFF520 to prepare a final preparation of capsid 620. Processing 13 of the collected pooled material containing rAAV with GOI3 pseudotyped with AAV9 capsids involves purifying 130 capsids from the collected pooled material and processing the capsids through AEX230, TFF330, ZUC430, and TFF530 to prepare a final preparation of capsid 620.

[0153] In steps 100, 101, 102, and 103 of Figure 13, rAAV is purified from the collected pool material for subsequent AEX and ZUC processing. For example, purification steps 110 and 120 involve processing the collected pool material for GOI1 and GOI2 using AVB immunochromatography for affinity purification of AAV5 capsids. In another example, purification step 130 involves processing the collected pool material for GOI3 using column immunochromatography for affinity purification of AAV9 capsids. Affinity column-purified AAV capsids are used for AEX and ZUC processing.

[0154] As shown in step 200 of Figures 14 and 13, the isolated capsids, comprising a mixture of heavy, partial, light, and empty AAV capsids, are subjected to anion exchange chromatography (AEX) using an AEX column, a polymeric, strong, or weak anion exchange column (AEX column). The AEX step 200 includes steps 201 to equilibrate the column, 202 to load the column with collected pool material, 203 to wash the column to remove impurities, and 204 to elute and isolate the AAV capsids from the AEX column. In the example for GOI1, the AEX step 210 includes steps 211 to equilibrate the column, 212 to load the column with collected pool material, 213 to wash the column to remove impurities, and 214 to elute and isolate the AAV capsids from the AEX column. In the example for GOI2, the AEX step 220 includes steps of equilibrating the column 221, loading the column with collected pool material 222, washing the column to remove impurities 223, and eluting and isolating the AAV capsids from the AEX column 224. In the example for GOI3, the AEX step 230 includes steps of equilibrating the column 231, loading the column with collected pool material 232, washing the column to remove impurities 233, and eluting and isolating the AAV capsids from the AEX column 234.

[0155] We analyzed the zeta potentials of the following capsids at different pH levels: heavy capsids for GOI1, GOI2, and GOI3, light capsids extracted after AEX elutions 204, 214, 224, and 234, and ZUC-treated capsids from steps 400, 410, 420, and 430. As shown in Figure 18 for GOI2, we found that there is a light capsid population that can be removed using AEX because the net negative charge of these light capsids is lower than that of heavy capsids. This light capsid population also cannot be detected by typical 260 nm / 280 nm absorbance measurements and requires more precise measurements (i.e., size exclusion chromatography) for detection.

[0156] Buffers and solutions used in AEX separations are known in the art, and examples of such buffers include AEX equilibration buffers having a conductivity of less than 1 mS / cm or between 1 and 7 mS / cm and a pH in the range of 7 to 10, AEX wash buffers having a conductivity in the range of 4 to 7 mS / cm and a pH in the range of 7 to 9, AEX elution buffers having a conductivity in the range of 5 to 10 mS / cm and a pH in the range of 7 to 9, AEX strip buffers having a conductivity in the range of 53.2 to 70.1 mS / cm, and AEX elution pool conditioning buffers having a pH in the range of 6 to 9.

[0157] For the AEX column, the following parameters are used: a loading capacity ranging from 0.1 x 10e16 to 10 x 10e16 vg / L, a column with a strong anion exchange resin and a bed height ranging from 7 to 15 cm, and a flow rate ranging from 50 to 160 cm / hr.

[0158] The isolated capsids are filtered through a 0.22 micron (μm) filter before and after AEX treatment.

[0159] Prior to the AEX separation step, the pH of the isolated capsids is adjusted with a conditioning buffer to a pH in the range of 7-9 and a conductivity below 3 mS / cm.

[0160] The AEX column is prepared with a fixed volume of AEX equilibration buffer. The post-column pH and conductivity are checked for a pH range of 7-10 and a conductivity of less than 1 mS / cm or 1-7 mS / cm.

[0161] The loading pool is applied and the column is washed with AEX equilibration buffer and AEX wash buffer. The absorbance of the eluted AEX wash buffer is 260 =A 280 Manually observe the column to determine when A 260 =A 280 If no crossover is observed, add additional AEX wash buffer to the AEX column.

[0162] AEX elution is achieved by adding a fixed volume of AEX elution buffer to the AEX column.

[0163] A of AEX processes 200, 210, 220, 230 for GOI1, GOI2, and GOI3 260 and A 280 The profile is visualized and analyzed. Figure 25A shows the AEX treatment profile for rAAV preparation for GOI2 during the wash, elution, and strip steps. 260 and A 280 Show the profile.

[0164] The pH of the elution pool was adjusted to 6-9 using a fixed volume of AEX elution pool adjustment buffer.

[0165] Generally, vg and capsid (cp) titers can be assessed by any method suitable for measuring vg and capsid, respectively. For example, quantitative polymerase chain reaction (qPCR) can be used to measure vg titer, and enzyme-linked immunosorbent assay (ELISA) can be used to measure Cp titer. Alternatively, SEC (size exclusion chromatography)-HPLC can be used to measure vg and cp titers. In addition, RP (reverse phase)-HPLC assays can be used to evaluate the potential impact of process parameters on the VP ratio.

[0166] Quantitative polymerase chain reaction (qPCR) can be used to quantify Vg using a standard qPCR system, such as the Applied Biosystems 7500 Fast Real-Time PCR System. Alternatively, digital droplet PCR (ddPCR) can be used to quantify Vg. Primers and probes can be designed to target AAV DNA, allowing for its quantification as it accumulates during PCR. Examples of ddPCR are described in Pasi, K. John, et al., "Multiyear Follow-Up of AAV5-hFVIII-SQ Gene Therapy for Hemophilia A," New England Journal of Medicine 382.1 (2020): 29-40; Regan, John F., et al., "A Rapid Molecular Approach for Chromosomal Phasing," PloS one 10.3 (2015): e0118270; and Furuta-Hanawa, Birei, Teruhide Yamaguchi, and Eriko Uchida, "Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors," Human Gene Therapy Methods 30.4 (2019): 127-136. Other systems for vg quantification include SEC, SEC-HPLC, and size exchange chromatography multi-angle light scattering, all of which are described in WO2021 / 062164, which is incorporated by reference in its entirety.

[0167] Capsid ELISA (cp-ELISA) assays, for example, measure intact capsids using the AAV5 capsid ELISA method and may utilize commercially available kits (e.g., Progen PRAAV5). This kit ELISA utilizes a monoclonal antibody specific for a conformational epitope on assembled AAV5 or other capsids. Capsids can be captured on a plate-bound monoclonal antibody, followed by binding of a detection antibody. Assay signals can be generated by adding conjugated streptavidin peroxidase, followed by the addition of a colorimetric TMB substrate solution and sulfuric acid to terminate the reaction. The titer of a test sample is interpolated from a four-parameter calibration curve of target capsid standards. Another system for quantifying capsid titer is SEC-MALS, which is described in WO 2021 / 062164.

[0168] Heavy and partial AAV capsids can be measured using techniques known in the art. For example, the total number of capsids can be measured using cp-ELISA with antibodies specific for the capsid protein. Heavy and partial capsids can be measured using qPCR to measure the vector genomes present.

[0169] Particle distribution profiles from preparations of GOI1, GOI2, and GOI3 after anion exchange chromatography 200, 210, 220, and 230 were analyzed. Figure 19 shows that treatment of the rAAV preparation of GOI2 with AEX reduced the concentration of light and empty capsids to 9.8%. Cryo-EM images from preparations of GOI1, GOI2, and GOI3 after anion exchange chromatography 200, 210, 220, and 230 were also analyzed. As shown in Figure 20, capsid counts from cryo-EM images of the rAAV preparation for GOI2 after AEX treatment were 57.7% dense particles (i.e., heavy and partial capsids) and 42.3% "non-dense" particles (i.e., light capsids). Therefore, AEX does not remove all of the light capsids from the rAAV preparation.

[0170] The removal of contaminating virus by AEX treatment 200, 210, 220, 230 is also assessed by adding a known concentration of contaminating virus to isolated capsids and processing the composition through an AEX column. As shown in Table 1, AEX treatment of rAAV preparations of GOI 1 resulted in a Log reduction of at least 2. 10 This reduction reduced the concentration of contaminating viruses. [Table 1]

[0171] After AEX, the collected elution pool is subjected to a first tangential flow filtration (TFF) ultrafiltration / diafiltration (UF / DF) in step 300 of Figure 13. The AEX column elution pool was concentrated and diafiltered into TFF buffer in preparation for zonal ultracentrifugation. As shown in Figure 15, TFF UF / DF 300 includes step 302 of providing a sample 301 (e.g., eluate 204) and diafiltering / ultrafiltering the sample into permeate / filtrate 303 or retentate 302, which can be returned to sample 301. For GOI 1, TFF UF / DF 310 includes step 312 of providing a sample 311 (e.g., eluate 214) and diafiltering / ultrafiltering the sample into permeate / filtrate 313 or retentate 312, which can be returned to sample 311. For GOI2, TFF UF / DF 320 includes a step 322 of providing a sample 321 (e.g., eluate 224) and diafiltration / ultrafiltration of the sample into a permeate / filtrate 323 or a retentate 322 that can be returned to sample 321. For GOI3, TFF UF / DF 330 includes a step 332 of providing a sample 331 (e.g., eluate 234) and diafiltration / ultrafiltration of the sample into a permeate / filtrate 333 or a retentate 332 that can be returned to sample 331.

[0172] 0.1×10e17vg / m 2 ~10×10e17vg / m 2Loads ranging from 0.1 to 1.0 were loaded into the ultrafiltration chamber and diafiltered with a 100 kD molecular weight cut-off (MWCO) membrane, the process being controlled by TMP and cross-flow.

[0173] The diafiltered pool is subjected to 0.2 μm filtration using a 0.22 uM PVDF filter to generate the final TFF pool.

[0174] The TFF pool can optionally be frozen at 60° C. or below prior to ZUC processing if a longer holding time is desired.

[0175] After TFF UF / DF 300, and as shown in FIG. 16, the TFF pool is processed by ZUC 400, which includes step 401 of loading a zonal rotor with composition 302 and the components used to form the gradient (e.g., cesium chloride, etc.), step 402 of centrifuging the loaded rotor, and step 403 of collecting identified fractions. For GOI 1, ZUC 410 includes step 411 of loading a zonal rotor with composition 312 and the components used to form the gradient (e.g., cesium chloride, etc.), centrifuging the loaded rotor 412, and collecting identified fractions 413. For GOI 2, ZUC 420 includes step 421 of loading a zonal rotor with composition 322 and the components used to form the gradient (e.g., cesium chloride, etc.), step 422 of centrifuging the loaded rotor, and step 423 of collecting identified fractions. For GOI3, ZUC430 includes step 431 of filling a zonal rotor with composition 332 and components used to form the gradient (e.g., cesium chloride, etc.), step 432 of centrifuging the filled rotor, and step 433 of collecting identified fractions.

[0176] rAAV (i.e., GOI1, GOI2, and GOI3) preparations were treated with AEX 200, 210, 220, and 230 and ZUC 400, 410, 420, and 430. The fractions containing ZUC light capsids were collected and re-treated with AEX. 260and A 280 The profiles were visualized. As shown in Figures 27A, 27B, and 27C for the rAAV preparation of GOI2, a population of light capsids was also found to elute with the heavy capsids during AEX. Because qPCR showed that these capsids had no quantifiable encapsulated DNA, despite having the same net negative charge as the heavy capsids, this population of light capsids could be further separated from the full capsids using zonal ultracentrifugation (ZUC). In step 400 of Figure 13, the TFF product was subjected to ZUC by diluting the TFF product pool with TFF-A buffer and adjusting the final CsCl concentration to a range of 15% to 75% with CsCl buffer. An overlay solution with a CsCl concentration (15%-75% CsCl) less than the cesium chloride of the CsCl-adjusted product pool was pumped into the centrifuge rotor, followed by the CsCl-adjusted product pool and then a cushion solution with a CsCl concentration (15%-75% CsCl) less than the cesium chloride of the CsCl-adjusted product pool. During the spinning, a CsCl gradient was formed, fractionating the product forms at different densities. Fractions from the gradient were collected from the rotor. ZUC was performed at ambient temperature. The product pool was collected automatically based on in-line density measurements or by analysis of the collected fractions. ZUC was run using the following parameters:

[0177] For GOI 1, a ZUC rotor is loaded with a composition having a titer ranging from 0.1 x 10e17 vg / load to 10 x 10e17 vg / load. ZUC parameters include centrifuging the composition at speeds ranging from 80,000 G to 125,000 G for 14 to 16 hours at temperatures ranging from 10 °C to 36 °C.

[0178] For GOI 2, a ZUC rotor is loaded with a composition having a titer ranging from 0.1 x 10e16 vg / load to 10 x 10e16 vg / load or 0.5 x 10e16 vg / load to 50 x 10e16 vg / load. ZUC parameters include centrifuging the composition at a speed ranging from 10,000 rpm to 50,000 rpm for 15 to 25 hours at a temperature ranging from 10°C to 36°C. Alternatively, one ZUC parameter includes centrifuging the composition at a speed ranging from 50,000 G to 100,000 G.

[0179] The Vg concentration versus density of different collected fractions 403, 413, 423, and 433 of rAAV (i.e., GOI1, GOI2, and GOI3) processed with ZUC400, 410, 420, and 430 was determined and analyzed. Figure 21 shows the increasing concentration of Vg up to fraction 22, with fractions 18-29 identified as having increasing concentrations of heavy and partial capsids for rAAV preparations of GOI2. Note that fractions 18-29 had densities ranging from greater than 1.30 g / mL to less than or equal to 1.45 g / mL. Particle distribution profiles from different collected fractions 403, 413, 423, and 433 of rAAV (i.e., GOI1, GOI2, and GOI3) processed with ZUC400, 410, 420, and 430 were also analyzed. Figure 22 shows that treatment of the rAAV preparation with ZUC reduced the light capsid concentration to 1.1%.

[0180] rAAV after ZUC treatment (i.e., GOI1, GOI2, and GOI3) were analyzed by Western blot, alkaline agarose gel, and cryo-electron microscopy. Figures 23A and 23B show that for rAAV preparations of GOI2, high-density fractions 18–24 have a significantly higher concentration of capsids with larger genome sizes (i.e., heavy and partial capsids) than lower-density fractions 10–16. As shown in Figure 24, capsid counts from cryo-electron microscopy images of rAAV preparations for GOI2 after ZUC treatment were 85.7% high-density particles (i.e., heavy and partial capsids) and 14.3% "non-high-density" particles (i.e., light capsids). Therefore, ZUC alone does not remove all of the light capsids from the rAAV preparations.

[0181] The Vg concentrations of different collected fractions 403, 413, 423, and 433 of rAAV (i.e., GOI1, GOI2, and GOI3) were treated with ZUC400, 410, 420, and 430 and determined by qPCR, ddPCR, SEC, SEC-HPLC, or SEC-MALS. The capsid titers of different collected fractions 403, 413, 423, and 433 of rAAV (i.e., GOI1, GOI2, and GOI3) were treated with ZUC400, 410, 420, and 430 and determined by cp-ELISA or SEC-MALS. For the rAAV preparation of GOI2, Figure 25B shows the capsid titer of each fraction as determined by cp-ELISA and the vg titer of each fraction as determined by qPCR. As shown in Figure 25B, fractions 15-26 have larger capsid and vg titers compared to the other fractions. The capsid titers of rAAV (i.e., GOI1, GOI2, and GOI3) treated with or without AEX 200, 210, 220, and 230 were determined by cp-ELISA. Figure 26 also shows that AEX substantially reduces light and empty capsids, such that ZUC treatment does not overload the cells with light and empty capsids.

[0182] Table 2 shows the characteristics of rAAV preparations of GOI2 after ZUC treatment at different titer loads. [Table 2]

[0183] Table 3 shows the characteristics of different rAAV preparations of GOI2 after ZUC treatment at different titer loads. [Table 3]

[0184] Analysis of the reduction of contaminating viruses by ZUC treatment is also evaluated for GOI 1, GOI 2, and GOI 3. In the case of baculovirus, ZUC treatment reduces the contaminating viruses by a Log reduction of 2 or more. 10 The concentrations (e.g., 2.09 and 2.46) were decreased.

[0185] In step 500 of Figure 13, the ZUC elution pool is concentrated and diafiltered into stabilized TFF-B buffer. As shown in Figure 17, TFF UF / DF 500 includes step 502 of providing a sample 501 (e.g., collected fraction 403) and diafiltering / ultrafiltering the sample into permeate / filtrate 503 or retentate 502, which can be returned to sample 501. For GOI 1, TFF UF / DF 310 includes step 512 of providing a sample 511 (e.g., collected fraction 413) and diafiltering / ultrafiltering the sample into permeate / filtrate 513 or retentate 512, which can be returned to sample 511. For GOI2, TFF UF / DF 520 includes steps 522 of providing a sample 521 (e.g., collected fraction 423) and diafiltration / ultrafiltration of the sample into a permeate / filtrate 523 or a retentate 522 that can be returned to sample 521. For GOI3, TFF UF / DF 530 includes steps 532 of providing a sample 531 (e.g., collected fraction 433) and diafiltration / ultrafiltration of the sample into a permeate / filtrate 533 or a retentate 532 that can be returned to sample 531. 2 ~10×10e17vg / m 2Loads ranging from 0.1 to 1.0 were loaded into the ultrafiltration chamber and diafiltered with a 100 kD molecular weight cut-off (MWCO) membrane, the process being controlled by TMP and cross-flow.

[0186] In step 600 of Figure 13, the combined TFF product pool material is diluted to a predetermined concentration in formulation buffer and filtered through a 0.2 μm filter.

[0187] Combined AEX (Figure 25A) and ZUC (Figure 25B) treatment of the rAAV preparation of GOI2 yields an essentially pure preparation of heavy and partial capsids with a Cp / Vg ratio of approximately 1.0.

[0188] Analytical ultracentrifugation analysis was performed on the rAAV product treated with AEX and ZUC for the rAAV preparation of GOI 1. The capsid composition of the treated rAAV product was 0% light capsids, 3.9% capsid aggregates, 7.68% intermediate or partially complete capsids, and 88.4% heavy capsids.

[0189] Example 3 - Removal of AAV production impurities: rAAV associated with Rep proteins and deamidated capsids It was discovered that Rep proteins, particularly Rep78 and Rep68, remain associated with the concentration of rAAV after production. This Rep78 / Rep68-associated rAAV may be an impurity unable to infect cells, or cells infected with Rep78 / Rep68-associated rAAV may be unable to express (e.g., by transcription and / or translation) the desired element (e.g., nucleotide sequence, protein, etc.). Rep78 / Rep68-associated rAAV may contribute to reduced efficacy per unit dose of capsid and increase the risk of immune response due to the necessary increase in foreign protein introduced into patients for an effective amount of heavy / full / partially complete capsid. Therefore, AEX and ZUC treatment reduce the concentration of Rep78 / Rep68-associated rAAV. Rep78 / Rep68-associated rAAV in GOI1, GOI2, and GOI3 preparations was quantified by liquid chromatography-mass spectrometry (LC-MS). This assay accurately measures Rep78 / Rep68 concentrations. Capsids are isolated after AVB, AEX, and ZUC treatments. The capsids are denatured to release viral proteins and digest them into peptides prior to LC-MS analysis. Peptides derived from Rep78 / Rep68 proteins are separated on LC, and signals from multiple fragments of the targeting peptide are analyzed by a triple quadrupole mass spectrometer.

[0190] As shown in Figure 28, during AVB immunochromatography for AAV5 capsids, the concentration of rAAV associated with Rep proteins elutes with therapeutically effective rAAV. For GOI1, AEX treatment substantially reduced the concentration of rAAV associated with Rep proteins, but a portion of rAAV associated with Rep proteins eluted with therapeutically effective rAAV. After AEX, ZUC treatment further reduced the concentration of rAAV associated with Rep proteins. For example, AEX and ZUC treatment removed the concentration of rAAV associated with Rep proteins, such that the final composition containing therapeutically effective rAAV was substantially devoid of rAAV associated with Rep proteins.

[0191] As shown in Figure 29 for GOI1, rAAV associated with Rep proteins remains in the AEX column after the washing and elution steps, and rAAV associated with Rep proteins exits the AEX column only when the column is regenerated.

[0192] As shown in Figure 30 for GOI1, ZUC treatment can also purify therapeutically effective rAAV from rAAV associated with Rep proteins by separating the therapeutically effective rAAV from rAAV associated with Rep proteins. Notably, the isolated "pooled" fraction contains little or no rAAV associated with Rep proteins, while the "post-pooled" fraction contains a sufficiently high concentration of rAAV associated with Rep proteins. Note also that the rAAV associated with Rep proteins is empty or light capsid.

[0193] Capsid deamidation has been shown to reduce rAAV infectivity for expression of transgenes delivered by rAAV (Giles, April R., et al. Molecular Therapy 26.12 (2018): 2848-2862) and Frederick, Amy, et al. Human Gene Therapy 31.13-14 (2020): 756-774). Therefore, rAAV with deamidated capsids (or deamidated rAAV) may be impurities that cannot infect cells, or cells infected with deamidated rAAV may be unable to express (e.g., by transcription and / or translation) the desired element (e.g., a nucleotide sequence, a protein, etc.). Deamidated rAAV may also contribute to reduced efficacy per unit dose of capsid and increase the risk of immune response due to the required increase in the amount of foreign protein introduced into the patient for an effective amount of heavy / full / partially complete capsid. Therefore, AEX and ZUC treatment reduces the concentration of rAAV with deamidated capsids. The deamidation level of VP1 protein at the N-termini of GOI1, GOI2, and GOI3 is quantified by liquid chromatography-mass spectrometry (LC-MS). This assay accurately measures the deamidation rate in the N-terminal region of VP1. Capsids are isolated after AVB, AEX, and ZUC treatment. The capsids are denatured to release viral proteins and digested into peptides prior to LC-MS analysis. The unmodified and deamidated forms of the target VP1 N-terminal peptide containing the targeted deamidation site are separated on LC, and the signals obtained from multiple fragments of the target peptide are analyzed by a triple quadrupole mass spectrometer.

[0194] As shown in Figure 31 for GOI1, the concentration of deamidated rAAV elutes with the therapeutically effective rAAV during AVB immunochromatography for AAV5 capsids. AEX treatment substantially reduced the concentration of deamidated rAAV, but some deamidated rAAV eluted with the therapeutically effective rAAV. After AEX, ZUC treatment further reduced the concentration of deamidated rAAV. For example, AEX and ZUC treatment eliminated the concentration of deamidated rAAV such that the final composition containing therapeutically effective rAAV was substantially devoid of deamidated rAAV.

[0195] As shown in Figure 32 for GOI1, deamidated rAAV is removed during the wash steps and exits the AEX column only when the column is regenerated. Note also that the AEX eluate has a substantially reduced concentration of deamidated rAAV.

[0196] As shown in Figure 33 for GOI1, ZUC treatment also separates therapeutically effective rAAV from deamidated rAAV, so that the therapeutically effective rAAV can be purified deamidated rAAV. Notably, the isolated "pooled" fraction contains a reduced concentration of deamidated rAAV, while the "post-pooled" fraction contains a sufficiently high concentration of deamidated rAAV. Note also that deamidated rAAV is an empty or light capsid.

[0197] While exemplary embodiments have been described above, it is not intended that these embodiments describe all possible forms of the invention. Rather, the words used herein are words of description rather than limitation, and it is understood that various changes can be made without departing from the spirit and scope of the invention. Furthermore, features of various embodiments may be combined to form further embodiments of the invention. The present disclosure includes the following embodiments. [1] A method for purifying therapeutically effective recombinant adeno-associated virus (rAAV) particles, the method comprising: providing a composition comprising therapeutically effective rAAV particles and AAV-produced impurities, wherein a first portion of the AAV-produced impurities comprises impurities having a different net charge than the AAV particles and a second portion of the AAV-produced impurities comprises impurities having a different density than the AAV particles; removing the first portion from the composition by anion exchange chromatography; removing the second portion from the composition by zonal ultracentrifugation; The method, wherein after anion exchange chromatography and zonal ultracentrifugation, the composition is substantially devoid of AAV-produced impurities. [2] The method of embodiment 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is at least 95% pure from AAV-produced impurities. [3] The method of embodiment 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is at least 99% pure from AAV-produced impurities. [4] The method of embodiment 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is 99+% pure from AAV-produced impurities. [5] The method of embodiment 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is at least 95% pure from therapeutically ineffective rAAV particles. [6] The method of embodiment 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is at least 99% pure from therapeutically ineffective rAAV particles. [7] The method of embodiment 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is 99+% pure from therapeutically ineffective rAAV particles. [8] The method of embodiment 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation contains no detectable therapeutically ineffective rAAV particles. [9] The method of any one of embodiments 5 to 8, wherein the therapeutically ineffective rAAV particles comprise a capsid associated with Rep proteins.

[10] The method of embodiment 9, wherein the capsid associated with the Rep protein capsid comprises capsid and Rep protein separated from therapeutically effective rAAV particles by anion exchange chromatography.

[11] The method of embodiment 9, wherein the capsid associated with the Rep protein capsid comprises capsid and Rep protein separated from therapeutically effective rAAV particles by zonal ultracentrifugation.

[12] The method of embodiment 9, wherein the capsid associated with a Rep protein capsid comprises a capsid having an associated Rep protein.

[13] The method of any one of embodiments 5 to 8, wherein the therapeutically ineffective rAAV particles comprise a capsid having one or more VP1, VP2, or VP3 capsid proteins with deamidated amino acids.

[14] The method of any one of embodiments 5 to 8, wherein the therapeutically ineffective rAAV particles comprise capsids that lack a vector genome or encapsulate undetectable concentrations of nucleotides.

[15] The method of any one of embodiments 5 to 8, wherein the therapeutically ineffective rAAV particles comprise capsids having a vector genome with one or more sizes that are insufficient for a cell infected by the capsid to produce a therapeutically effective nucleotide sequence.

[16] The method of any one of embodiments 5 to 8, wherein the therapeutically ineffective rAAV particles comprise a capsid having a vector genome with one or more sizes that reduce expression of an element by a cell infected with the capsid compared to expression of the element by a cell infected under the same conditions but lacking infection by the capsid, and a therapeutically effective rAAV encoding the element.

[17] The method of embodiment 1, wherein the anion exchange chromatography is a polystyrene / divinylbenzene resin.

[18] The method of embodiment 17, wherein the resin is modified with quaternary ammonium groups.

[19] The method of embodiment 1, wherein the zonal ultracentrifugation uses a cesium chloride gradient.

[20] The zonal ultracentrifugation in a cesium chloride gradient adding a concentration of cesium chloride to the eluate; overlaying a first cesium chloride solution in a rotating centrifuge rotor, the first cesium chloride solution having a cesium chloride concentration less than the cesium chloride concentration of the eluate; adding the eluate from the anion exchange ion chromatography; adding a second cesium chloride solution, the second cesium chloride solution having a cesium chloride concentration greater than the cesium chloride concentration of the eluate; centrifuging the rotating centrifuge rotor to form a density gradient within the eluate; collecting fractions from said density gradient.

[21] A method for purifying therapeutically effective recombinant adeno-associated virus (rAAV) particles, the method comprising: providing a composition comprising therapeutically active and therapeutically inactive rAAV particles; removing at least a portion of the therapeutically ineffective rAAV particles from the composition by anion exchange chromatography; and processing the composition by zonal ultracentrifugation; The method, wherein after anion exchange chromatography and zonal ultracentrifugation, the composition is substantially devoid of therapeutically ineffective rAAV particles.

[22] The method of embodiment 21, wherein the removing step allows for subsequent processing of the composition by zonal ultracentrifugation.

[23] The method of embodiment 21, wherein the removing step enables the composition of the providing step to have a greater amount of therapeutically effective rAAV particles that are processed by zonal ultracentrifugation.

[24] The method of embodiment 21, wherein the removal step removes at least 0.1% of the therapeutically ineffective rAAV particles from the composition.

[25] The method of embodiment 21, wherein the removal step removes at least 50% of the therapeutically ineffective rAAV particles from the composition.

[26] The method of embodiment 21, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is at least 95% pure from therapeutically ineffective rAAV particles.

[27] The method of embodiment 21, wherein the composition after anion exchange chromatography and zonal ultracentrifugation contains no detectable therapeutically ineffective rAAV particles.

[28] The method of any one of embodiments 21 to 27, wherein the therapeutically ineffective rAAV particles comprise a capsid associated with Rep proteins.

[29] The method of embodiment 28, wherein the capsid associated with the Rep protein capsid comprises capsid and Rep protein separated from therapeutically effective rAAV particles by anion exchange chromatography.

[30] The method of embodiment 28, wherein the capsid associated with the Rep protein capsid comprises capsid and Rep protein separated from therapeutically effective rAAV particles by zonal ultracentrifugation.

[31] The method of embodiment 28, wherein the capsid associated with a Rep protein capsid comprises a capsid having an associated Rep protein.

[32] The method of any one of embodiments 21 to 27, wherein the therapeutically ineffective rAAV particles comprise capsids having VP1, VP2, or VP3 capsid proteins with deamidated amino acids.

[33] The method of any one of embodiments 21 to 27, wherein the therapeutically ineffective rAAV particles comprise capsids that lack a vector genome or encapsulate undetectable concentrations of nucleotides.

[34] The method of any one of embodiments 21 to 27, wherein the therapeutically ineffective rAAV particles comprise capsids having a vector genome with one or more sizes that are insufficient for a cell infected by the capsid to produce a therapeutically effective nucleotide sequence. 35. The method of any one of embodiments 21 to 27, wherein the therapeutically ineffective rAAV particles comprise a capsid having a vector genome with one or more sizes that reduce expression of an element by a cell infected with the capsid compared to expression of the element by a cell infected under the same conditions but lacking infection by the capsid, and a therapeutically effective rAAV encoding the element.

Claims

1. 1. A method for purifying therapeutically effective recombinant adeno-associated virus (rAAV) particles, said method comprising: providing a composition comprising therapeutically effective rAAV particles and AAV-produced impurities, wherein a first portion of the AAV-produced impurities comprises impurities having a different net charge than the AAV particles, and a second portion of the AAV-produced impurities comprises impurities having a different density than the AAV particles; removing the first portion from the composition by anion exchange chromatography, wherein therapeutically ineffective AAV particles pass through the anion exchange chromatography column in a wash buffer, and therapeutically effective AAV particles bind to the anion exchange chromatography column; removing the second portion from the composition by zonal ultracentrifugation; processing the composition via tangential flow filtration (TFF) between anion exchange chromatography and zonal ultracentrifugation; Including, The method, wherein after anion exchange chromatography and zonal ultracentrifugation, the composition is at least 95% pure from AAV-producing impurities.

2. 10. The method of claim 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is at least 99% pure from AAV-producing impurities.

3. 10. The method of claim 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is 99+% pure from AAV-produced impurities.

4. 10. The method of claim 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is at least 99% pure from therapeutically ineffective rAAV particles.

5. 10. The method of claim 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation is 99+% pure from therapeutically ineffective rAAV particles.

6. 2. The method of claim 1, wherein the composition after anion exchange chromatography and zonal ultracentrifugation contains no detectable therapeutically ineffective rAAV particles.

7. The method of any one of claims 4 to 6, wherein the therapeutically ineffective rAAV particles comprise a capsid associated with Rep proteins.

8. 8. The method of claim 7, wherein the capsid associated with a Rep protein capsid comprises capsid and Rep proteins separated from therapeutically effective rAAV particles by anion exchange chromatography.

9. 8. The method of claim 7, wherein the capsid associated with a Rep protein capsid comprises capsid and Rep proteins separated from therapeutically effective rAAV particles by zonal ultracentrifugation.

10. The method of claim 7 , wherein the capsid associated with a Rep protein capsid comprises a capsid having an associated Rep protein.

11. 7. The method of any one of claims 4 to 6, wherein the therapeutically ineffective rAAV particles comprise a capsid having one or more VP1, VP2, or VP3 capsid proteins with deamidated amino acids.

12. 7. The method of any one of claims 4 to 6, wherein the therapeutically ineffective rAAV particles comprise capsids that lack a vector genome or encapsulate undetectable concentrations of nucleotides.

13. 7. The method of any one of claims 4 to 6, wherein the therapeutically ineffective rAAV particles comprise capsids having a vector genome with one or more sizes insufficient for a cell infected by the capsid to produce a therapeutically effective nucleotide sequence.

14. 7. The method of any one of claims 4 to 6, wherein the therapeutically ineffective rAAV particles comprise capsids having a vector genome with one or more sizes that reduce expression of an element by a cell infected with a therapeutically effective rAAV encoding the element and the capsid, compared to expression of the element by a cell infected under the same conditions but lacking infection by the capsid.

15. 10. The method of claim 1, wherein the anion exchange chromatography is a polystyrene / divinylbenzene resin.

16. 10. The method of claim 1, wherein the zonal ultracentrifugation uses a cesium chloride gradient.

17. said zonal ultracentrifugation through a cesium chloride gradient comprising: adding a concentration of cesium chloride to the eluate; overlaying a first cesium chloride solution in a rotating centrifuge rotor, the first cesium chloride solution having a cesium chloride concentration less than the cesium chloride concentration of the eluate; adding the eluate from the anion exchange ion chromatography; adding a second cesium chloride solution, the second cesium chloride solution having a cesium chloride concentration greater than the cesium chloride concentration of the eluate; centrifuging the rotating centrifuge rotor to form a density gradient within the eluate; and collecting fractions from the density gradient.

18. 1. A method for purifying therapeutically effective recombinant adeno-associated virus (rAAV) particles, said method comprising: providing a composition comprising therapeutically active and therapeutically inactive rAAV particles; removing at least a portion of the therapeutically ineffective rAAV particles from the composition by anion exchange chromatography, wherein the therapeutically ineffective AAV particles pass through the anion exchange chromatography column in a wash buffer and the therapeutically effective AAV particles bind to the anion exchange chromatography column; processing said composition by zonal ultracentrifugation; processing the composition via tangential flow filtration (TFF) between anion exchange chromatography and zonal ultracentrifugation; Including, The method, wherein after anion exchange chromatography and zonal ultracentrifugation, the composition is at least 95% pure from therapeutically ineffective rAAV particles.

19. 20. The method of claim 18, wherein the anion exchange step allows for subsequent processing of the composition by zonal ultracentrifugation.

20. 20. The method of claim 18, wherein the anion exchange step enables the composition of the providing step to have a greater amount of therapeutically effective rAAV particles that are processed by zonal ultracentrifugation.

21. 19. The method of claim 18, wherein the anion exchange step removes at least 0.1% of the therapeutically ineffective rAAV particles from the composition.

22. 19. The method of claim 18, wherein the anion exchange step removes at least 50% of the therapeutically ineffective rAAV particles from the composition.

23. 19. The method of claim 18, wherein the composition after anion exchange chromatography and zonal ultracentrifugation contains no detectable therapeutically ineffective rAAV particles.

24. 24. The method of any one of claims 18 to 23, wherein the therapeutically ineffective rAAV particles comprise a capsid associated with Rep proteins.

25. 25. The method of claim 24, wherein the capsid associated with a Rep protein capsid comprises capsid and Rep proteins separated from therapeutically effective rAAV particles by anion exchange chromatography.

26. 25. The method of claim 24, wherein the capsid associated with a Rep protein capsid comprises capsid and Rep proteins separated from therapeutically effective rAAV particles by zonal ultracentrifugation.

27. 25. The method of claim 24, wherein the capsid associated with a Rep protein capsid comprises a capsid having an associated Rep protein.

28. 24. The method of any one of claims 18 to 23, wherein the therapeutically ineffective rAAV particles comprise capsids having VP1, VP2, or VP3 capsid proteins with deamidated amino acids.

29. 24. The method of any one of claims 18 to 23, wherein the therapeutically ineffective rAAV particles comprise capsids that lack a vector genome or encapsulate undetectable concentrations of nucleotides.

30. 24. The method of any one of claims 18 to 23, wherein the therapeutically ineffective rAAV particles comprise capsids having a vector genome with one or more sizes that are insufficient for a cell infected by the capsid to produce a therapeutically effective nucleotide sequence.

31. 24. The method of any one of claims 18-23, wherein the therapeutically ineffective rAAV particles comprise capsids having a vector genome with one or more sizes that reduce expression of an element by a cell infected with a therapeutically effective rAAV encoding the element and the capsid, compared to expression of the element by a cell infected under the same conditions but lacking infection by the capsid.

32. 1. A method for producing a composition comprising purified therapeutically effective recombinant adeno-associated virus (rAAV) particles, said method comprising: providing a composition comprising therapeutically effective rAAV particles and AAV-produced impurities, wherein a first portion of the AAV-produced impurities comprises impurities having a different net charge than the AAV particles, and a second portion of the AAV-produced impurities comprises impurities having a different density than the AAV particles; removing the first portion from the composition by anion exchange chromatography, wherein therapeutically ineffective AAV particles pass through the anion exchange chromatography column in a wash buffer, and therapeutically effective AAV particles bind to the anion exchange chromatography column; removing the second portion from the composition by zonal ultracentrifugation; processing the composition via tangential flow filtration (TFF) between anion exchange chromatography and zonal ultracentrifugation; Including, The method, wherein after anion exchange chromatography and zonal ultracentrifugation, the composition is at least 95% pure from AAV-produced impurities, and the therapeutically effective rAAV particles contained in the composition are purified.

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