Purified matrix containing AAV-bound polypeptide and method of use thereof

Abstract

An improved AAV purification method is needed. [Solution] This specification discloses a purified matrix containing an adeno-associated virus (AAV) conjugated polypeptide and a method for using the same.

Description

Technical Field

[0001] Cross - Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 081,405, filed on September 22, 2020, and U.S. Provisional Patent Application No. 62 / 978,616, filed on February 19, 2020. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0002] Description of Electronically Submitted Text Files

[0002] The contents of the text file electronically submitted with this specification are incorporated herein by reference in their entirety: a computer - readable copy of the sequence listing (file name: ISOL_003_02WO_SeqList_ST25.txt, recording date: February 19, 2021; file size: 15 megabytes).

[0003] Technical Field

[0003] This disclosure generally relates to compositions and methods for the purification of biopharmaceuticals. More specifically, this disclosure relates to a purification matrix comprising an adeno - associated virus binding polypeptide and methods of using the same.

Background Art

[0004] Background of the Invention

[0004] Adeno - associated virus (AAV) is a promising vehicle for the delivery of one or more therapeutic genes in gene therapy. AAV is a small, replication - defective DNA virus that has the ability to integrate into the genome of infected cells. AAV promotes the persistent expression of therapeutic genes and reduces the need for repeated administration of gene therapy vectors.

Summary of the Invention

Problems to be Solved by the Invention

[0005]

[0005] However, there are limitations to the methods for producing and purifying AAV, which make the widespread use of AAV in gene therapy difficult. The proliferation of AAV requires the use of helper viruses such as adenoviruses. Because helper viruses are required, the purification of AAV particles is complicated. Current AAV particle purification methods involve lysing AAV-infected cells using repeated freeze-thaw cycles, followed by fractionation of the cell lysate using density gradient centrifugation to obtain infectious AAV particles that are free of cellular contaminants and substantially free of helper viruses. Standard purification techniques generally result in very low yields of active (infectious) virus (0.3-5%). Furthermore, it is difficult to obtain an AAV composition that is completely free of helper viruses. Therefore, there is a need for improved AAV purification methods in the art. [Means for solving the problem]

[0006] Summary of the Invention

[0006] This disclosure provides a purified matrix containing an AAV-conjugated polypeptide and a method for using the same.

[0007]

[0007] In some embodiments, the disclosure provides a purified matrix comprising an AAV-binding polypeptide coupled to a polypeptide having phase behavior, wherein the AAV-binding polypeptide comprises the ectodomain of an AAV receptor (AAVR), or an AAV-binding fragment or derivative thereof. In some embodiments, the AAV-binding polypeptide is reversibly coupled to the polypeptide having phase behavior. In some embodiments, the AAV-binding polypeptide is covalently coupled to the polypeptide having phase behavior.

[0008]

[0008] In some embodiments, AAVR is human AAVR. In some embodiments, AAVR is monkey AAVR. In some embodiments, AAVR is orangutan AAVR. In some embodiments, AAVR is mouse AAVR. In some embodiments, AAVR is a derivative of any one of human, monkey, orangutan, or mouse AAVR.

[0009]

[0009] In some embodiments, AAVR is wild-type AAVR. In some embodiments, AAVR is mutant AAVR.

[0010]

[0010] In some embodiments, the AAV-binding polypeptide comprises one of the sequences of SEQ ID NOs: 29, 33, 52, or 64. In some embodiments, the AAV-binding polypeptide comprises one of the sequences of SEQ ID NOs: 29, 33, 52, or 64, which have up to 25 amino acid mutations.

[0011]

[0011] In some embodiments, the AAV-binding polypeptide comprises amino acids 411-499 of Sequence ID No. 35 having at least one, at least two, at least three, at least four, or at least five mutations. In some embodiments, each mutation is individually selected from the group consisting of V440H, S431H, Q432H, T434H, Y442H, I462H, D435H, D436H, K438H, and I439H.

[0012]

[0012] In some embodiments, the AAV-binding polypeptide comprises one of SEQ ID NOs: 28-32, 37-41, 43-47, and 52-86. In some embodiments, the AAV-binding polypeptide comprises at least two, at least three, at least four, or at least five of SEQ ID NOs: 28-32, 37-41, 43-47, and 52-86.

[0013]

[0013] In some embodiments, the AAV-binding polypeptide comprises a polycystic kidney disease 1 (PKD1) domain or a fragment thereof. In some embodiments, the AAV-binding polypeptide comprises a polycystic kidney disease 2 (PKD2) domain or a fragment thereof. In some embodiments, the AAV-binding polypeptide comprises a polycystic kidney disease 3 (PKD3) domain or a fragment thereof. In some embodiments, the AAV-binding polypeptide comprises a polycystic kidney disease 4 (PKD4) domain or a fragment thereof. In some embodiments, the AAV-binding polypeptide comprises a polycystic kidney disease 5 (PKD5) domain or a fragment thereof.

[0014]

[0014] In some embodiments, the AAV-binding polypeptide includes the sequence of SEQ ID NO: 29. In some embodiments, the AAV-binding polypeptide includes the sequence of SEQ ID NO: 29 in which at least one amino acid (i.e., a non-histidine amino acid) is mutated to histidine.

[0015]

[0015] In some embodiments, the disclosure provides a purified matrix comprising an AAV-binding polypeptide coupled to a support, wherein the AAV-binding polypeptide comprises the ectodomain of the AAV receptor (AAVR), or an AAV-binding fragment or derivative thereof. In some embodiments, the support comprises beads, resins, membranes, fibers, polymers, plates, or chips. In some embodiments, the support comprises beads comprising Sepharose, agarose, cellulose, polystyrene, polymethacrylate, and / or polyacrylamide. In some embodiments, the support comprises magnetic beads. In some embodiments, the support comprises a polymer. In some embodiments, the support comprises a synthetic polymer.

[0016]

[0016] In some embodiments, the AAV-conjugated polypeptide is reversibly coupled to a support. In some embodiments, the AAV-conjugated polypeptide is reversibly coupled to a polypeptide having phase behavior. In some embodiments, the AAV-conjugated polypeptide is covalently coupled to a support. In some embodiments, the AAV-conjugated polypeptide is covalently coupled to a polypeptide having phase behavior. In some embodiments, the AAV-conjugated polypeptide is non-covalently coupled to a support. In some embodiments, the AAV-conjugated polypeptide is non-covalently coupled to a polypeptide having phase behavior.

[0017]

[0017] In some embodiments, the AAV-conjugated polypeptide is coupled to a support via a linker. In some embodiments, the AAV-conjugated polypeptide is coupled to a polypeptide with phase behavior via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker includes a protease cleavage site. In some embodiments, the linker is a chemical linker.

[0018]

[0018] In some embodiments, the fusion protein comprises an AAV-binding polypeptide and a polypeptide having phase behavior.

[0019]

[0019] In some embodiments, the polypeptide having phase behavior is an elastin-like polypeptide. In some embodiments, the polypeptide having phase behavior is a resilin-like polypeptide.

[0020]

[0020] In some embodiments, the polypeptide exhibiting phase behavior comprises a pentapeptide repeat having the sequence (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 10), or a randomized scrambled analog thereof; where Xaa may be any amino acid other than proline. In some embodiments, polypeptides exhibiting phase behavior include (GRGDSPY)n (SEQ ID NO: 1); (GRGDSPH)n (SEQ ID NO: 2); (GRGDSPV)n (SEQ ID NO: 3); (GRGDSPYG)n (SEQ ID NO: 4); (RPLGYDS)n (SEQ ID NO: 5); (RPAGYDS)n (SEQ ID NO: 6); (GRGDSYP)n (SEQ ID NO: 7); (GRGDSPYQ)n (SEQ ID NO: 8); (GRGNSPYG)n (SEQ ID NO: 9); (GVGVP)n (SEQ ID NO: 11); (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12); (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 13); (GVGVPGWGVPGVGVPGWGVPGVGVP)m (SEQ ID NO: 14); (GVGVPGVGVPGVGVPGVGVPGVGVGVPGEGVPGFGVPGVGVP)m (SEQ ID NO: 15); The polypeptide comprises an amino acid sequence selected from (GVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)m (SEQ ID NO: 16); and (GAGVPGVGVPGAGVPGVGVPGAGVP)m (SEQ ID NO: 17); or a randomized scrambled analog thereof; where n is an integer in the range of 20 to 360; and m is an integer in the range of 4 to 25. In some embodiments, the polypeptide with phase behavior comprises the amino acid sequence (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 144) or (GVGVPGVGVPGLGVPGVGVPGVGVP)m (SEQ ID NO: 146), where m is an integer between 2 and 32.In some embodiments, the polypeptide exhibiting phase behavior comprises the amino acid sequence (GVGVPGAGVP)m (SEQ ID NO: 145), where m is an integer between 5 and 80. In some embodiments, the polypeptide exhibiting phase behavior comprises the amino acid sequence (GXGVP)m (SEQ ID NO: 147), where m is an integer between 10 and 160, and where X is independently selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine. In some embodiments, the phase-behaving polypeptide comprises an amino acid sequence selected from (GVGVP)m (SEQ ID NO: 143), (ZZPXXXXGZ)m (SEQ ID NO: 148), (ZZPXGZ)m (SEQ ID NO: 149), (ZZPXXGZ)m (SEQ ID NO: 150), or (ZZPXXXGZ)m (SEQ ID NO: 151), where m is an integer between 10 and 160, where X is any amino acid except proline or glycine if present, and where Z is any amino acid if present. In some embodiments, the phase-behaving polypeptide comprises the amino acid sequence (GRGDXPZX)m (SEQ ID NO: 152) or (XZPXDGRG)m (SEQ ID NO: 153), where X is glutamine or serine, Z is tyrosine or valine, and m is an integer between 10 and 160.

[0021]

[0021] In some embodiments, the purified matrix comprises an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 52, or SEQ ID NO: 64, and a polypeptide exhibiting phase behavior having the amino acid sequence of (GVGVPGLGVPGVGVPGLGVPGVGVP)m(SEQ ID NO: 12) (wherein m is 16). In some embodiments, the purified matrix comprises the amino acid sequence of SEQ ID NO: 87.

[0022]

[0022] In some embodiments, the present disclosure provides a method for purifying AAV, which includes contacting the AAV with a purification matrix described herein.

[0023]

[0023] In some embodiments, the AAV comprises a wild-type AAV capsid protein. In some embodiments, the AAV comprises a mutant AAV capsid protein. In some embodiments, the AAV comprises a capsid protein of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV or bovine AAV.

[0024]

[0024] In some embodiments, the AAV reversibly binds to the purification matrix to form an AAV-purification matrix complex. In some embodiments, the method comprises separating the AAV-purification matrix complex from one or more impurities. In some embodiments, the AAV-purification matrix complex is separated from one or more impurities by washing the AAV-purification matrix complex.

[0025]

[0025] In some embodiments, the method comprises eluting the AAV from the AAV-purification matrix complex. In some embodiments, the method comprises eluting the AAV by changing the pH of the composition comprising the AAV-purification matrix complex. In some embodiments, the method comprises eluting the AAV by changing the temperature of the composition comprising the AAV-purification matrix complex. In some embodiments, the method comprises eluting the AAV by changing the ionic strength of the composition comprising the AAV-purification matrix complex. In some embodiments, the method comprises eluting the AAV by adding a reducing agent to the composition comprising the AAV-purification matrix complex.

[0026]

[0026] In some embodiments, the method includes increasing the size of the AAV-purification matrix complex using a first environmental factor. In some embodiments, the first environmental factor includes one or more of: a.) a change in one or more of temperature, pH, salt concentration, or pressure; b.) the addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, enzymes, denaturants; or c.) the application of electromagnetic waves or sound waves.

[0027]

[0027] In some embodiments, the method includes separating the AAV-purification matrix complex from at least one impurity based on size. In some embodiments, the AAV-purification matrix complex is separated from at least one impurity based on diameter. In some embodiments, the AAV-purification matrix complex is separated from at least one impurity based on mass. In some embodiments, the separation based on size is performed using tangential flow filtration, analytical ultracentrifugation, membrane chromatography, high performance liquid chromatography, normal flow filtration, acoustic separation, centrifugation, countercurrent centrifugation, and high performance protein liquid chromatography.

[0028]

[0028] In some embodiments, a second environmental factor is used to elute the AAV from the AAV-purification matrix complex. In some embodiments, the second environmental factor includes one or more of: a change in one or more of temperature, pH, salt concentration, or pressure; the addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, denaturants, enzymes; or the application of electromagnetic waves or sound waves. In some embodiments, the method of the present disclosure is completed in about 2 hours to about 10 hours.

[0029]

[0029] In some embodiments, the purification method described herein is completed in about 4 hours to about 8 hours.

[0030]

[0030] In some embodiments, the composition comprises AAV particles purified by the method of the present disclosure. In some embodiments, the composition is free of impurities by at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0031]

[0031] In some embodiments, the AAV-binding polypeptide comprises one of the sequences of SEQ ID NOs. 28-47, 52, or 64, or one of the sequences of SEQ ID NOs. 28-47, 52, or 64 having at least one amino acid mutation.

[0032]

[0032] The Specified also provides nucleic acids encoding one or more AAV-binding polypeptides described herein.

[0033]

[0033] The Specified also provides vectors comprising nucleic acids encoding one or more AAV-binding polypeptides described herein.

[0034]

[0034] The Specified also provides compositions comprising an AAV-binding polypeptide disclosed herein, a nucleic acid encoding an AAV-binding polypeptide, and / or a vector comprising a nucleic acid encoding an AAV-binding polypeptide.

[0035]

[0035] The Specified also provides a kit comprising an AAV-binding polypeptide disclosed herein, a nucleic acid encoding an AAV-binding polypeptide, and / or a vector comprising a nucleic acid encoding an AAV-binding polypeptide.

[0036]

[0036] The Spectrum also provides a method for increasing the yield of AAV particles during their production, comprising culturing AAV-producing cells in the presence of a purified matrix. In some embodiments, at least about 10 μM of purified matrix is ​​present. In some embodiments, at least about 10 μM of purified matrix is ​​present. In some embodiments, the AAV particles are wild-type AAV particles of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32,33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the AAV particles are mutant AAV particles. In some embodiments, the AAV particles reversibly bind to the purified matrix to form an AAV-purified matrix complex.

[0037]

[0037] The Spectrum also provides a method for stabilizing AAV particles during their production, comprising culturing AAV-producing cells in the presence of a purified matrix. In some embodiments, at least about 10 μM of purified matrix is ​​present. In some embodiments, at least about 10 μM of purified matrix is ​​present. In some embodiments, the AAV particles are wild-type AAV particles of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32,33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the AAV particles are mutant AAV particles. In some embodiments, the AAV particles reversibly bind to the purified matrix to form an AAV-purified matrix complex.

[0038]

[0038] The Spectrum also provides a method for stabilizing AAV particles during their purification, which includes contacting the AAV particles with a purified matrix during their purification. In some embodiments, at least about 10 μM of purified matrix is ​​present during the purification of the AAV particles. In some embodiments, at least about 50 μM of purified matrix is ​​present during the purification of the AAV particles. In some embodiments, the AAV particles are wild-type AAV particles of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32,33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the AAV particles are mutant AAV particles. In some embodiments, the AAV particles reversibly bind to the purified matrix to form an AAV-purified matrix complex.

[0039]

[0039] The Spectrum also provides a method for stabilizing AAV particles during storage, which includes storing the AAV particles in the presence of a purified matrix. In some embodiments, at least about 10 μM of purified matrix is ​​present when purifying the AAV particles. In some embodiments, at least about 50 μM of purified matrix is ​​present when purifying the AAV particles. In some embodiments, the AAV particles are wild-type AAV particles of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32,33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the AAV particles are mutant AAV particles. In some embodiments, the AAV particles reversibly bind to the purified matrix to form an AAV-purified matrix complex.

[0040]

[0040] The Spectrum also provides a method for increasing the shelf life of AAV particles, which includes storing the AAV particles in the presence of a purified matrix. In some embodiments, at least about 10 μM of purified matrix is ​​present when purifying the AAV particles. In some embodiments, at least about 50 μM of purified matrix is ​​present when purifying the AAV particles. In some embodiments, the AAV particles are wild-type AAV particles of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32,33, AAVrh74, avian AAV, or bovine AAV. In some embodiments, the AAV particles are mutant AAV particles. In some embodiments, the AAV particles reversibly bind to the purified matrix to form an AAV-purified matrix complex.

[0041]

[0041] These and other embodiments will be described in detail in the following detailed description, examples, and claims. [Brief explanation of the drawing]

[0042] [Figure 1]

[0042] The percentage of AAV particles captured from the cell supernatant using purified matrix, as determined by quantitative polymerase chain reaction (qPCR), is shown. [Figure 2]

[0043] This shows the percentage of AAV particles eluted from the purified matrix using various elution conditions (e.g., a second environmental factor) as determined by qPCR. [Figure 3]

[0044] Images are provided of (1) a sample of unpurified cell supernatant and (2) a silver-stained gel containing a sample containing AAV particles purified by the method of this disclosure. In the sample containing AAV particles, bands for VP1, VP2, and VP3 proteins were observed at the expected sizes (i.e., 87 kDa, 72 kDa, and 62 kDa, respectively). [Figure 4]

[0045] Western blot images are provided, which show that the AAV VP1, VP2, and VP3 capsid proteins present in the cell supernatant were successfully captured and removed from the cell supernatant using the purified matrix described herein. [Figure 5A]

[0046] The images show photographs of cells infected with control AAV particles (Pos Ctrl), or AAV8 particles carrying the tdTomato transgene, either (i) not purified by the method of this disclosure (Pos Ctrl), or (ii) purified with the purification matrix of this disclosure (e.g., ViraTag®). Both control AAV and AAV purified with ViraTag® are infectious. Images labeled "Neg Ctrl" show cells not infected with any AAV particles. [Figure 5B]

[0047] The fluorescence intensity of cells infected with control AAV particles (Pos Ctrl) or AAV particles purified with a purified matrix as described herein (e.g., ViraTag®) is shown. Both types of AAV particles tested carried the tdTomato trans gene. [Figure 6]

[0048] This graph shows the tangential flow filtration flux and intermembrane pressure differential during the separation of the AAV9 purified matrix complex from impurities. The purified matrix can be efficiently purified of AAV9 particles by tangential flow filtration (TFF) in a concentrate-diafiltration-concentrate-diafiltration (CDCD) mode. This process is carried out in a permeation-controlled mode with a high flux and a low, stable intermembrane pressure differential (TMP). [Figure 7]

[0049] This is a Western blot, showing the presence or absence of AAV VP1, VP2, and VP3 proteins at various steps of the tangential flow filtration (TFF) process. The first lane, labeled SM (Starting Material), indicates the presence of AAV particles in the starting material. SM is the supernatant from cultured HEK293 cells producing a recombinant AAV9 vector packaging the tdTomato trans gene, treated with 10 U / mL benzonase and 0.01% pluronic acid. The second lane, labeled C(P), indicates the absence of AAV particles in the permeate. The third lane, labeled C(R), indicates the presence of AAV particles in the retention solution. The fourth lane, labeled W(P), indicates the absence of AAV particles in the washing solution containing the removed contaminants. The fifth lane, labeled E(P), shows the eluted AAV particles. [Figure 8]

[0050] This graph shows the quantification of contaminated host cell proteins (HCPs) in a composition containing an AAV-purified matrix complex during TFF concentration and diafiltration. The composition containing the AAV-purified matrix complex is referred to as SM or "starting material" and contains AAV particles of serotype AAV9. A 1 mL fraction is collected throughout the entire TFF concentration and diafiltration process. DV1, DV2, DV3-4, DV5-6, DV7-8, and DV9-10 refer to the fractions collected during the TFF concentration phase. W1-2, W3-4, and W5-6 refer to the fractions collected during the washing phase of diafiltration. EC, E1, E2, E3, and E4 refer to the fractions collected during the elution phase of diafiltration. [Figure 9]

[0051] This graph shows the concentration of double-stranded DNA (dsDNA) impurities in a composition containing AAV9 particles before purification by the purification matrix (referred to as the starting material (SM)) and after purification by the purification matrix (referred to as "eluted"). [Figure 10]

[0052] This graph shows the effect of AAV8 particle compositions on the "AAV capture efficiency" of the purified matrix. The compositions containing AAV8 particles included a composition referred to as "lysate," which was derived from the cell lysate of HEK293 cells producing AAV8 particles, and a composition referred to as "supernatant," which contained the culture medium recovered from HEK293 cells producing AAV8 particles. The compositions contained viral titers of 1 × 10⁷ virus particles per microliter (vp / uL) (referred to as "E7"), 1 × 10⁸ vp / uL, or 1 × 10¹⁰ vp / uL. The compositions were either clarified (+) or unclarified (-). The compositions were either exposed to nuclease (+) or unexposed to nuclease (-). AAV8 particles were captured from each composition using the purified matrix. The AAV capture efficiency for each sample was calculated using the following formula: 100 × (number of AAV8 particles captured by the purification matrix / number of AAV8 particles in the composition before purification). [Figure 11]

[0053] Images of silver-stained SDS-PAGE gels are provided, showing that purification of AAV8 particles with a purification matrix yields high-purity AAV8 particles, regardless of pretreatment with benzonase nuclease. (-) indicates that pretreatment with benzonase nuclease was not performed, and (+) indicates that pretreatment was performed. [Figure 12]

[0054] This graph shows the dsDNA concentration in AAV8 samples with and without benzonase nuclease pretreatment (+). When evaluated by the Quant-iT picogreen assay, the composition containing purified AAV8 eluted from the purified matrix had similar levels of dsDNA, regardless of whether pretreatment was performed or not. Lysified SM: Starting material containing clarified cell lysate; Capture: Supernatant from the purified matrix capture step; Elution: Supernatant from the purified matrix elution step. [Figure 13]

[0055] This figure shows the AAV capture efficiency after centrifugation at various speeds. It demonstrates that over 95% of AAV8 particles are captured when using centrifugal speeds of 500 relative centrifugal force (RCF) or higher, including 3500 and 16000 RCF. [Figure 14]

[0056] This shows a comparison of AAV2 titers produced by HEK293 cell cultures under standard conditions (control) and with purified matrix, as quantified by reverse-terminal repeat (ITR) quantitative polymerase chain reaction (qPCR). This data indicates that the titer can increase by more than 8% in the presence of purified matrix. Student's t-test, p=0.077. [Figure 15]

[0057] This shows the percentage change in total AAV8 capsid after repeated freeze-thaw cycles in a composition containing AAV8 particles and a purified matrix, and in a composition containing AAV8 particles and PBS (negative control). [Figure 16]

[0058] This graph shows the effect of the purified matrix concentration on the capture efficiency of AAV8 particles. The capture efficiency was calculated using the following formula: 100 × (number of AAV8 particles captured by the purified matrix / number of AAV8 particles in the composition before purification). [Modes for carrying out the invention]

[0043] definition

[0059] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context specifically indicates otherwise. For example, when we say “a protein,” it refers to one protein or a mixture of such proteins; when we say “the method,” it includes reference to equivalent steps and / or methods known to those skilled in the art.

[0044]

[0060] When used herein, the terms “about” or “approximately” when preceding a number indicate a range of ±10% of the value. For example, “about 100” includes 90 and 110.

[0045]

[0061] Furthermore, as used herein, “and / or” means and encompasses any possible combination of one or more of the related enumerated items, as well as the absence of any combination as interpreted otherwise ("or").

[0046]

[0062] Unless otherwise indicated in the context, it is specifically intended that the various features described herein may be used in any combination.

[0047]

[0063] Furthermore, this disclosure also intends that in some embodiments, any feature or combination of features shown herein may be excluded or omitted. For example, where it is indicated herein that a particular amino acid may be selected from A, G, I, L and / or V, the wording also indicates that the amino acid may be selected from any part of those amino acids, e.g., A, G, I or L; A, G, I or V; A or G; L only, etc., where each of such partial combinations is expressly indicated herein. Furthermore, the wording also indicates that one or more of the designated amino acids may be disclaimed. For example, in a detailed embodiment, the amino acid may be not A, G or I; not A; not G or V, etc., where each of such possible disclaimers is expressly indicated herein.

[0048]

[0064] Adeno-associated viruses (AAVs) are small, replication-deficient parvoviruses. As used herein, AAV can refer to any wild-type or mutant AAV of any one of the following serotypes: AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh8, AAVrh10, AAVrh74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAVs currently known or to be discovered. In some embodiments, AAVs may have a single-stranded genome or a double-stranded genome (e.g., self-complementary AAVs).

[0049]

[0065] An "AAV particle" typically comprises a capsid and a nucleic acid (e.g., a nucleic acid containing a trans gene) encapsulated within the capsid by a protein capsid. The "capsid" is a nearly spherical protein shell containing individual "capsid protein subunits" or "capsid proteins" (e.g., about 60 capsid protein subunits) associated and arranged in icosahedral symmetry with T=1. Thus, the capsid of an AAV vector described herein contains multiple capsid proteins. When an AAV particle is described as containing capsid proteins, it will be understood that the AAV particle contains a capsid, where the capsid contains one or more AAV capsid proteins. When an AAV particle is described as bound to a binding domain, it will be understood that the binding domain may be bound to one or more capsid proteins within the capsid. The terms "empty AAV particle" or "empty capsid" refer to an AAV particle or capsid that does not contain any expression cassette or vector genome or nucleic acid containing a trans gene.

[0050]

[0066] As used herein, the term “AAV sample” is used synonymously with “AAV composition” and refers to a composition containing AAV particles. In some embodiments, “AAV sample” refers to a composition containing a specific serotype of AAV. For example, “AAV8 sample” refers to a composition containing AAV8 particles.

[0051]

[0067] As used herein, the terms “contaminant” and “impurity” are used synonymously. A contaminant may refer to any substance undesirable in the purified composition. In some embodiments, a contaminant is any substance other than the biological product that is to be purified. Non-limiting examples of contaminants include, but are not limited to, solvents, proteins, peptides, carbohydrates, nucleic acids, viruses, cells (e.g., bacteria, yeast, or mammalian cells), lipids, or lipopolysaccharides. In some embodiments, a contaminant is an endotoxin or mycotoxin.

[0052]

[0068] As used herein, the term “fragment” includes, when it refers to a protein or polypeptide, a truncated form of the protein or polypeptide. For example, a fragment of AAVR may contain about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or about 99% of the amino acids of full-length AAVR.

[0053]

[0069] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used synonymously and refer to compounds containing amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be contained in a protein sequence. The term “peptide” may refer to a short chain of amino acids, including, for example, natural peptides, recombinant peptides, synthetic peptides, or combinations thereof. Proteins and peptides may include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins.

[0054]

[0070] A "polynucleotide" is a sequence of nucleotide bases and may be RNA, DNA, or a DNA-RNA hybrid sequence (including both naturally occurring and non-naturally occurring nucleotides). In some embodiments, the polynucleotide is either a single-stranded or double-stranded DNA sequence.

[0055]

[0071] As used herein, “isolating” or “purifying” (or grammatical equivalents) a virus particle means that the virus particle is separated at least partially from at least some of the other components in the starting material containing the virus particle (e.g., a cell lysate). In typical embodiments, the “isolated” or “purified” virus particle is concentrated at least about 10 times, about 100 times, about 1000 times, about 10,000 times, or more compared to the starting material.

[0056]

[0072] As used herein, the term “amino acid” encompasses any naturally occurring amino acid, its modified forms, and synthetic amino acids. Table 1 lists naturally occurring levorotatory (L-)amino acids.

[0057] [Table 1]

[0058]

[0073] Alternatively, the amino acid may be a modified amino acid residue (non-limiting examples are shown in Table 2), and / or an amino acid modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation).

[0059] [Table 2]

[0060] [Table 3]

[0061]

[0074] Furthermore, amino acids that do not exist naturally can be called "unnatural" amino acids.

[0062]

[0075] As used herein, the term “environmental factor” is any factor that alters one or more properties of a composition when applied to a composition containing a protein-based purification matrix. Non-limiting examples of environmental factors include changes in one or more of the following: temperature, pH, salt concentration, concentration of the purification matrix, concentration of the bioagent, or pressure; the addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, denaturants, reducing agents, or oxidizing agents; or the application of electromagnetic or sound waves.

[0063]

[0076] As used herein, the term “phase-behaving polypeptide” refers to any polypeptide capable of undergoing a phase transition. In some embodiments, such polypeptides undergo a phase transition due to the application of environmental factors. Exemplary phase-behaving polypeptides include elastin-like polypeptides (ELPs) and resilin-like polypeptides (RLPs).

[0064] As used herein, the term “fusion protein” refers to a polypeptide produced when two heterogeneous nucleotide sequences or fragments thereof, which encode two (or more) different polypeptides that are not found fused together in nature, are fused together in the correct translational reading frame.

[0065] Purified matrix containing AAV-bound polypeptide.

[0077] This disclosure provides a purification matrix comprising recombinant adeno-associated virus (AAV) conjugated polypeptide. In some embodiments, this disclosure provides a method for purifying adeno-associated virus (AAV) particles, comprising contacting the AAV particles with the purification matrix described herein. In some embodiments, the method described herein produces high-titer AAV particles and removes one or more contaminants from a composition containing the AAV particles.

[0066] Adeno-associated virus (AAV)

[0078] In some embodiments, the purification matrix described herein is used for the purification of AAV particles. AAV belongs to the genus Dependoparvovirus, which is part of the family Parvoviridae. AAV has a linear single-stranded DNA (ssDNA) genome of approximately 4.7 kilobases with two 145-nucleotide reverse-ended repeats (ITRs) at its ends. AAV does not encode polymerase and relies on cellular polymerase for genome replication.

[0067]

[0079] AAV can replicate as a lytic virus or be integrated into host cell DNA and maintained as a provirus. Under certain conditions, AAV can replicate even without a helper virus, but efficient replication requires co-infection with a helper virus such as adenovirus, cytomegalovirus, Epstein-Barr virus, or vaccinia virus.

[0068]

[0080] The compositions and methods disclosed herein may be used to purify wild-type or mutant AAV particles of any one of the following serotypes: AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh8, AAVrh10, AAVrh74, AAVhu.68, bird AAV, cattle AAV, dog AAV, horse AAV, sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, or any other currently known or subsequently discovered AAV.

[0069] AAV-bound polypeptide

[0081] In some embodiments, the disclosure provides a purified matrix containing an AAV-binding polypeptide. In some embodiments, the AAV-binding polypeptide contains the ectodomain of the AAV receptor (AAVR), or an AAV-binding fragment or derivative thereof.

[0070]

[0082] AAVR, also known as KIAA0319L (see, for example, Uniprot accession number Q8IZA0), is a 150 kDa glycoprotein that binds to the capsids of several AAV serotypes, including AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9. The ectodomain of AAVR contains eight cysteine ​​motifs (MANEC) at the N-terminus and five immunoglobulin domains known as polycystic kidney disease (PKD) domains. AAV particles bind to the PKD domains to facilitate transduction. Therefore, in some embodiments, the AAV-binding polypeptides described herein contain 1, 2, 3, 4, or 5 PKD domains, or fragments or derivatives thereof.

[0071]

[0083] In some embodiments, the AAV-conjugated polypeptide comprises human AAVR (SEQ ID NO: 35), mouse AAVR (SEQ ID NO: 36), or orangutan AAVR (SEQ ID NO: 42), or a fragment or derivative thereof. In some embodiments, the AAV-conjugated polypeptide comprises a sequence having at least 90% or at least 95% identity with any one of SEQ ID NOs: 35, 36, or 42. In some embodiments, the AAV-conjugated polypeptide comprises the ectodomain of AAVR, or a fragment or derivative thereof. In some embodiments, the AAV-conjugated polypeptide comprises a fragment of AAVR containing an N-terminal methionine. In some embodiments, the AAV-conjugated polypeptide comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the AAV-conjugated polypeptide comprises the amino acid sequence of SEQ ID NO: 34.

[0072]

[0084] In some embodiments, the AAV-binding polypeptide includes the sequence of SEQ ID NO: 33 or SEQ ID NO: 34 having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more amino acid mutations. In some embodiments, the AAV-binding polypeptide includes the sequence of SEQ ID NO: 33 or SEQ ID NO: 34, or a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto. Unless otherwise specified, sequence identity is determined using the Basic Local Alignment Search Tool (BLAST®) from the National Center for Biotechnology Information (NCBI), available at blast.ncbi.nlm.nih.gov / Blast.cgi. In some embodiments, sequence identity is calculated over the entire length of the sequences being compared. In some embodiments, sequence identity is calculated over 20-amino acid, 50-amino acid, 75-amino acid, 100-amino acid, 250-amino acid, 500-amino acid, 750-amino acid, or 1000-amino acid fragments of each sequence being compared.

[0073]

[0085] In some embodiments, the AAV-conjugated polypeptide described herein comprises one or more PKDs, such as two, three, four, five, or more PKDs. In some embodiments, each PKD is individually selected from the PKDs listed in Table 3. For example, in some embodiments, each PKD is individually selected from SEQ ID NOs. 28-32, 37-41, and 43-47. In some embodiments, the AAV-conjugated polypeptide comprises multiple PKDs, which are linked together by a linker. Non-limiting examples of linkers are described throughout this disclosure. In some embodiments, the AAV-conjugated polypeptide comprises multiple PKD domains, where each PKD domain has the same or substantially the same sequence. In some embodiments, the AAV-conjugated polypeptide comprises multiple PKD domains, where each PKD has a different sequence.

[0074] [Table 4]

[0075]

[0086] In some embodiments, the AAV-binding polypeptide contains a polycystic kidney disease 1 (PDK1) domain. In some embodiments, the AAV-binding polypeptide contains a polycystic kidney disease 2 (PDK2) domain. In some embodiments, the AAV-binding polypeptide contains a polycystic kidney disease 3 (PDK3) domain. In some embodiments, the AAV-binding polypeptide contains a polycystic kidney disease 4 (PDK4) domain. In some embodiments, the AAV-binding polypeptide contains a polycystic kidney disease 5 (PDK5) domain.

[0076]

[0087] In some embodiments, the AAV-conjugated polypeptide includes PKD1 and PKD2 domains. In some embodiments, the AAV-conjugated polypeptide includes PKD1 and PKD2 domains having the amino acid sequence of SEQ ID NO: 92. In some embodiments, the AAV-conjugated polypeptide includes PKD1 and PKD3 domains. In some embodiments, the AAV-conjugated polypeptide includes PKD1 and PKD4 domains. In some embodiments, the AAV-conjugated polypeptide includes PKD1 and PKD5 domains. In some embodiments, the AAV-conjugated polypeptide includes PKD2 and PKD3 domains. In some embodiments, the AAV-conjugated polypeptide includes PKD2 and PKD4 domains. In some embodiments, the AAV-conjugated polypeptide includes PKD2 and PKD5 domains. In some embodiments, the AAV-conjugated polypeptide includes PKD3 and PKD4 domains. In some embodiments, the AAV-conjugated polypeptide includes PKD3 and PKD5 domains. In some embodiments, the AAV-conjugated polypeptide includes PKD4 and PKD5 domains. In some embodiments, the AAV-conjugated polypeptide comprises three PKD domains, where each PKD domain is independently selected from PKD1 to PKD5. In some embodiments, the AAV-conjugated polypeptide comprises four PKD domains, where each PKD domain is independently selected from PKD1 to PKD5. In some embodiments, the AAV-conjugated polypeptide comprises five PKD domains, where each PKD domain is independently selected from PKD1 to PKD5. In some embodiments, the AAV-conjugated polypeptide comprises more than five PKD domains, where each PKD domain is independently selected from PKD1 to PKD5.

[0077]

[0088] In any embodiment of the preceding paragraph, each PKD domain may be independently selected from wild-type or mutant PKD domains. In some embodiments, each PKD may have at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with wild-type PKD. In some embodiments, the AAV-binding polypeptide disclosed herein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% sequence identity with wild-type PKD. In some embodiments, the AAV-binding polypeptide binds to AAV using one or more PKDs.

[0078]

[0089] In some embodiments, the AAV-binding polypeptides described herein contain at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 amino acid mutations compared to their wild-type AAVR or PKD. In some embodiments, the AAV-binding polypeptides contain up to about 25 amino acid mutations or more compared to their wild-type AAVR or PKD. For example, the AAV-binding polypeptides may contain about 25–35, about 35–45, about 45–55, about 55–65, or about 65–75 amino acid mutations compared to their wild-type AAVR.

[0079]

[0090] In some embodiments, the AAV-binding polypeptides described herein contain at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, and at least about 25 amino acid mutations compared to their wild-type AAVR or PKD, where each mutation includes a change from a native amino acid residue to histidine.

[0080]

[0091] In some embodiments, the AAV-binding polypeptide includes the sequence of SEQ ID NO: 29. In some embodiments, the AAV-binding polypeptide includes the sequence of SEQ ID NO: 29 in which at least one amino acid (i.e., a non-histidine amino acid) is mutated to histidine.

[0081]

[0092] In some embodiments, the AAV-binding polypeptide includes SEQ ID NO: 35 having at least one, at least two, at least three, at least four, or at least five mutations. In some embodiments, each mutation is individually selected from the group consisting of V440H, S431H, Q432H, T434H, Y442H, I462H, D435H, D436H, K438H, and I439H.

[0082]

[0093] In some embodiments, the AAV-binding polypeptide comprises amino acids 411-499 of SEQ ID NO: 35 having at least one, at least two, at least three, at least four, or at least five mutations, where each mutation is individually selected from the group consisting of V440H, S431H, Q432H, T434H, Y442H, I462H, D435H, D436H, K438H, and I439H.

[0083]

[0094] In some embodiments, the AAV-binding polypeptide of SEQ ID NO: 35 or a fragment thereof has one or more of the mutation combinations listed in Table 4. Each row in Table 4 represents a mutation combination.

[0084] [Table 5]

[0085] [Table 6]

[0086] [Table 7]

[0087] [Table 8]

[0088] [Table 9]

[0089] [Table 10]

[0090] [Table 11]

[0091] [Table 12]

[0092] [Table 13]

[0093] [Table 14]

[0094] Table 15

[0095] Table 16

[0096] Table 17

[0097] Table 18

[0098] Table 19

[0099] Table 20

[0100] Table 21

[0101] Table 22

[0102] Table 23

[0103] Table 24

[0104] Table 25

[0105] Table 26

[0106] Table 27

[0107] Table 28

[0108] Table 29

[0109] Table 30

[0110] Table 31

[0111] Table 32

[0112]

[0095] In some embodiments, the AAV-binding polypeptide comprises SEQ ID NO: 29 having at least one, at least two, at least three, at least four, or at least five mutations, where each mutation is individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H. In some embodiments, the AAV-binding polypeptide comprises SEQ ID NO: 29 having V32H and V34H mutations. In some embodiments, the AAV-binding polypeptide comprises SEQ ID NO: 29 having S23H and Q24H mutations. In some embodiments, the AAV-binding polypeptide comprises SEQ ID NO: 29 having at least one, at least two, at least three, at least four, or at least five mutations, where each mutation is individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H. In some embodiments, the AAV-binding polypeptide contains one of the amino acid sequences from SEQ ID NOs. 76 to 86.

[0113]

[0096] In some embodiments, the AAV-binding polypeptide includes SEQ ID NO: 29 having an additional 5 amino acids having the amino acid sequence of GNRPP (SEQ ID NO: 89) at the N-terminus and an additional 5 amino acids having the amino acid sequence of VDYPG (SEQ ID NO: 90) at the C-terminus. In some embodiments, the AAV-binding polypeptide includes the amino acid sequence of SEQ ID NO: 52. In some embodiments, the AAV-binding polypeptide includes SEQ ID NO: 29 having at least 1, at least 2, at least 3, at least 4, or at least 5 mutations, each individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H, an additional 5 amino acids having the amino acid sequence of GNRPP (SEQ ID NO: 89) at the N-terminus and an additional 5 amino acids having the amino acid sequence of VDYPG (SEQ ID NO: 90) at the C-terminus. In some embodiments, the AAV-binding polypeptide comprises Sequence ID No. 52 having at least one, at least two, at least three, at least four, or at least five mutations, where each mutation is individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H.

[0114]

[0097] In some embodiments, the AAV-binding polypeptide is encoded by one nucleic acid sequence from SEQ ID NOs: 93 to 116. In some embodiments, the AAV-binding polypeptide comprises SEQ ID NOs: 29 having V32H and V34H mutations and an additional 5 amino acids having the amino acid sequence of GNRPP (SEQ ID NOs: 89) at the N-terminus, and an additional 5 amino acids having the amino acid sequence of VDYPG (SEQ ID NOs: 90) at the C-terminus. In some embodiments, the AAV-binding polypeptide comprises SEQ ID NOs: 29 having S23H and Q24H mutations and an additional 5 amino acids having the amino acid sequence of GNRPP (SEQ ID NOs: 89) at the N-terminus, and an additional 5 amino acids having the amino acid sequence of VDYPG (SEQ ID NOs: 90) at the C-terminus. In some embodiments, the AAV-binding polypeptide includes five additional amino acids having the amino acid sequence of SEQ ID NO: 29 and GNRPP (SEQ ID NO: 89) at the N-terminus, and five additional amino acids having the amino acid sequence of VDYPG (SEQ ID NO: 90) at the C-terminus, each having at least one, at least two, at least three, at least four, or at least five mutations, each individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H, respectively, and five additional amino acids having the amino acid sequence of VDYPG (SEQ ID NO: 90) at the C-terminus. In some embodiments, the AAV-binding polypeptide includes one amino acid sequence from SEQ ID NOs: 53 to 63.

[0115]

[0098] In some embodiments, the AAV-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 29, plus five additional amino acids having the amino acid sequence of GNRPP (SEQ ID NO: 89) at the N-terminus, and four additional amino acids having the amino acid sequence of VDYP (SEQ ID NO: 91) at the C-terminus. In some embodiments, the AAV-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, the AAV-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 29 having at least one, at least two, at least three, at least four, or at least five mutations, each individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H, plus five additional amino acids having the amino acid sequence of GNRPP (SEQ ID NO: 89) at the N-terminus, and four additional amino acids having the amino acid sequence of VDYP (SEQ ID NO: 91) at the C-terminus. In some embodiments, the AAV-binding polypeptide comprises five additional amino acids having the SEQ ID NO: 29+V32H and V34H mutations and the amino acid sequence of GNRPP (SEQ ID NO: 89) at the N-terminus, and four additional amino acids having the amino acid sequence of VDYP (SEQ ID NO: 91) at the C-terminus. In some embodiments, the AAV-binding polypeptide comprises five additional amino acids having the SEQ ID NO: 29 having the S23H and Q24H mutations and the amino acid sequence of GNRPP (SEQ ID NO: 89) at the N-terminus, and four additional amino acids having the amino acid sequence of VDYP (SEQ ID NO: 91) at the C-terminus. In some embodiments, the AAV-binding polypeptide comprises the amino acid sequence of SEQ ID NO: 29 having at least one, at least two, at least three, at least four, or at least five mutations, each individually selected from the group consisting of S23H, Q24H, T26H, D29H, K30H, I31H, V32H, Y34H, and I54H, plus five additional amino acids having the amino acid sequence of GNRPP (SEQ ID NO: 89) at the N-terminus, and four additional amino acids having the amino acid sequence of VDYP (SEQ ID NO: 91) at the C-terminus. In some embodiments, the AAV-binding polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 65 to 75.

[0116]

[0099] In some embodiments, the AAV-binding polypeptide described herein comprises one or more MANEC motifs (see, for example, SEQ ID NOs. 49-51). In some embodiments, the AAV-binding polypeptide described herein comprises one or more recombinant MANEC motifs. In some embodiments, the AAV-binding polypeptide described herein comprises an amino acid sequence having at least about 80%, at least about 90%, at least about 95%, or at least about 95% identity with a wild-type MANEC motif. In some embodiments, the AAV-binding polypeptide comprises a MANEC motif having at least about 80%, at least about 90%, or at least about 95% identity with any one of SEQ ID NOs. 49-51. In some embodiments, the AAV-binding polypeptide binds to an AAV particle via one or more MANEC motifs.

[0117]

[0100] In some embodiments, the AAV-binding polypeptide described herein contains an N-terminal methionine. In some embodiments, the N-terminal methionine initiates translation of the AAV-binding polypeptide described herein. In some embodiments, the AAV-binding polypeptide described herein lacks an N-terminal methionine.

[0118]

[0101] In some embodiments, the AAV-binding polypeptide described herein binds to AAV particles of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, and / or AAVrh74. In some embodiments, the AAV-binding polypeptide binds to one or more AAV particles of the following serotypes: AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9. In some embodiments, the AAV-binding polypeptide binds to AAV1 particles. In some embodiments, the AAV-binding polypeptide binds to AAV2 particles. In some embodiments, the AAV-binding polypeptide binds to AAV3B particles. In some embodiments, the AAV-binding polypeptide binds to AAV5 particles. In some embodiments, the AAV-binding polypeptide binds to AAV6 particles. In some embodiments, the AAV-binding polypeptide binds to AAV8 particles. In some embodiments, the AAV-binding polypeptide binds to the AAV9 particle.

[0119]

[0102] In some embodiments, AAVR is human AAVR. In some embodiments, AAVR is primate AAVR. In some embodiments, AAVR is wild-type AAVR. In some embodiments, AAVR is mutant AAVR.

[0120]

[0103] In some embodiments, AAVR is a glycoprotein. In some embodiments, the AAV-binding polypeptide described herein includes one or more glycosylation sites. In some embodiments, the AAV-binding polypeptide includes an O-linked glycosylation site. In some embodiments, the AAV-binding polypeptide includes an N-linked glycosylation site. In some embodiments, AAVR includes N-linked glycosylation. In some embodiments, AAVR has one or more asparagine and / or glutamine residues glycosylated.

[0121]

[0104] In some embodiments, the purified matrix contains about 1 to about 100 AAV-binding polypeptides. In some embodiments, the number of AAV-binding polypeptides is about 1, about 5, about 10, about 20, about 30, about 40, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100. In some embodiments, a single polypeptide with phase behavior may be coupled to multiple AAV-binding polypeptides, such as about 1 to about 100 AAV-binding polypeptides.

[0122]

[0105] In some embodiments, the AAV-binding polypeptide comprises one of the sequences of SEQ ID NOs: 28-34, 37-41, 43-47, and 52-86. In some embodiments in which a single polypeptide exhibiting phase behavior is coupled to multiple AAV-binding polypeptides, each AAV-binding polypeptide may be independently selected from SEQ ID NOs: 28-34, 37-41, 43-47, and 52-86.

[0123]

[0106] In some embodiments, the nucleic acid encodes one or more AAV-binding polypeptides described herein. In some embodiments, the vector comprises a nucleic acid encoding one or more AAV-binding polypeptides described herein.

[0124]

[0107] In some embodiments, the kit includes an AAV-binding polypeptide, a nucleic acid encoding the AAV-binding polypeptide, and / or a vector comprising the nucleic acid encoding the AAV-binding polypeptide.

[0125]

[0108] In some embodiments, the purified matrix contains an AAV-conjugated polypeptide. In some embodiments, the kit contains a purified matrix.

[0126]

[0109] In some embodiments, the AAV-binding polypeptide described herein binds to one or more AAV particles. In some embodiments, a purified matrix containing the AAV-binding polypeptide binds to one or more AAV particles.

[0127]

[0110] In some embodiments, the AAV-bonded polypeptide has a Kd of about 1 nM to about 500 nM. In some embodiments, the AAV-bonded polypeptide has a Kd of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 23 The Kd is approximately 0 nM, 240 nM, 250 nM, 260 nM, 270 nM, 280 nM, 290 nM, 300 nM, 310 nM, 320 nM, 330 nM, 340 nM, 350 nM, 360 nM, 370 nM, 380 nM, 390 nM, 400 nM, 410 nM, 420 nM, 430 nM, 440 nM, 450 nM, 460 nM, 470 nM, 480 nM, 490 nM, or 500 nM (including all values ​​and ranges in between). In some embodiments, the AAV-bound polypeptide has an affinity of 50 nM or more. In some embodiments, the AAV-binding polypeptide has affinities of approximately 50 nM, 40 nM, 30 nM, 20 nM, 2 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM.

[0128]

[0111] In some embodiments, the binding of AAV-binding polypeptides to AAV particles is broken at pH 2.5 or higher. In some embodiments, the binding of AAV-binding polypeptides to AAV particles is broken at pH approximately 2.5, approximately 3.0, approximately 3.5, approximately 4.0, approximately 4.5, approximately 5.0, approximately 5.5, approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, approximately 8.0, approximately 8.5, approximately 9.0, approximately 9.5, approximately 10.0, approximately 10.5, approximately 11.0, approximately 11.5, approximately 12.0, approximately 12.5, approximately 13.0, or approximately 13.5.

[0129] support

[0112] In some embodiments, the Disclosure provides a purification matrix comprising an AAV-conjugated polypeptide coupled to a support (e.g., a solid support). In some embodiments, the Disclosure provides a purification matrix comprising an AAV-conjugated polypeptide and a peptide having phase behavior coupled to a support. In some embodiments, the support is beads, resins, plates, chips, membranes, fibers, or polymers. In some embodiments, the support comprises silica, agarose, soft agarose, cellulose, cellulose acetate, polystyrene, Sepharose, heparin Sepharose, celluline sulfate, hydroxyapatite, ceramic hydroxyapatite, agarose, dextran, latex, polymethacrylate, polyacrylamide, nitrocellulose, nylon, polyester, polyethersulfone, poly(styrenedivinyl)benzene, ceramic particles, polyacrylamide, polyolefin, and / or polyvinylidene fluoride, sulfopropyl immobilized on agarose, and / or combinations thereof.

[0130]

[0113] In some embodiments, the support is a polymer. Non-limiting examples of polymers include polyamylic acid, polyacrylonitrile, polyallyamine, polyacrylates, butyl polyacrylate, polymethyl methacrylate, alkyl polyacrylates, polyalkyl methacrylate, polybutadiene, polycarbomethylsilane, polystyrene, polypeptides, polynucleic acids, and poly(carbonate)urethanes. In some embodiments, the support is a synthetic polymer. In some embodiments, the support is a recombinant polymer.

[0131]

[0114] In some embodiments, the support is a resin. In some embodiments, the resin is positively charged. In some embodiments, a cation is attached to the resin. In some embodiments, the cation is a quaternary amino group. In some embodiments, the positively charged resin includes diethylaminoethylcellulose, magnetic amines, magnetic propylamines, magnetic quaternary ammonium, magnetic poly-D-lysine, poly-D-lysine functionalized polyurethane, spermine latex, tris(2-aminoethyl)amine latex, tris(2-aminoethyl)amine beaded agarose, tris(2-aminoethyl)acrylamide, and tris(2-aminoethyl)polyurethaneamine. In some embodiments, the resin is negatively charged. In some embodiments, anions are attached to the resin. In some embodiments, anions are covalently attached to the resin. Non-limiting examples of anions include sulfonates or carboxylates.

[0132]

[0115] In some embodiments, the support is porous. In some embodiments, the porous support is a resin or beads. In some embodiments, the porous support is magnetic beads. In some embodiments, the porous support has pore sizes ranging from about 50 nm to about 5000 nm. In some embodiments, the pore sizes are about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1000 nm, about 1100 nm, about 1200 nm, about 1300 nm, about 1400 nm, about 1500 nm, about 1600 nm, about 1700 nm, about 1800 nm, about 1900 nm, about 2000 nm, about 2100 nm, about 2200 nm, about 2300 nm, about 2400 nm, These are approximately 2500nm, 2600nm, 2700nm, 2800nm, 2900nm, 3000nm, 3100nm, 3200nm, 3300nm, 3400nm, 3500nm, 3600nm, 3700nm, 3800nm, 3900nm, 4000nm, 4100nm, 4200nm, 4300nm, 4400nm, 4500nm, 4600nm, 4700nm, 4800nm, 4900nm, or 5000nm. In some embodiments, pore diameter refers to the radius of the pore. In some embodiments, pore diameter refers to the diameter of the pore.

[0133]

[0116] In some embodiments, the support comprises one or more crosslinking materials. In some embodiments, the porous support comprises one or more crosslinking materials. In some embodiments, the crosslinking prevents interaction between the AAV and the pores of the support. In some embodiments, the support is a static fiber network. In some embodiments, the support is a solid network.

[0134]

[0117] In some embodiments, the support is pretreated before coupling to the AAV-binding polypeptide. In some embodiments, the support is pretreated with an RNase solution.

[0135]

[0118] In some embodiments, the support is coupled to the AAV-binding polypeptide via a linker. In some embodiments, the linker is coupled to the support. In some embodiments, the linker is coupled to the AAV-binding polypeptide. Examples of linkers are provided throughout this disclosure.

[0136]

[0119] In some embodiments, the support is separated from the solution by decantation, centrifugation, or filtration. If the particles are magnetic, magnetic field separation can be used.

[0137]

[0120] In some embodiments, the support is housed in a vessel. Non-limiting examples of vessels include centrifuge tubes, spin tubes, syringes, cartridges, chambers, tips, multiwell plates, beakers, chromatography columns, or test tubes.

[0138] Polypeptides exhibiting phase behavior

[0121] In some embodiments, the disclosure provides a purified matrix comprising a polypeptide having phase behavior. In some embodiments, the disclosure provides a purified matrix comprising a polypeptide having phase behavior that is coupled to an AAV-binding protein. In some embodiments, the disclosure provides a purified matrix comprising a support that is coupled to a fusion protein comprising (i) an AAV-binding protein and (ii) a polypeptide having phase behavior.

[0139]

[0122] In some embodiments, the polypeptide exhibiting phase behavior is a resilin-like polypeptide (RLP). A resilin-like polypeptide is an elastic polypeptide having mechanical properties including desirable elastic recovery, compressive modulus, tensile modulus, stiffness, elongation at break, maximum tensile strength, hardness, rebound elasticity, and compression set. In some embodiments, the resilin-like polypeptide described herein is a polymer comprising one or more repeats. In some embodiments, the polymer repeat may have an amino acid sequence selected from any one of SEQ ID NOs: 1 to 9.

[0140]

[0123] In some embodiments, the resilin-like polypeptide includes two or more repeats, for example, the repeat of SEQ ID NO: 1 and the repeat of SEQ ID NO: 3.

[0141]

[0124] In some embodiments, the resilin-like polypeptides described herein include repeats that appear up to 500 times within a given RLP. In some embodiments, the repeat appears approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 450, or 500 times.

[0142]

[0125] In some embodiments, the RLP comprises one or more partial repeats. In some embodiments, the length of the partial repeat is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the RLP comprises one or more additional amino acids at the N-terminus or C-terminus of the RLP that is not part of the repeat.

[0143]

[0126] In some embodiments, one or more RLP repeats are scrambled, i.e., they contain different amino acid sequences but retain the same amino acid composition. For example, a repeat may have a different amino acid sequence from SEQ ID NO: 8 but retain the same amino acid composition.

[0144]

[0127] In some embodiments, the polypeptide exhibiting phase behavior is an elastin-like polypeptide. Elastin-like polypeptides (ELPs) are biopolymers derived from tropoelastin. In some embodiments, the elastin-like polypeptides described herein are polymers comprising a pentapeptide repeat having the sequence (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 10).

[0145]

[0128] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 ,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,14 9, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 (including all values ​​and ranges in between).

[0146]

[0129] In some embodiments, the pentapeptide repeat is scrambled, for example, to include a different amino acid sequence but maintain the same amino acid composition. For example, ELP may include a different amino acid sequence than SEQ ID NO: 10 but maintain the same amino acid composition, for example, 40% of the sequence is glycine, 20% of the sequence is Xaa (e.g., any amino acid other than proline), 20% of the sequence is proline, and 20% of the sequence is valine.

[0147]

[0130] In some embodiments, the ELP comprises one or more partial repeats. In some embodiments, the length of the partial repeat is 1, 2, 3, or 4 amino acids. In some embodiments, the ELP comprises one or more additional amino acids at the N-terminus or C-terminus of the ELP that is not part of the repeat.

[0148]

[0131] ELP and RLP undergo phase transitions in response to environmental factors. ELP and RLP retain the ability to undergo phase transitions when coupled to one or more polypeptides (such as one or more AAV-binding polypeptides) or when expressed as fusion proteins with one or more other polypeptides (such as one or more AAV-binding polypeptides). Polymers like ELP and RLP exhibit a transition temperature (Tt), also called the cloud point temperature (Tc). In some embodiments, ELP and RLP undergo a reversible phase transition from a soluble phase to an insoluble phase at Tt. ELP, which transitions from a soluble phase to an insoluble phase with heating or an increase in salt concentration, has a Tt called the lower critical eutectic temperature (LCST). RLP, which transitions from a soluble phase to an insoluble phase with cooling or a decrease in salt concentration, has a Tt called the lower critical eutectic temperature (UCST). In some embodiments, the phase transition occurs as a result of a change in the secondary structure of ELP and / or RLP. For example, the phase transition of ELP occurs as a result of a change in secondary structure from a random coil (less than Tt) to a type II β-turn. In some embodiments, the change in secondary structure is characterized by a method selected from circular dichroism spectroscopy, small-angle X-ray scattering, ultraviolet-visible spectrophotometry, static light scattering, dynamic light scattering, nuclear magnetic resonance spectroscopy, solid-state nuclear magnetic resonance spectroscopy, infrared spectroscopy, Fourier transform infrared spectroscopy (FTIR), small-angle neutron scattering, microscopy, and cryo-electron microscopy. In some embodiments, the phase transition of ELP does not occur due to a change in secondary structure.

[0149]

[0132] In some embodiments, the RLP and ELP described herein have a transition temperature between about 0°C and about 100°C. In some embodiments, the RLP and ELP described herein have a transition temperature between about 10°C and about 50°C. In some embodiments, the transition temperature is about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24℃, approximately 25℃, approximately 26℃, approximately 27℃, approximately 28℃, approximately 29℃, approximately 30℃, approximately 31℃, approximately 32℃, approximately 33℃, approximately 34℃, approximately 35℃, approximately 36℃, approximately 37℃, approximately 38℃, approximately 39℃, approximately 40℃, approximately 41℃, approximately 42℃, approximately 43℃, approximately 44℃, approximately 45℃, approximately 46℃, approximately 47℃, approximately 48℃, approximately 49℃, approximately 50℃, approximately 51℃, approximately 52℃, approximately 53℃, approximately 54℃, approximately 55℃, approximately 56℃, approximately 57℃, approximately 58℃, approximately 59℃, approximately 60℃, approximately 61℃, approximately 62℃, approximately 63℃, approximately 64℃, approximately 65℃, approximately 66℃, approximately 67℃, approximately 68℃, approximately 69℃, approximately 70℃, approximately 71℃, approximately 72℃, approximately 73℃, approximately 74℃, approximately 75℃, approximately The transition temperatures are 76°C, approximately 77°C, approximately 78°C, approximately 79°C, approximately 80°C, approximately 81°C, approximately 82°C, approximately 83°C, approximately 84°C, approximately 85°C, approximately 86°C, approximately 87°C, approximately 88°C, approximately 89°C, approximately 90°C, approximately 91°C, approximately 92°C, approximately 93°C, approximately 94°C, approximately 95°C, approximately 96°C, approximately 97°C, approximately 98°C, approximately 99°C, or approximately 100°C. In some embodiments, the RLP described herein has a transition temperature of approximately 10°C to approximately 100°C.

[0150]

[0133] In some embodiments, the Tt of RLP and ELP described herein is regulated by manipulating the primary structure (e.g., amino acid sequence) of RLP and ELP. In some embodiments, the hydrophobicity of ELP or RLP is regulated. In some embodiments, the hydrophobicity of ELP is modified by changing the identity of the guest residue Xaa. In some embodiments, increasing the hydrophobicity of ELP or RLP results in a decrease in Tt. In some embodiments, decreasing the hydrophobicity of ELP or RLP results in an increase in Tt. In some embodiments, the polarity of ELP or RLP is regulated. In some embodiments, the polarity of ELP is regulated by changing the identity of the guest residue Xaa. In some embodiments, increasing the polarity of ELP or RLP results in an increase in Tt. In some embodiments, decreasing the polarity of ELP or RLP results in a decrease in Tt.

[0151]

[0134] In some embodiments, adjusting the number (n) of the ELP pentapeptide repeats changes Tt. In some embodiments, n in the pentapeptide repeat (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 10) is an integer between 1 and 500. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 ,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,14 9, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 (including all values ​​and ranges in between).

[0152]

[0135] In some embodiments, Xaa is a "guest residue," i.e., any amino acid that does not cause the phase behavior of the ELP to disappear. In some embodiments, Xaa is any amino acid except proline. In some embodiments, Xaa is independently selected for each repeat. For example, a given ELP may contain the guest residues alanine, glycine, and valine in a ratio of 8:7:1. In some embodiments, Xaa is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline (praline), serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, Xaa is a non-classical amino acid selected from the group consisting of Table 2 and / or 2,4-diaminobutyric acid, α-aminoisobutyric acid, alloisoleucine, 4-aminobutyric acid, 2-aminobutyric acid (Abu), ε-Ahx, 6-aminohexanoic acid, 2-aminoisobutyric acid (Aib), 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids, such as β-methylamino acids, Cα-methylamino acids, Na-methylamino acids, etc., and amino acid analogs in general. In some embodiments, Xaa is a D-isomer of a natural or non-classical amino acid.

[0153]

[0136] In some embodiments, the Tt of RLP and ELP described herein is adjusted by introducing one or more environmental factors into a composition containing RLP and / or ELP. In some embodiments, the Tt of ELP and / or RLP is adjusted by adjusting the ionic strength of the solvent. In some embodiments, the ionic strength of the solvent is adjusted by adding a salt. In some embodiments, ELP and / or RLP have a lower Tt in a solvent containing anions classified as cosmotropes. Anions that are cosmotropes are highly hydrated and affect the shielding of water on ELP and / or RLP. In some embodiments, the Tt of ELP and / or RLP may be adjusted by adding anions that are chaotropes. At low concentrations, the addition of chaotropes increases the Tt of ELP and / or RLP. At high concentrations, the addition of chaotropes decreases the Tt of ELP and / or RLP. In some embodiments, the Tt of ELP and / or RLP may be adjusted by introducing one or more reagents that break hydrogen bonds. Non-exclusive examples of reagents that disrupt hydrogen bonding include sodium dodecyl sulfate (SDS) and urea. In some embodiments, Tt is adjusted using reagents that enhance hydrogen bond formation. In some embodiments, Tt is adjusted using reagents that enhance hydrophobic interactions. Trifluoroethanol is a reagent that lowers Tt by enhancing both hydrophobic interactions and hydrogen bond formation.

[0154]

[0137] In some embodiments, Tt can be adjusted by adjusting the ELP and / or RLP concentrations. In some embodiments, as the ELP and / or RLP concentrations increase, Tt decreases. In some embodiments, as the ELP and / or RLP concentrations decrease, Tt increases.

[0155]

[0138] In addition, Tt can also be adjusted by controlling pH, light, and ion concentration.

[0156]

[0139] In some embodiments, Tt can be adjusted by pH adjustment by adjusting the number (e.g., adding or removing) and identity (e.g., having a positive or negative charge) of charged amino acids (e.g., histidine, lysine, arginine, glutamic acid, aspartic acid, ornithine, or other unnatural charged amino acids).

[0157]

[0140] In some embodiments, the ELP and / or RLP described herein are block copolymers. A block copolymer comprises two or more sequence domains or blocks, where the two or more blocks have different properties. Non-limiting examples of properties that can be tuned include hydrophilicity, hydrophobicity, polarity, and secondary structure. In some embodiments, the block copolymer is amphiphilic, for example, comprising at least one hydrophobic block and at least one hydrophilic block.

[0158]

[0141] In some embodiments, the ELP and / or RLP described herein assemble into various forms. Non-limiting examples of forms include spherical aggregates, micelles, vesicles, fibrils, nanofibrils, nanotubes, and hydrogels. In some embodiments, the RLP and / or ELP described herein assemble into various forms after the addition of environmental factors. In some embodiments, the RLP and / or ELP described herein change from one form to another after the addition of environmental factors. In some embodiments, the RLP and / or ELP described herein change from one form to another after the addition of AAV particles.

[0159]

[0142] In some embodiments, the RLP and / or ELP undergo a phase transition by adding environmental factors. In some embodiments, during the RLP and / or ELP phase transition, the RLP and / or ELP are transformed from one form to another.

[0160]

[0143] In some embodiments, the phase transition of RLP and / or ELP results in the formation of a dense body, a liquid, or droplets.

[0161]

[0144] In some embodiments, polypeptides exhibiting phase behavior are (a)(GRGDSPY)n(Sequence ID 1) (b)(GRGDSPH)n(Sequence ID 2) (c)(GRGDSPV)n(Sequence ID 3) (d)(GRGDSPYG)n(Sequence ID 4) (e)(RPLGYDS)n(Sequence ID 5) (f)(RPAGYDS)n(Sequence ID 6) (g)(GRGDSYP)n(Sequence ID 7) (h)(GRGDSPYQ)n(Sequence ID 8) (i)(GRGNSPYG)n(Sequence ID 9) (j)(GVGVP)n(Sequence ID 11); (k)(GVGVPGLGVPGVGVPGLGVPGVGVP)m(Sequence ID 12); (l)(GVGVPGVGVPGAGVPGVGVPGVGVP)m(Sequence ID 13); (m)(GVGVPGWGVPGVGVPGWGVPGVGVP)m(Sequence ID 14); (n)(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)m(SEQ ID NO: 15); (o)(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)m(Sequence No. 16); and (p)(GAGVPGVGVPGAGVPGVGVPGAGVP)m(Sequence ID 17) An amino acid sequence selected from the group consisting of the following; or their randomized scramble analogues (In the formula: n is an integer in the range of 1 to 500; and m is an integer in the range of 4 to 25.

[0162]

[0145] In some embodiments, the polypeptide exhibiting phase behavior is (GVGVPGLGVPGVGVPGLGVPGVGVP)m(SEQ ID NO: 12) (wherein m is 16). In some embodiments, the polypeptide exhibiting phase behavior comprises the amino acid sequence of (GVGVPGLGVPGVGVPGLGVPGVGVP)m(SEQ ID NO: 12) (wherein m is 16) and up to 10 additional amino acids at the N-terminus or C-terminus. In some embodiments, the polypeptide exhibiting phase behavior comprises the amino acid sequence of (GVGVPGLGVPGVGVPGLGVPGVGVP)m(SEQ ID NO: 12) (wherein m is 16) and an additional C-terminal glycine. In some embodiments, the polypeptide exhibiting phase behavior comprises the amino acid sequence of (GVGVPGLGVPGVGVPGLGVPGVGVP)m(SEQ ID NO: 12) (wherein m is 16) and an additional N-terminal methionine. In some embodiments, the polypeptide exhibiting phase behavior comprises the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16), an additional C-terminal glycine, and an additional N-terminal methionine.

[0163]

[0146] In some embodiments, the polypeptide exhibiting phase behavior has the amino acid sequence of SEQ ID NO: 88.

[0164]

[0147] In some embodiments, the polypeptide exhibiting phase behavior includes the amino acid sequence (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 144) or (GVGVPGVGVPGLGVPGVGVPGVGVP)m (SEQ ID NO: 146) (wherein m is an integer between 2 and 32). In some embodiments, the polypeptide exhibiting phase behavior includes the amino acid sequence (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 144) (wherein m is 8 or 16). In some embodiments, the polypeptide exhibiting phase behavior includes the amino acid sequence (GVGVPGAGVP)m (SEQ ID NO: 145) (wherein m is an integer between 5 and 80). In some embodiments, the polypeptide exhibiting phase behavior comprises the amino acid sequence (GXGVP)m (SEQ ID NO: 147) (wherein m is an integer between 10 and 160, and in each repeat, X is independently selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine).

[0165]

[0148] In some embodiments, polypeptides exhibiting phase behavior are (a)(GVGVP)m(Sequence ID 143); (b)(ZZPXXXXGZ)m(Sequence ID 148); (c)(ZZPXGZ)m(Sequence ID 149); (d)(ZZPXXGZ)m(sequence number 150); or (e)(ZZPXXXGZ)m(Sequence ID 151) The formula includes an amino acid sequence selected from (wherein m is an integer between 10 and 160, X is any amino acid other than proline or glycine if present, and Z is any amino acid if present).

[0166]

[0149] In some embodiments, the polypeptide exhibiting phase behavior comprises the amino acid sequence (GVGVP)m (SEQ ID NO: 143) (wherein m is 20, 40, or 80). In some embodiments, the polypeptide exhibiting phase behavior comprises the amino acid sequence (GRGDXPZX)m (SEQ ID NO: 152) or (XZPXDGRG)m (SEQ ID NO: 153) (wherein X is glutamine or serine, Z is tyrosine or valine, and m is an integer between 10 and 160).

[0167]

[0150] In some embodiments, the phase-behaving polypeptide comprises a first set of repeat sequences and a second set of repeat sequences. The first set of repeat sequences and the second set of repeat sequences may each individually contain a sequence that is repeated one or more times. In some embodiments, the first set of repeat sequences and / or the second set of repeat sequences contain a repeat sequence which includes one of the sequence numbers 1-17 and 143-153. In some embodiments, the phase-behaving polypeptide comprises a first set of repeat sequences and a second set of repeat sequences, where the first set of repeat sequences comprises the amino acid sequence (GRGDXPZX)40 (sequence number 154) and the second set of repeat sequences comprises the amino acid sequence (GVGVP)80 (sequence number 155) (wherein X is glutamine and Z is tyrosine). In some embodiments, a polypeptide exhibiting phase behavior comprising a first set of repeat sequences and a second set of repeat sequences comprises the sequence of SEQ ID NO: 156. In some embodiments, the polypeptide exhibiting phase behavior comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 sets of distinct repeat sequences. In some embodiments, each pair of repeat sequences within a polypeptide exhibiting phase behavior occurs approximately 5 to 400 times, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190 , including sequences that repeat approximately 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, or approximately 400 times.

[0168]

[0151] In some embodiments, a phase-behaving polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-17, 88, and 143-153 also comprises up to 10 additional N-terminal and / or C-terminal amino acids. In some embodiments, a phase-behaving polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-17, 88, and 143-153 also comprises an additional N-terminal methionine. In some embodiments, a phase-behaving polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-17, 88, and 143-153 also comprises an additional C-terminal glycine.

[0169]

[0152] In some embodiments, the phase-behaving polypeptide has the same amino acid composition as ELP and / or RLP, but does not contain repeats. In some embodiments, the phase-behaving polypeptide contains an amino acid sequence that is about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to ELP and / or RLP. In some embodiments, the phase-behaving polypeptide contains an amino acid composition that is about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to ELP and / or RLP. In some embodiments, the phase-behaving polypeptide contains a hydrophobic amino acid composition that is about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical to ELP and / or RLP.

[0170]

[0153] In some embodiments, the polypeptide exhibiting phase behavior includes a non-repeating unstructured polypeptide. In some embodiments, the non-repeating unstructured polypeptide has an amino acid sequence comprising at least 50 amino acids. In some embodiments, the non-repeating unstructured polypeptide has an amino acid sequence comprising at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids. In some embodiments, the sequence of the non-repeating unstructured polypeptide is at least about 10% proline (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%) and at least 20% glycine (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%). In some embodiments, the non-repeating unstructured polypeptide has a sequence containing at least about 40% of amino acids selected from the group consisting of valine, alanine, leucine, lysine, threonine, isoleucine, tyrosine, serine, and phenylalanine.

[0171]

[0154] In some embodiments, the non-repeating unstructured polypeptide comprises a sequence that does not contain three consecutive identical amino acids, wherein no 5-10 amino acid subsequence appears more than once in the non-repeating unstructured polypeptide, and the non-repeating unstructured polypeptide comprises a subsequence that begins with proline and ends with proline, wherein the subsequence further comprises at least one glycine.

[0172]

[0155] In some embodiments, the ELP and / or RLP described herein are expressed as components of a fusion protein. In some embodiments, the fusion protein comprises the ELP and / or RLP and an AAV-binding polypeptide such as an AAVR ectodomain or a fragment or derivative thereof. In some embodiments, the fusion protein is expressed in bacterial or mammalian cells. In some embodiments, the fusion protein is expressed in Escherichia coli. In some embodiments, the fusion protein is expressed in insect cells. In some embodiments, the sequence of the non-repeating unstructured polypeptide is at least about 10% proline (e.g., at least 10%, at least 20%, at least 30%, at least 40%) and at least 20% glycine (e.g., at least 20%, at least 30%, at least 40%, or at least 50%), and at least 40% (e.g., at least 40%, at least 50%, at least 60%, or at least 70%) are amino acids selected from the group consisting of valine, alanine, leucine, lysine, threonine, isoleucine, tyrosine, serine, and phenylalanine.

[0173]

[0156] In some embodiments, the non-repeating unstructured polypeptide does not contain three consecutive identical amino acids. In some embodiments, the non-repeating unstructured polypeptide includes a subsequence that appears only once in the non-repeating unstructured polypeptide sequence (e.g., a fragment of the non-repeating unstructured polypeptide). In some embodiments, the non-repeating unstructured polypeptide includes a subsequence that begins and ends with proline. In some embodiments, the non-repeating unstructured polypeptide includes a subsequence that contains at least one glycine molecule.

[0174]

[0157] In some embodiments, the polypeptide exhibiting phase behavior includes the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16). In some embodiments, the polypeptide exhibiting phase behavior includes the amino acid sequence of SEQ ID NO: 164. In some embodiments, the polypeptide exhibiting phase behavior includes the amino acid sequence of SEQ ID NO: 170.

[0175]

[0158] In some embodiments, the polypeptide exhibiting phase behavior includes a signal peptide.

[0176] Binding of AAV to the purified matrix

[0159] In some embodiments, the disclosure provides a purified matrix comprising an AAV-conjugated polypeptide, wherein the AAV-conjugated polypeptide is coupled to a polypeptide and / or support exhibiting phase behavior.

[0177]

[0160] In some embodiments, the disclosure provides a purified matrix comprising a fusion protein comprising (i) an AAV-binding polypeptide and (ii) a polypeptide exhibiting phase behavior, wherein the fusion protein is coupled to a support (e.g., a solid support). In some embodiments, the fusion protein is coupled to the support via residues in the AAV-binding polypeptide. In some embodiments, the fusion protein is coupled to the support via residues in the polypeptide exhibiting phase behavior. In some embodiments, the fusion protein is coupled to the support via residues in a linker between the AAV-binding polypeptide and the support. In some embodiments, the binding to and / or coupling to the support is reversible.

[0178]

[0161] In some embodiments, the purified matrix is ​​brought into contact with AAV particles. In some embodiments, the purified matrix binds to the AAV particles to form a complex. In some embodiments, the purified matrix comprising a fusion protein containing (i) an AAV-binding polypeptide and (ii) a polypeptide exhibiting phase behavior binds to the AAV particles to form a complex. In some embodiments, the purified matrix comprising a fusion protein containing (i) an AAV-binding polypeptide and (ii) a polypeptide exhibiting phase behavior binds to the AAV particles to form a complex.

[0179]

[0162] In some embodiments, the purified matrix reversibly binds to the AAV. Reversible binding and / or reversible coupling means that the complex can dissociate into its individual components, for example, it can be separated. For example, if a complex is reversibly formed between the purified matrix and the AAV particles, the purified matrix and the AAV particles can subsequently dissociate. In some embodiments, reversible binding makes it possible to separate the AAV particles from the purified matrix. In some embodiments, the dissociation is caused by environmental factors. In some embodiments, reversible binding makes it possible to separate contaminants and / or impurities from the purified matrix. In some embodiments, reversible binding makes it possible to separate other molecules from the purified matrix.

[0180]

[0163] In some embodiments, the reversible bonding is non-covalent, i.e., no covalent bonds are formed between the interacting components of the complex (such as between the purified matrix and contaminants, bioagents, and / or other molecules). In some embodiments, non-covalent interactions result in bonding between the purified matrix and contaminants, bioagents, and / or other molecules. Non-limiting examples of non-covalent interactions include dipole forces, van der Waals forces, London dispersion forces, hydrogen bonds, hydrophobic interactions, and electrostatic interactions. In some embodiments, non-covalent bonding is broken by the addition of environmental factors.

[0181]

[0164] In some embodiments, the bond between the purified matrix and the AAV particles is covalent. In some embodiments, the covalent bond between the purified matrix and the AAV particles may be cleaved, for example, using a protease.

[0182]

[0165] In some embodiments, the purification matrix is ​​recyclable, meaning it can be reused after one or more uses. In some embodiments, the purification matrix is ​​regenerated after the first use before it can be used again. In some embodiments, the purification matrix is ​​regenerated by incubation with guanidine hydrochloride. In some embodiments, the purification matrix is ​​regenerated by incubation in guanidine hydrochloride at a concentration in the range of about 1 M to about 10 M, such as about 6 M. In some embodiments, the purification matrix is ​​regenerated by incubation in sodium hydroxide. In some embodiments, the purification matrix is ​​regenerated by incubation in sodium hydroxide at a concentration in the range of about 0.1 M to about 10 M, such as about 1 M. The incubation may be, for example, about 1 to about 30 minutes, about 5 to about 10 minutes, or about 10 to about 10 minutes. In some embodiments, the incubation is about 5 minutes long.

[0183]

[0166] In some embodiments, the purified matrix is ​​regenerated by incubating the purified matrix at a high temperature, such as above 80°C, above 85°C, above 90°C, above 95°C, or above 100°C. The incubation may be, for example, about 1 to about 30 minutes, about 5 to about 10 minutes, or about 10 to about 10 minutes. In some embodiments, the incubation is about 5 minutes long. In some embodiments, the purified matrix is ​​regenerated by incubating the purified matrix at 95°C for about 5 minutes.

[0184]

[0167] In some embodiments, after purifying AAV particles from a first composition containing AAV particles, the purification matrix is ​​regenerated and used to capture AAV particles from further compositions containing AAV particles. In some embodiments, the purification matrix can be reused for at least five purification cycles, e.g., at least about five cycles, at least about six cycles, at least about seven cycles, at least about eight cycles, at least about nine cycles, and at least about ten cycles. Each purification cycle refers to the use of the purification matrix to purify AAV particles from a composition containing AAV particles. Subsequent cycles refer to further use of the same purification matrix to purify AAV particles from further compositions containing AAV particles. For example, in the third purification cycle, the purification matrix is ​​used for the third time to purify AAV particles from a third composition containing AAV particles; the same purification matrix has already been used in the first cycle to purify AAV particles from a first composition containing AAV particles and in the second cycle to purify AAV particles from a second composition containing AAV particles.

[0185]

[0168] In some embodiments, the purification matrix retains the ability to capture at least 95% of AAV particles from the composition even after at least five purification cycles.

[0186] Linker

[0169] In some embodiments, the AAV-conjugated polypeptide is coupled to a polypeptide or support having phase behavior via a linker. In some embodiments, any linker that does not interfere with the function of the purified matrix may be used.

[0187]

[0170] In some embodiments, the linker connects the AAV-binding polypeptide to the phase-behaving polypeptide. In some embodiments, the linker enables cooperative interaction between the phase-behaving polypeptide and the AAV-binding polypeptide. In some embodiments, the linker is a peptide. In some embodiments, the linker preserves the phase behavior of the phase-behaving polypeptide. In some embodiments, the linker preserves the Tt of the phase-behaving polypeptide. In some embodiments, the linker preserves the structure of the capture domain. In some embodiments, the linker contains 1 to 50 amino acids. In some embodiments, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, or 50 amino acids.

[0188]

[0171] In some embodiments, the rigidity of the linker is increased by including proline in the linker amino acid sequence.

[0189]

[0172] In some embodiments, the flexibility of the linker is increased by including small polar amino acids, including threonine, serine, and glycine.

[0190]

[0173] In some embodiments, the linker may take on a variety of secondary structures, including, but not limited to, α-helices, β-strands, and random coils. In some embodiments, the linker may take on an α-helix and include an amino acid repeat of (EAAAK)n (SEQ ID NO: 18) (wherein n is the number of repeats in the range of 1 to 20).

[0191]

[0174] In some embodiments, the linker includes (G4S)n (SEQ ID NO: 19) [wherein n can be an integer from 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)]. In embodiments, the polypeptide linker has a repeat of (SGGG)n (SEQ ID NO: 20) [wherein n is an integer from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)]. In an embodiment, the polypeptide linker has a repeat of (GGGS)n (SEQ ID NO: 21) [wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)].

[0192]

[0175] In some embodiments, the linker has the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 22). In some embodiments, the linker has the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 23). In some embodiments, the linker contains only glycine.

[0193]

[0176] In some embodiments, the peptide linker includes a protease cleavage site. In some embodiments, the protease cleavage site is a furin cleavage site.

[0194]

[0177] In some embodiments, the polypeptide linker is a poly-(Gly)n linker [wherein n is 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)] (SEQ ID NO: 48). In other embodiments, the linker is selected from the group consisting of dipeptides, tripeptides, and quadripeptides. In embodiments, the linker is a dipeptide selected from the group consisting of alanine-serine (AS), leucine-glutamic acid (LE), and serine-arginine (SR).

[0195]

[0178] In some embodiments, the linker is selected from GKSSGSGSESKS (SEQ ID NO: 157), GTSGSGKSSEGKG (SEQ ID NO: 158), GTSGSGKSSEGSGSTKG (SEQ ID NO: 159), GTSGSGKPGSGEGSTKG (SEQ ID NO: 160), EGKSSGSGSESKEF (SEQ ID NO: 161), SRSSG (SEQ ID NO: 162), and SGSSC (SEQ ID NO: 163).

[0196]

[0179] In some embodiments, the linker is a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A peptide. A 2A peptide is a class of 18-22 amino acid long peptides that induce ribosome skipping during protein translation in cells. In some embodiments, the 2A peptide is a T2A peptide having the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 164), a P2A peptide having the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 165), an E2A peptide having the amino acid sequence QCTNYALLKLAGDVESNPGP (SEQ ID NO: 166), or an F2A peptide having the amino acid sequence VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 167). In some embodiments, the 2A peptide has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity with any one of SEQ ID NOs. In some embodiments, the 2A peptide further comprises GSG (SEQ ID NO: 168) at its N-terminus.

[0197]

[0180] In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is selected from the group consisting of carbohydrate linkers, lipid linkers, fatty acid linkers, and polyether linkers.

[0198]

[0181] In some embodiments, the linker is a direct covalent link between the support and the AAV-binding polypeptide. In some embodiments, the linker is a direct covalent link between an amino acid residue of the AAV-binding polypeptide and an amino acid residue of the phase-behaving polypeptide. In some embodiments, the fusion protein comprises a phase-behaving polypeptide and an AAV-binding polypeptide. In some embodiments, the fusion protein further comprises one or more linkers as described herein. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a phase-behaving polypeptide, a linker, and an AAV-binding polypeptide. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, an AAV-binding polypeptide, a linker, and a phase-behaving polypeptide.

[0199]

[0182] In some embodiments, the AAV-binding polypeptide comprises one or more polycystic kidney disease domains (PKDs), where the PKDs are individually selected from PKD1, PKD2, PKD3, PKD4, and PKD5. In some embodiments, two or more PKDs are separated by a linker. In some embodiments, the AAV-binding polypeptide comprises a first PKD domain, a linker, and a second PKD domain from the N-terminus to the C-terminus. In some embodiments, the AAV-binding polypeptide comprises a first PKD domain, a linker, a second PKD domain, a linker, and at least one additional PKD domain from the N-terminus to the C-terminus.

[0200]

[0183] In some embodiments, the linker comprises a fragment of AAVR. In some embodiments, the linker is between 1 and 1200 amino acids in length. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, It contains 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, or 1250 amino acids. In some embodiments, the linker has an amino acid sequence selected from any one of SEQ ID NOs. 24-27.

[0201] AAV purification method

[0184] In some embodiments, the present disclosure provides a method for purifying AAV particles using the disclosed purification matrix.

[0202]

[0185] In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16) and (ii) an AAV-conjugated polypeptide comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16) and (ii) an AAV-conjugated polypeptide comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16) and (ii) an AAV-conjugated polypeptide comprising the amino acid sequence of SEQ ID NO: 64.

[0203]

[0186] In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16) and (ii) an AAV-conjugated polypeptide comprising the amino acid sequence of SEQ ID NO: 169. In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence of SEQ ID NO: 170 and (ii) an AAV-conjugated polypeptide comprising the amino acid sequence of SEQ ID NO: 169. In some embodiments, the purified matrix comprises the amino acid sequence of SEQ ID NO: 171, where the phase-behaving polypeptide comprises the amino acid sequence of SEQ ID NO: 170 and the AAV-conjugated polypeptide comprises the amino acid sequence of SEQ ID NO: 169.

[0204]

[0187] In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide containing the amino acid sequence of SEQ ID NO: 88 and (ii) an AAV-conjugated polypeptide containing the amino acid sequence of SEQ ID NO: 29. In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide containing the amino acid sequence of SEQ ID NO: 88 and (ii) an AAV-conjugated polypeptide containing the amino acid sequence of SEQ ID NO: 52. In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide containing the amino acid sequence of SEQ ID NO: 88 and (ii) an AAV-conjugated polypeptide containing the amino acid sequence of SEQ ID NO: 64.

[0205]

[0188] In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16), and (ii) an AAV-binding polypeptide comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16), (ii) an AAV-binding polypeptide comprising the amino acid sequence of SEQ ID NO: 29, (iii) optionally up to about 10 additional amino acids at the N-terminus and / or C-terminus of the phase-behaving polypeptide and / or the AAV-binding polypeptide, (iv) optionally C-terminal glycine, and (v) optionally N-terminal methionine.

[0206]

[0189] In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m(SEQ ID NO: 12) (wherein m is 16) and (ii) an AAV-binding polypeptide comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m(SEQ ID NO: 12) (wherein m is 16) and (ii) an AAV-binding polypeptide comprising the amino acid sequence of SEQ ID NO: 52 and (iii) an optional N-terminal methionine, (iv) an optional C-terminal glycine, and (v) an optional up to about 10 additional amino acids at the N-terminus and / or C-terminus of the phase-behaving polypeptide and / or the AAV-binding polypeptide.

[0207]

[0190] In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16), and (ii) an AAV-binding polypeptide comprising the amino acid sequence of SEQ ID NO: 64. In some embodiments, the purified matrix comprises (i) a phase-behaving polypeptide comprising the amino acid sequence (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16), (ii) an AAV-binding polypeptide comprising the amino acid sequence of SEQ ID NO: 64, (iii) an optional N-terminal methionine, (iv) an optional C-terminal glycine, and (v) an optional up to about 10 additional amino acids at the N-terminus and / or C-terminus.

[0208]

[0191] In some embodiments, the purified matrix contains a polypeptide having phase behavior that includes the amino acid sequence of SEQ ID NO: 88.

[0209]

[0192] In some embodiments, the purified matrix comprises an AAV-binding polypeptide having one of the amino acid sequences of SEQ ID NOs: 29, 52, or 64.

[0210]

[0193] In some embodiments, the purified matrix contains an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 64.

[0211]

[0194] In some embodiments, the purified matrix has the amino acid sequence of SEQ ID NO: 87. In some embodiments, the purified matrix has the amino acid sequence of SEQ ID NO: 171. In some embodiments, the purified matrix has one of the amino acid sequences of SEQ ID NOs: 171-174 or 87.

[0212]

[0195] In some embodiments, the method allows for the separation of AAV particles from one or more impurities (also referred to herein as contaminants). In some embodiments, the impurities are any chemical or biological product that is undesirable in the AAV composition. In some embodiments, the impurities are viruses, proteins, nucleic acids, lipopolysaccharides, lipids, mycotoxins, carbohydrates, and / or cells. In some embodiments, the cells include bacterial cells, animal cells, and human cells. In some embodiments, the cells are selected from the group consisting of bacterial cells, yeast cells, or animal cells such as mammalian cells. In some embodiments, the cells are chicken cells, mouse cells, guinea pig cells, rat cells, rabbit cells, goat cells, horse cells, sheep cells, dog cells, cat cells, or bovine cells. In some embodiments, the cells are human cells.

[0213]

[0196] In some embodiments, the Disclosure provides a method for purifying wild-type or mutant AAVs selected from AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh8, AAVrh10, AAVrh74, AAVhu.68, bird AAV, cattle AAV, dog AAV, horse AAV, sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAVs currently known or to be discovered later.

[0214]

[0197] In some embodiments, the Disclosure provides a method for purifying AAV particles, comprising contacting the AAV particles with a purification matrix comprising an AAV-binding polypeptide and a support, wherein the AAV-binding polypeptide is coupled to the support.

[0215]

[0198] In some embodiments, the present disclosure provides a method for purifying AAV particles, comprising contacting the AAV particles with a purification matrix comprising an AAV-binding polypeptide and a polypeptide exhibiting phase behavior, wherein the AAV-binding polypeptide is coupled to the polypeptide exhibiting phase behavior.

[0216]

[0199] In some embodiments, a method for purifying AAV particles comprises contacting the AAV particles with a purification matrix, the purification matrix comprising (i) an AAV-binding polypeptide and (ii) a polypeptide having phase behavior; the AAV particles bind to the purification matrix to form a complex; the size of the complex increases by a first environmental factor; the complex is separated from at least one contaminant based on size; and the AAV particles are separated from the purification matrix by a second environmental factor.

[0217]

[0200] In some embodiments, a method for purifying AAV particles comprises contacting the AAV particles with a purification matrix, the purification matrix comprising (i) an AAV-binding polypeptide and (ii) a polypeptide having phase behavior; the AAV particles bind to the matrix to form a complex; the size of the complex increases; the complex is separated from at least one contaminant based on size; and the AAV particles are separated from the matrix by an environmental factor.

[0218]

[0201] In some embodiments, the methods described herein involve the formation of a complex between AAV particles and a purified matrix. In some embodiments, the AAV particles reversibly bind to the purified matrix. In some embodiments, the AAV particles bind to the purified matrix described herein through non-covalent interactions.

[0219]

[0202] In some embodiments, a complex is formed between the purified matrix described herein and wild-type or mutant AAV particles of a serotype selected from AAV1, AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh8, AAVrh10, AAVrh74, AAVhu.68, bird AAV, cattle AAV, dog AAV, horse AAV, sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV serotypes currently known or to be discovered later.

[0220]

[0203] In some embodiments, a complex is formed between the purified matrix and AAV particles having wild-type AAV capsid protein. In some embodiments, the complex is formed between the purified matrix and AAV particles having mutant AAV capsid protein. In some embodiments, the complex is formed between the purified matrix and human AAV particles. In some embodiments, the complex is formed between the purified matrix and primate AAV particles. In some embodiments, the complex is formed between the purified matrix and bovine AAV particles. In some embodiments, the complex is formed between the purified matrix and bird AAV particles. In some embodiments, the complex is formed between the purified matrix and orangutan AAV particles. In some embodiments, the complex is formed between the purified matrix and monkey AAV particles. In some embodiments, the complex is formed between the purified matrix and mouse AAV particles.

[0221]

[0204] In some embodiments, environmental factors are used to increase the size of the AAV-purified matrix complex. As used herein, the term “increase in size” may refer to an increase in the diameter of the complex or an increase in the mass of the complex. In some embodiments, the increase in size is an increase in the molar mass of the complex. In some embodiments, the increase in size is an increase in the hydrodynamic radius of the complex.

[0222]

[0205] In some embodiments, the size of the complexes described herein increases after the application of environmental factors. In some embodiments, the size of the complexes formed between the purified matrix and the bioagent, contaminant, and / or other molecules increases. In some embodiments, the size of the initial complex increases as a result of aggregation of multiple complexes. In some embodiments, multiple complexes aggregate due to the self-assembly of the purified matrix. In some embodiments, multiple complexes aggregate due to the application of environmental factors. In some embodiments, the size increase is stabilized by non-covalent interactions between multiple protein-based purified matrix molecules. In some embodiments, the size increase is stabilized by non-covalent interactions between polypeptides with phase behavior. In some embodiments, non-covalent interactions are dipole forces, van der Waals forces, London dispersion forces, hydrogen bonds, hydrophobic interactions, and / or electrostatic interactions.

[0223]

[0206] In some embodiments, the method of the present disclosure provides the formation of a plurality of complexes in a mixture. In some embodiments, the size of all complexes increases. In some embodiments, the size of some complexes increases while the size of other complexes remains constant. In some embodiments, the size of one complex increases while the size of other complexes remains constant.

[0224]

[0207] In some embodiments, the size of the complex increases by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, at least about 100-fold, or more. In some embodiments, the size of the initial complex increases by at least about 2-fold. In some embodiments, the size of the initial complex increases by at least about 5-fold. In some embodiments, the size of the initial complex increases by at least about 10-fold. In some embodiments, the size of the initial complex increases by at least about 25-fold.

[0225]

[0208] In some embodiments, the increase in the size of the complex can be visually observed with the naked eye. For example, the increase in the size of the complex may cause a change in the color, clarity, viscosity of the composition containing the complex, and / or a change in the solubility of the complex (e.g., precipitation from a solution), where such changes can be observed without the use of any special equipment by a human.

[0226]

[0209] In some embodiments, one of ordinary skill in the art can measure the increase in the size of the complex by methods known in the art. In some embodiments, the increase in the size of the complex can be measured using a technique selected from the group consisting of X-ray scattering, small-angle X-ray scattering, wide-angle X-ray scattering, dynamic light scattering, analytical ultracentrifugation, size exclusion chromatography, and photon correlation spectroscopy.

[0227]

[0210] In some embodiments, the environmental factor is a first environmental factor added to a composition containing AAV particles and a purification matrix. Examples of environmental factors are provided throughout this disclosure.

[0228]

[0211] In some embodiments, the AAV-purified matrix complex is separated from one or more impurities by washing the AAV-purified matrix complex. In some embodiments, washing the AAV-purified matrix does not prevent the AAV particles from binding to the purified matrix. In some embodiments, the AAV-purified matrix complex is washed with a buffer. Non-limiting examples of buffers include sodium acetate, physiological saline, glycine HCl, cacodylate buffer, tris-HCl, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), citrate, phosphate buffer, tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), and tris(hydroxymethyl)aminomethane (Tris). In some embodiments, the buffer comprises one or more of arginine, histidine, urea, pluronic acid, and triton-x-100. In some embodiments, the AAV-purified matrix complex is washed with a solvent. Non-limiting examples of solvents include acetone, acetonitrile, dimethylformamide, water, ethanol, toluene, methyl acetate, and ethyl acetate.

[0229]

[0212] In some embodiments, the AAV-purified matrix complex is separated from at least one impurity based on size. In some embodiments, the AAV-purified matrix complex is separated from at least one impurity based on diameter. In some embodiments, the AAV-purified matrix is ​​separated from at least one impurity based on radius. In some embodiments, the AAV-purified matrix is ​​separated from at least one impurity based on mass. In some embodiments, the AAV-purified matrix is ​​separated from at least one impurity based on molar mass. In some embodiments, the AAV-purified matrix is ​​separated from at least one impurity based on size by using a technique selected from the group consisting of centrifugation, tangential flow filtration, analytical ultracentrifugation, membrane chromatography, high-performance liquid chromatography, size exclusion chromatography, normal flow filtration, ultrasonic separation, centrifugation, countercurrent centrifugation, and high-performance protein liquid chromatography.

[0230] In some embodiments, the AAV-purified matrix complex is separated from at least one impurity based on size using centrifugation. In some embodiments, between about 100 relative centrifugal force (RCF) and about 16,000 RCF, such as between about 500 and about 16,000 RCF, between about 1,000 RCF and 16,000 RCF, is applied to separate the AAV-purified matrix complex from at least one impurity. In some embodiments, at least 500 relative centrifugal force (RCF), such as at least about 500 RCF, at least about 600 RCF, at least about 700 RCF, at least about 800 RCF, at least about 900 RCF, at least about 1000 RCF, at least about 2000 RCF, at least about 3000 RCF, at least about 3500 RCF, at least about 4000 RCF, at least about 5000 RCF, at least about 6000 RCF, at least about 7000 RCF, at least about 8000 RCF, at least about 9000 RCF, at least about 10,000 RCF, at least about 11,000 RCF, at least about 12,000 RCF, at least about 13,000 RCF, at least about 14,000 RCF, at least about 15,000 RCF, at least about 16,000 RCF, at least about 17,000 RCF, at least about 18,000 RCF, at least about 19,000 RCF, or at least about 20,000 RCF is applied to separate the AAV-purified matrix complex from at least one impurity.

[0231]

[0214] In some embodiments, the AAV-purified matrix is ​​separated from at least one impurity on a size basis using TFF. In some embodiments, the AAV-purified matrix may be separated from at least one impurity on a size basis using TFF, a step also referred to herein as “diafiltration”. Diafiltration includes both washing and elution steps. Washing removes impurities contained in the composition containing the AAV-purified matrix. Elution separates the purified AAV particles from the purified matrix. In some embodiments, the AAV-purified matrix is ​​concentrated using TFF. In some embodiments, the concentration of the AAV-purified matrix in the composition may be increased using TFF, a step also referred to herein as “concentration”.

[0232]

[0215] Tangential flow filtration employs both microfiltration and ultrafiltration membranes for molecular separation and / or concentration. Microfiltration membranes typically have pore sizes between 0.1 μm and 10 μm. Ultrafiltration membranes typically have smaller pore sizes than microfiltration membranes, between 0.001 μm and 0.1 μm. In some embodiments, the methods of the present disclosure utilize membranes with pore sizes between approximately 0.001 μm and approximately 10 μm. In some embodiments, the film has pore diameters of approximately 0.001 μm, 0.01 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm (including all values ​​and ranges in between). In some embodiments, the film has a pore diameter of approximately 0.1 μm. In some embodiments, the film has a pore diameter of approximately 0.2 μm.

[0233]

[0216] In some embodiments, the film is made of hydrophilic polyvinylidene polyfluoride (poly(vinylildene difluoride)) (PVDF), polyetheresulfone (PES), cellulose phosphate, diethylaminoethylcellulose, polysufone, regenerated cellulose, nylon, cellulose nitrate, cellulose acetate, PEGylated PES, and sulfonated PES.

[0234]

[0217] In TFF, the membrane is placed tangentially to the flow of the mixed fluid, and the mixed fluid flows tangentially over the first surface of the membrane. At the same time, the fluid medium is placed in contact with the second surface of the membrane. The differential pressure between the membranes is the force that pushes the fluid through the membrane and carries the permeable molecules along with it.

[0235]

[0218] In some embodiments, size-based separation of AAV-purified matrix complexes from one or more contaminants or impurities is performed using a TFF with a transmembrane pressure differential between approximately 0.1 bar and approximately 3 bar. In some embodiments, the intermembrane differential pressure is approximately 0.1 bar, approximately 0.2 bar, approximately 0.3 bar, approximately 0.4 bar, approximately 0.5 bar, approximately 0.6 bar, approximately 0.7 bar, approximately 0.8 bar, approximately 0.9 bar, approximately 1.0 bar, approximately 1.1 bar, approximately 1.2 bar, approximately 1.3 bar, approximately 1.4 bar, approximately 1.5 bar, approximately 1.6 bar, approximately 1.7 bar, approximately 1.8 bar, approximately 1.9 bar, approximately 2.0 bar, approximately 2.1 bar, approximately 2.2 bar, approximately 2.3 bar, approximately 2.4 bar, approximately 2.5 bar, approximately 2.6 bar, approximately 2.7 bar, approximately 2.8 bar, approximately 2.9 bar, or approximately 3.0 bar (including all values ​​and ranges in between). In some embodiments, the differential pressure between membranes is approximately 1.5 bar.

[0236]

[0219] In some embodiments, the cross-flow velocity is adjusted to improve the separation of the AAV particles described herein and the purified matrix from one or more contaminants. The cross-flow velocity is the rate at which the solution flows through the supply channel and across the membrane. This provides a force that pushes molecules that may restrict the flow of the filtrate. In some embodiments, the cross-flow velocity is between about 500 L / m² / h and about 2000 L / m² / h. In some embodiments, the cross-flow velocity is between approximately 500 L / m² / h, 600 L / m² / h, 700 L / m² / h, 800 L / m² / h, 900 L / m² / h, 1000 L / m² / h, 1100 L / m² / h, 1200 L / m² / h, 1300 L / m² / h, 1400 L / m² / h, 1500 L / m² / h, 1600 L / m² / h, 1700 L / m² / h, 1800 L / m² / h, 1900 L / m² / h, or 2000 L / m² / h (including all values ​​and ranges in between). In some embodiments, the cross-flow velocity is approximately 960 L / m² / h. In some embodiments, TFF separation is performed by using a membrane that retains a complex containing a purified matrix and AAV particles, but allows contaminants to pass through.

[0237]

[0220] In some embodiments, AAV particles are eluted from the AAV-purified matrix complex by changing the pH of the composition containing the AAV-purified matrix complex. In some embodiments, the pH is changed to about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3. Increase by 0, approximately 3.1, approximately 3.2, approximately 3.3, approximately 3.4, approximately 3.5, approximately 3.6, approximately 3.7, approximately 3.8, approximately 3.9, approximately 4.0, approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, or approximately 6.0 units. In some embodiments, the pH is set to approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3. The pH is reduced by approximately 0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 units. In some embodiments, AAV particles are eluted from the AAV-purified matrix complex at a pH of approximately 2. In some embodiments, AAV particles are eluted from the AAV-purified matrix complex at a pH of approximately 3.

[0238]

[0221] In some embodiments, AAV particles are eluted from the AAV-purified matrix complex by changing the temperature of the composition containing the AAV-purified matrix complex. In some embodiments, the temperature is raised by 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In some embodiments, the temperature is lowered by approximately 0.5°C, 1°C, 2°C, 3°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, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0239]

[0222] In some embodiments, AAV particles are eluted from the AAV-purified matrix complex by changing the ionic strength of the composition containing the AAV-purified matrix complex. In some embodiments, the change in ionic strength is brought about by increasing the salt concentration. In some embodiments, the change in ionic strength is brought about by decreasing the salt concentration. Non-limiting examples of salts include sodium chloride, potassium chloride, ammonium chloride, sodium acetate, sodium citrate, glycine, arginine, copper sulfate, sodium iodide, ammonium sulfate, and sodium sulfate. In some embodiments, dialysis is used to change the salt concentration in a composition containing a protein-based purified matrix and a bioagent, contaminant, and / or molecule.

[0240]

[0223] In some embodiments, AAV particles are eluted from the AAV-purified matrix complex by adding a reducing agent to a composition containing the AAV-purified matrix complex. In some embodiments, one or more reducing agents are selected from the group consisting of dithiothreitol (DTT), 2-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP), hydrazine, boron hydride, amine boranes, lower alkyl-substituted amine boranes, triethanolamine, and N,N,N',N'-tetramethylethylenediamine (TEMED).

[0241]

[0224] In some embodiments, an environmental factor is used to elute AAV particles from the AAV-purified matrix complex. In some embodiments, the environmental factor is a first environmental factor added to a composition comprising AAV particles and the AAV-purified matrix complex. In some embodiments, the environmental factor is a second environmental factor added to a composition comprising AAV particles and the AAV-purified matrix complex. Non-limiting examples of environmental factors are provided throughout this disclosure.

[0242]

[0225] In some embodiments, the method for purifying AAV particles using the purification matrix described herein is completed in about 30 minutes to about 24 hours. In some embodiments, the method described herein is completed in about 30 minutes to about 24 hours. In some embodiments, the method is completed in about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. In some embodiments, the method for purifying AAV particles using the purification matrix described herein is completed in about 2 hours to about 10 hours.

[0243]

[0226] In some embodiments, the purification yield of AAV particles is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0244]

[0227] In some embodiments, the AAV particles are purified to a purity of at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0245]

[0228] In some embodiments, the methods described herein allow for the purification of at least 0.1 kg, at least about 0.2 kg, at least about 0.3 kg, at least about 0.4 kg, at least about 0.5 kg, at least about 0.6 kg, at least about 0.7 kg, at least about 0.8 kg, at least about 0.9 kg, at least about 1 kg, at least about 2 kg, at least about 3 kg, at least about 4 kg, at least about 5 kg, at least about 6 kg, at least about 7 kg, at least about 8 kg, at least about 9 kg, at least about 10 kg, or more of AAV (including all values ​​and ranges in between).

[0246]

[0229] In some embodiments, the AAV particles retain their biological activity and / or structure. In some embodiments, purified AAV particles exhibit enhanced biological activity.

[0247]

[0230] In some embodiments, purified AAV particles retain about 95% of their infectivity after purification. In some embodiments, purified AAV particles retain about 96% of their infectivity after purification. In some embodiments, purified AAV particles retain about 97% of their infectivity after purification. In some embodiments, purified AAV particles retain about 98% of their infectivity after purification. In some embodiments, purified AAV particles retain about 99% of their infectivity after purification. In some embodiments, purified AAV particles retain about 100% of their infectivity after purification.

[0248]

[0231] In some embodiments, compositions comprising AAV particles purified using the matrix disclosed herein are enriched with “encapsulated” AAV particles. Encapsulated AAV particles contain desired genomic material (e.g., AAV genome or transfer cassette) encapsulated within an AAV capsid. Empty AAV particles lack desired genomic material.

[0249]

[0232] There are several techniques for determining the relative amounts of encapsulated particles and empty particles in a sample. For example, the total number of AAV particles present in a sample eluted from the purified matrix of this disclosure may be determined using ELISA. In some embodiments, the number of viral genomes present in a sample eluted from the purified matrix of this disclosure may be determined using quantitative polymerase chain reaction (qPCR). In some embodiments, the qPCR primers target reverse terminal repeat (ITR) sequences. qPCR using primers that target ITR sequences is referred to herein as "ITR qPCR". The ratio of the number of viral genomes to the total number of AAV particles provides an approximation of the ratio of encapsulated particles to the total number of particles. In some embodiments, the relative ratio of encapsulated particles to the total number of particles is approximated using qPCR:ELISA values ​​(e.g., number of viral genomes present in the sample:total number of AAV particles present in the sample).

[0250]

[0233] In some embodiments, the qPCR:ELISA value increases after purification with the purification matrix of the present disclosure. In some embodiments, after purification with the purification matrix of the present disclosure, the qPCR:ELISA value of the sample is at least about 25%, such as at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% increased compared to the qPCR:ELISA value of the sample before purification. In some embodiments, after purification with the purification matrix of the present disclosure, the qPCR:ELISA value of the sample is at least about 50% increased compared to the qPCR:ELISA value of the sample before purification.

[0251] Method for stabilizing AAV particles

[0234] The inventors have discovered that the purification matrix described herein can unexpectedly help in the stabilization of AAV particles during their production, purification, and / or storage. As used herein in connection with AAV particles, the term "stabilize" or "stabilization" refers to the ability of the purification matrix to reduce the degradation or aggregation of an AAV sample containing multiple AAV particles, prevent the AAV particles from binding to other proteins (e.g., the AAV receptor of cells), or enhance the synthesis by the production strain cells.

[0252]

[0235] Accordingly, in some embodiments, AAV particles are brought into contact with a purification matrix during their production, purification, or storage. In some embodiments, a method for increasing the yield of AAV particles during their production includes culturing AAV-producing cells in the presence of a purification matrix. In some embodiments, a method for stabilizing AAV particles during their production includes culturing AAV-producing cells in the presence of a purification matrix. In some embodiments, a method for stabilizing AAV particles during their purification includes bringing AAV particles into contact with a purification matrix during their purification. In some embodiments, a method for stabilizing AAV particles during their storage includes storing AAV particles in the presence of a purification matrix. In some embodiments, a method for increasing the shelf life of AAV particles includes storing AAV particles in the presence of a purification matrix.

[0253]

[0236] For example, the purified matrix may be brought into contact with the AAV particles during production under culture. The AAV particles are typically produced in a production cell line such as HEK293 cells or Sf9 cells. In some embodiments, the production cell line is transfected with one or more plasmids containing various genes necessary for AAV production (e.g., a triple transfection protocol). In some embodiments, the production cell line is infected with a baculovirus construct containing various genes necessary for AAV production. Adding the purified matrix to the AAV-producing cells under culture (e.g., by adding it to tissue culture medium) may increase the yield and / or quality of the AAV particles obtained in this step. In some embodiments, the purified matrix is ​​approximately 1 μM to approximately 1 mM, for example, approximately 1 μM, approximately 2 μM, approximately 3 μM, approximately 4 μM, approximately 5 μM, approximately 6 μM, approximately 7 μM, approximately 8 μM, approximately 9 μM, approximately 10 μM, approximately 20 μM, approximately 30 μM, approximately 40 μM, approximately 50 μM, approximately 60 μM, approximately 70 μM, approximately 80 μM, approximately 90 μM, approximately 100 μM, approximately 150 μM, approximately The purified matrix may be added to the culture at concentrations of 200 μM, approximately 250 μM, approximately 300 μM, approximately 350 μM, approximately 400 μM, approximately 450 μM, approximately 500 μM, approximately 550 μM, approximately 600 μM, approximately 650 μM, approximately 700 μM, approximately 750 μM, approximately 800 μM, approximately 850 μM, approximately 900 μM, approximately 950 μM, or approximately 1 mM (including all values ​​and ranges in between). In some embodiments, the purified matrix is ​​added to the culture at a concentration of approximately 10 μM. In some embodiments, the purified matrix is ​​added to the culture at a concentration of approximately 100 μM. Without being constrained by any theory, it is thought that the purified matrix can bind to and / or physically surround AAV particles as they are being produced, thereby preventing AAV particles from binding to other proteins, including cellular receptors for AAV particles. Therefore, AAV produced by cultured cells and secreted into the culture medium will not be able to reinfect the producing cell line in the presence of a purified matrix. In some embodiments, adding a purified matrix to AAV-producing cell lines under culture may increase the yield of AAV particles.For example, adding purified matrix to AAV-producing cell lines under culture may result in an increase in viral titer of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, or more, compared to cells cultured without the purified matrix.

[0254]

[0237] As another example, after a purified AAV sample has been prepared (by the methods described herein or by other methods known in the art), the purified matrix may be added to the sample before storage (e.g., before freezing). Without being constrained by any theory, it is thought that the purified matrix binds to and / or physically surrounds the AAV particles, thereby preventing them from agglomerating with other AAV particles, and also helps protect the AAV particles from degradation, particularly during multiple freeze-thaw cycles. Aggregation of AAV particles may be observed visually by microscopy and / or by techniques selected from the group consisting of X-ray scattering, laser diffraction, analytical ultracentrifugation, dynamic light scattering, nanoparticle tracking analysis, resonance mass spectrometry, size exclusion chromatography, gel permeation chromatography, light shielding methods, and combinations thereof. In some embodiments, AAV particles may be frozen and stored in the presence of a purification matrix at temperatures between approximately -80°C and approximately 40°C, for example, approximately -80°C, approximately -75°C, approximately -70°C, approximately -65°C, approximately -60°C, approximately -55°C, approximately -50°C, approximately -45°C, approximately -40°C, approximately -35°C, approximately -30°C, approximately -25°C, approximately -20°C, approximately -15°C, approximately -10°C, approximately -5°C, approximately 0°C, approximately 4°C, approximately 5°C, approximately 10°C, approximately 15°C, approximately 20°C, approximately 25°C, approximately 30°C, approximately 35°C, or approximately 40°C.

[0255]

[0238] In some embodiments, when AAV particles are stored in the presence of a purification matrix, the shelf life of the AAV particles is extended by at least about 10% compared to samples stored in the absence of a purification matrix. For example, in some embodiments, the shelf life is extended by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% compared to the shelf life of AAV particles stored in the absence of a purification matrix at approximately the same temperature.

[0256]

[0239] In some embodiments, AAV particles are stored at about -80°C in the presence of a purification matrix. In some embodiments, AAV particles are stored at about -20°C in the presence of a purification matrix. In some embodiments, AAV particles are stored at about 4°C in the presence of a purification matrix. In some embodiments, when AAV particles are stored at about -80°C, about -20°C, or about 4°C in the presence of a purification matrix, the shelf life of the AAV is extended by at least about 10% compared to when it is stored in the absence of a purification matrix. For example, the shelf life of AAV particles may be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% longer than the shelf life of AAV stored at the same temperature in the absence of a purification matrix. As used herein, increased shelf life may refer to the increased length of time that AAV particles are stored and still retain substantially the same level of infectivity.

[0257]

[0240] In some embodiments, stabilizing AAV particles with a purification matrix during their production, purification, and / or storage may increase the yield of "encapsulated" AAV particles. Thus, in some embodiments, the improved stabilization of AAV particles when in contact with a purification matrix can be measured by quantifying the number of encapsulated AAV particles compared to empty particles. In some embodiments, using a purification matrix during the production, purification, and / or storage of AAV samples may result in increased qPCR:ELISA values ​​compared to AAV samples not produced, purified, and / or stored in the presence of a purification matrix.

[0258] environmental factors

[0241] In some embodiments, the methods of the present disclosure provide one or more environmental factors to a composition comprising a purified matrix and AAV particles. In some embodiments, one or more environmental factors are applied to a composition comprising a purified matrix and AAV particles. The application of the environmental factors causes a change in the composition comprising the purified matrix and AAV particles. In some embodiments, the environmental factors are used to increase the size of the AAV-purified matrix complex. In some embodiments, the environmental factors are used to remove one or more impurities from a composition comprising the AAV-purified matrix complex. In some embodiments, the environmental factors are used to elute one or more AAV particles from the purified matrix complex. In some embodiments, one or more environmental factors cause an increase in the size of a complex between a protein-based purified matrix and a biologic, contaminant, and / or molecule. In some embodiments, one or more environmental factors cause aggregation of polypeptides with phase behavior. In some embodiments, one or more environmental factors allow the AAV particles to retain their innate structure, function, and activity. In some embodiments, one or more environmental factors allow the AAV particles to enhance their innate structure, function, and activity.

[0259]

[0242] In some embodiments, the environmental factor is a change in temperature. In some embodiments, the temperature is increased by about 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, or about 40°C. In some embodiments, the temperature is lowered by approximately 0.5°C, 1°C, 2°C, 3°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, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0260]

[0243] In some embodiments, the environmental factor is a change in pH. In some embodiments, the pH is set to about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3. Increase by 0, approximately 3.1, approximately 3.2, approximately 3.3, approximately 3.4, approximately 3.5, approximately 3.6, approximately 3.7, approximately 3.8, approximately 3.9, approximately 4.0, approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, or approximately 6.0 units.

[0261]

[0244] In some embodiments, the pH is set to approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1.0, approximately 1.1, approximately 1.2, approximately 1.3, approximately 1.4, approximately 1.5, approximately 1.6, approximately 1.7, approximately 1.8, approximately 1.9, approximately 2.0, approximately 2.1, approximately 2.2, approximately 2.3, approximately 2.4, approximately 2.5, approximately 2.6, approximately 2.7, approximately 2.8, approximately 2.9, approximately 3. Reduce by 0, approximately 3.1, approximately 3.2, approximately 3.3, approximately 3.4, approximately 3.5, approximately 3.6, approximately 3.7, approximately 3.8, approximately 3.9, approximately 4.0, approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, or approximately 6.0 units.

[0262]

[0245] In some embodiments, the environmental factor is a change in ionic strength. In some embodiments, the change in ionic strength is brought about by increasing the concentration of the salt. In some embodiments, the change in ionic strength is brought about by decreasing the concentration of the salt. Non-limiting examples of salts include sodium chloride, potassium chloride, magnesium chloride, calcium chloride, ammonium chloride, sodium acetate, sodium citrate, copper sulfate, sodium iodide, and sodium sulfate. In some embodiments, the salt concentration is between about 0.1M and about 5M, for example, about 0.1M, about 0.2M, about 0.3M, about 0.4M, about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1M, about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.8M, about 1.9M, about 2M, about 2.1M, about 2.2M, about 2.3M. The concentrations are approximately 2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M, 3 M, 3.1 M, 3.2 M, 3.3 M, 3.4 M, 3.5 M, 3.6 M, 3.7 M, 3.8 M, 3.9 M, 4 M, 4.1 M, 4.2 M, 4.3 M, 4.4 M, 4.5 M, 4.6 M, 4.7 M, 4.8 M, 4.9 M, or 5 M. In some embodiments, the salt has a concentration of 0.6 M. In some embodiments, dialysis is used to change the concentration of the salt in a composition comprising a protein-based purification matrix and a bioagent, contaminant, and / or molecule.

[0263]

[0246] In some embodiments, the environmental factor is the addition of a cofactor. Non-limiting examples of cofactors include calcium, magnesium, cobalt, copper, zinc, iron, manganese, selenium, molybdenum, potassium, coenzyme A (CoA), nucleoside triphosphates, and vitamins (e.g., vitamins A, B, C, D, or F). In some embodiments, the cofactor is calcium. In some embodiments, the nucleoside triphosphate is adenosine triphosphate, uridine triphosphate, guanosine triphosphate, cytidine triphosphate, or thymidine triphosphate. In some embodiments, the vitamin is a fat-soluble substance. In some embodiments, the vitamin is water-soluble. Non-exclusive examples of vitamins include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine, or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid), vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, K1, and K2, folic acid, and biotin.

[0264]

[0247] In some embodiments, the environmental factor is a change in the concentration of the protein-based purified matrix. In some embodiments, the environmental factor is a change in the concentration of the bioagent, contaminants, and / or other molecules.

[0265]

[0248] In some embodiments, the environmental factor is a change in pressure of the composition comprising a protein-based purification matrix and a bioagent, contaminants, and / or molecules. In some embodiments, the change in pressure may be achieved by increasing or decreasing the volume of the composition.

[0266]

[0249] In some embodiments, the environmental factor is the addition of one or more surfactants. In some embodiments, the one or more surfactants are free fatty acid salts, soaps, fatty acid sulfonates such as sodium lauryl sulfate, ethoxylated compounds such as ethoxylated propylene glycol, lecithin, polygluconates, quaternary ammonium salts, lignin sulfonates, 3-((3-coramidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), sugars including sucrose and glucose, Triton X-100, and NP-40. In some embodiments, the surfactants are anionic, nonionic, or amphoteric.

[0267]

[0250] In some embodiments, the environmental factor is the addition of one or more molecular crowding agents. Non-limiting examples of molecular crowding agents include polyethylene glycol, dextran, and Ficol. PEG may include PEG400, PEG1450, PEG3000, PEG8000, and PEG10000.

[0268]

[0251] In some embodiments, the environmental factor is the addition of one or more oxidizing agents. Non-limiting examples of oxidizing agents include hydrogen peroxide, hydrophilic or hydrophobic activated hydrogen peroxide, pre-formed peracids, monopersulfates, or hypochlorites.

[0269]

[0252] In some embodiments, the environmental factor is the addition of one or more reducing agents. In some embodiments, the one or more reducing agents are selected from the group consisting of dithiothreitol (DTT), 2-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP), hydrazine, boron hydride, amine boranes, lower alkyl-substituted amine boranes, triethanolamine, and N,N,N',N'-tetramethylethylenediamine (TEMED). In some embodiments, the environmental factor is the addition of one or more denaturing agents. Non-limiting examples of denaturing agents include urea, guanidine hydrochloride, guanidine, sodium salicylate, dimethyl sulfoxide, and propylene glycol.

[0270]

[0253] In some embodiments, the environmental factor is the addition of one or more enzymes. Non-limiting examples of enzymes include nucleases such as proteases, kinases, phosphatases, synthetases, transferases, restriction endonucleases, lyases, isomerases, dehydrogenases, decarboxylases, and lipases.

[0271]

[0254] In some embodiments, the environmental factor is the application of electromagnetic waves. In some embodiments, the environmental factor is the application of light. In some embodiments, the electromagnetic waves have wavelengths between about 0.0001 nm and about 100 m. In some embodiments, the electromagnetic waves are selected from the group consisting of gamma rays, X-rays, ultraviolet rays, visible light, infrared rays, and radio waves. In some embodiments, the electromagnetic waves are gamma rays. In some embodiments, the gamma rays have wavelengths between about 0.0001 nm and about 0.01 nm, for example, 0.0001 nm, 0.0005 nm, 0.001 nm, 0.002 nm, 0.003 nm, 0.004 nm, 0.005 nm, 0.006 nm, 0.007 nm, 0.008 nm, 0.009 nm, and 0.01 nm. In some embodiments, the X-rays have wavelengths between approximately 0.01 nm and approximately 10 nm, for example, approximately 0.01 nm, 0.02 nm, 0.03 nm, 0.04 nm, 0.05 nm, 0.06 nm, 0.07 nm, 0.08 nm, 0.09 nm, 0.10 nm, 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or approximately 10 nm. In some embodiments, ultraviolet radiation has wavelengths between approximately 10 nm and approximately 400 nm, for example, approximately 10 nm, approximately 20 nm, approximately 30 nm, approximately 40 nm, approximately 50 nm, approximately 60 nm, approximately 70 nm, approximately 80 nm, approximately 90 nm, approximately 100 nm, approximately 150 nm, approximately 200 nm, approximately 250 nm, approximately 280 nm, approximately 300 nm, approximately 350 nm, or approximately 400 nm. In some embodiments, visible light waves have wavelengths between approximately 400 nm and approximately 800 nm, for example, approximately 400 nm, approximately 450 nm, approximately 500 nm, approximately 550 nm, approximately 600 nm, approximately 650 nm, approximately 700 nm, approximately 750 nm, or approximately 800 nm.In some embodiments, the infrared radiation has wavelengths between approximately 800 nm and approximately 0.1 cm, for example, approximately 800 nm, approximately 1 μm, approximately 2 μm, approximately 3 μm, approximately 4 μm, approximately 5 μm, approximately 6 μm, approximately 7 μm, approximately 8 μm, approximately 9 μm, approximately 10 μm, approximately 20 μm, approximately 30 μm, approximately 40 μm, approximately 50 μm, approximately 60 μm, approximately 70 μm, approximately 80 μm, approximately 90 μm, approximately 100 μm, approximately 200 μm, approximately 300 μm, approximately 400 μm, approximately 500 μm, approximately 600 μm, approximately 700 μm, approximately 800 μm, approximately 900 μm, or approximately 0.1 cm. In some embodiments, radio waves have wavelengths between approximately 0.1 cm and 100 m, for example, approximately 0.1 cm, approximately 1 cm, approximately 10 cm, approximately 100 cm, approximately 1000 cm, approximately 2000 cm, approximately 3000 cm, approximately 4000 cm, approximately 5000 cm, approximately 6000 cm, approximately 7000 cm, approximately 8000 cm, approximately 9000 cm, or approximately 100 m.

[0272]

[0255] In some embodiments, the environmental factor is the application of sound waves. In some embodiments, the sound waves have frequencies between approximately 1 Hz and 2000 kHz. In some embodiments, the sound waves have frequencies of approximately 1 Hz, approximately 5 Hz, approximately 10 Hz, approximately 20 Hz, approximately 30 Hz, approximately 40 Hz, approximately 50 Hz, approximately 60 Hz, approximately 70 Hz, approximately 80 Hz, approximately 90 Hz, approximately 100 Hz, approximately 200 Hz, approximately 300 Hz, approximately 400 Hz, approximately 500 Hz, approximately 600 Hz, approximately 700 Hz, approximately 800 Hz, approximately 900 Hz, approximately 1 kHz, approximately 100 kHz, approximately 200 kHz, It has frequencies of approximately 300kHz, 400kHz, 500kHz, 600kHz, 700kHz, 800kHz, 900kHz, 1000kHz, 1100kHz, 1200kHz, 1300kHz, 1400kHz, 1500kHz, 1600kHz, 1700kHz, 1800kHz, 1900kHz, or 2000kHz. [Examples]

[0273] Example 1. Development of AAV-binding polypeptides

[0256] Site-directed mutagenesis of the wild-type AAVR ectodomain is performed. More specifically, an AAV-binding polypeptide containing a certain amino acid sequence is selected from one of sequence numbers 33-47, and it is subjected to site-directed mutagenesis of one or more amino acid residues using standard cloning technology. The affinity of the AAV-binding polypeptide is characterized. An AAV-binding polypeptide having a binding affinity of at least 50 nM or more to AAV is selected. The ability of various environmental factors, including changes in pH and the addition of salt, to disrupt the binding of the AAV-binding polypeptide to AAV is characterized.

[0274] Example 2. Purification of AAV using a purification matrix containing AAV-binding polypeptide and a polypeptide exhibiting phase behavior.

[0257] Prepare and characterize a purified matrix containing the AAV-binding polypeptide of Example 1. More specifically, a fusion protein containing (i) the AAV-binding polypeptide of Example 1 and (ii) a polypeptide exhibiting phase behavior (e.g., ELP) is expressed in Escherichia coli according to a standard protocol. Characterize the affinity of the fusion protein to AAV particles using isothermal titration calorimetry. Determine the transition temperature of the fusion protein using ultraviolet-visible spectrophotometry. Next, prepare a purified matrix by covalently coupling the fusion protein to a support in a linker using a standard technique. The support is a bead, such as a magnetic bead or a porous bead.

[0275]

[0258] Next, the purified matrix is ​​incubated separately with various purified AAV samples, including AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9. The purified matrix forms complexes with the various AAV particles.

[0276]

[0259] Test various molar ratios of the purified matrix to AAV particles to determine the optimal ratio for purification (i.e., complete capture of AAV particles). Apply environmental factors (e.g., addition of salts such as sodium chloride or ammonium sulfate) to the composition containing the purified matrix and the bioagent to increase the size of the protein-based purified matrix.

[0277]

[0260] AAV particles are separated from the protein-based purification matrix using both tangential flow filtration and centrifugation. Although both centrifugation and tangential flow filtration can separate the biologics from the protein-based purification matrix, tangential flow filtration is preferred because it allows for rapid purification of thousands of liters of sample volume without the need for a special centrifuge.

[0278]

[0261] TFF is carried out under standard conditions—for example, a membrane differential pressure of 1.5 bar and a cross-flow velocity of 960 L / m2 / h. Standard TFF membranes are used, such as a 0.1 μm hydrophilic poly(vinylidene difluoride) (PVDF) membrane.

[0279]

[0262] Adjust the pH (e.g., to an acidic pH such as 4.5) to separate (i.e., elute) the purified matrix from the AAV. Characterize the purity of the AAV by size exclusion chromatography. The protein-based purified matrix is ​​then reused for further purification rounds.

[0280] Example 3. Purification of AAV from cell lysates using a purification matrix containing AAV-binding polypeptide and support.

[0263] AAV is produced in a production cell line (e.g., HEK293) according to a standard protocol. The cells are lysed and cell debris is removed by centrifugation. The cell supernatant is contacted with one of the purified matrices from Example 2 for a certain period of time to form a complex. An environmental factor is applied to increase the size of the complex. The purified matrix with bound AAV is separated from impurities based on size. AAV is eluted from the purified matrix by applying a second environmental factor. The titer of the AAV is then determined and it is frozen at -80°C for later use.

[0281] Example 4. Purification of multiple AAV serotypes using a purified matrix.

[0264] Recombinant AAV particles, including AAV1, AAV2, AAV6, AAV8, and AAV9 particles that package the tdTomato transgene, were produced in a production cell line (e.g., HEK293) according to a standard protocol. Cells were lysed and cell debris was removed by centrifugation. The cell supernatant was contacted with a purified matrix having the amino acid sequence of SEQ ID NO: 171 for a period of time to form a complex. The purified matrix contained an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 169 and a phase-behaving polypeptide having the amino acid sequence of SEQ ID NO: 170. The size of the complex was then increased by applying an environmental factor (e.g., 0.5M-2M NaCl, MgCl2, or CaCl2). The purified matrix, environmental factor, and cell supernatant were incubated at room temperature for 15 minutes. Subsequently, the purified matrix, environmental factor, and cell supernatant were concentrated (5-10 times) using a 13cm² hollow filter (0.2μm pore size). Six wash diavolutes were performed using phosphate buffer solution and sodium chloride. This protocol allowed for the separation of the purified matrix bound to AAV from impurities based on size. AAV was eluted from the purified matrix by applying a buffer (e.g., a second environmental factor). The purified matrix was collected from the retention solution. Various buffers were evaluated as shown in Table 5. Next, the AAV was titrated and frozen at -80°C for later use.

[0282] [Table 33]

[0283]

[0265] The amount of AAV captured from the solution and the amount of AAV obtained after elution were evaluated using quantitative real-time polymerase chain reaction (qPCR). The purified matrix captured over 99% of AAV particles of multiple serotypes (AAV1, AAV2, AAV6, AAV8, and AAV9) (Figure 1). Each buffer evaluated eluted more than 65% of bound AAV particles in a single diavolume (Figure 2).

[0284]

[0266] After elution of AAV particles, the purified matrix was recycled to determine whether it could be used for future purification. Recycling was performed by incubating the purified matrix at 95°C for 5 minutes or by immersing the purified matrix in 1M NaOH or 6M guanidine hydrochloride for 5 minutes.

[0285]

[0267] As shown in Table 6, the purified matrix can be regenerated and reused for repeated capture. After 5 cycles of purification / regeneration, the purified matrix captures 98% of AAV.

[0286] [Table 34]

[0287]

[0268] The purity of the eluted AAV sample was evaluated using sodium dodecycl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, any contaminants in the sample were visualized by silver staining of the gel. As shown in Figure 3, the major AAV structural proteins Vp1, Vp2, and Vp3 were visible on the gel, but no other major bands were observed. Western blotting confirmed the presence of capsid proteins eluted from the purified matrix in the sample (Figure 4). Furthermore, after the AAV particles were captured by the purified matrix, no AAV particles remained in the capture supernatant (Capture Sup in Figure 4). In summary, this data indicates that virtually all contaminants were removed from the sample after elution from the purified matrix, and the isolated AAV had high purity.

[0288]

[0269] In subsequent experiments, we evaluated whether the purified matrix could capture encapsulated capsids, empty capsids, or both. The total number of AAV capsids present in the sample eluted from the purified matrix was estimated using an ELISA-based assay. The eluted sample was also evaluated using qPCR to determine the number of viral genomes. The qPCR:ELISA values ​​were used to approximate the ratio of encapsulated capsids to total capsids. As shown in Table 7, the purified matrix enriched with encapsulated capsids.

[0289] [Table 35]

[0290]

[0270] The eluted AAV samples were also assayed to determine whether they retained infectivity after purification. HEK293 cells (10,000 cells / well) in culture were administered AAV8 carrying the tdTomato trans gene at a multiple of infection (MOI) of 1 × 10⁶ or 1 × 10⁷. After 48 incubations, the cells were visualized using fluorescence microscopy for tdTomato fluorescence. As shown in Figure 5A, AAV purified by the purification matrix (ViraTag®) infected cells to the same extent as AAV purified according to the standard protocol (Pos Ctrl). As shown in Figure 5B, this data was quantified. There was no statistically significant difference in infectivity levels between AAV8 purified by the standard protocol (Pos Ctrl) and AAV8 purified by the purification matrix (ViraTag®). Therefore, this data indicates that AAV particles purified using the tested purification matrix (ViraTag®) retain a high level of infectivity.

[0291] Example 5. Purification of AAV9 using a purification matrix and tangential flow filtration (TFF).

[0271] HEK293 cells producing a recombinant AAV9 vector packaging the tdTomato trans gene were grown in suspension and collected by centrifugation. 200 mL of supernatant was treated with 10 U / mL benzonase and 0.01% pluronic acid and filtered through a 0.2 micron bottle-top filter for sterile filtration. Next, this starting material (SM) was mixed with 1 μM purified matrix and 0.6 M NaCl salt (i.e., the first environmental factor) from Example 4 to form an AAV-purified matrix complex.

[0292]

[0272] This SM was processed in continuous flow culture mode using a Repligen KR2i tangential flow filtration (TFF) unit. The TFF was set up with a 20 mL holding solution vessel prepared with 50 μM purified matrix and 0.6 M NaCl, and a 13 cm² hollow fiber filter with 0.2 micron pores. Concentration-diafiltration (CD) mode runs (10x concentration factor (CF), 6x diavolute (DV)) were performed using a permeate control and permeate pump containing SM set to equal flow rates. Once all 200 mL of SM feed solution had been processed, the retained material was rinsed with 6 DV washing buffer (20 mM Tris, 0.5 M NaCl). The AAV9 material, now free of contaminants, was then redissolved on ice and mixed with an equal volume of 2 × elution buffer (i.e., second environmental factor) with the permeate valve closed. The elution buffer contained 100 mM glycine and 0.6 M NaCl at pH 3. After 20 minutes, this recirculated sample was warmed to room temperature and phase-separated with NaCl. The permeate valve was opened, and the pure AAV9 was eluted with elution buffer diavolute (100 mM glycine, pH 3, 0.6 M NaCl). The samples were collected using a second CD mode (2×CF, 4×DV) with NaCl. Flux and intermembrane pressure (TMP) were tracked throughout the run (Figure 6), demonstrating a stable, efficient, and scalable process. Permeate and retention samples were collected throughout the run for analysis of AAV loss by anti-AAV Western blotting (Figure 7), and purity analysis by quantification of host cell proteins (HCP) using HEK HCP ELISA (Cygnus®) and dsDNA quantification using Quant-iT® picogreen assay (Thermo Fisher®). This process was able to remove 2-3 log-number HCP (Figure 8) and double-stranded DNA (dsDNA) exceeding 3 log-number (Figure 9).

[0293] Example 6. Effects of potency, clarification, and nuclease treatment on AAV purification

[0273] The ability of the purified matrix of Example 4 to capture AAV8 particles from cell lysate or suspension culture medium (referred to as "supernatant" in Figure 10) of HEK293 cells producing AAV8 particles was tested. Cell lysate was prepared by resuspending a pellet of HEK293 cells producing AAV8 particles in 0.5% Triton-X-100.

[0294]

[0274] The cell lysates and culture media evaluated contained AAV particles with titers ranging from 1 × 10⁸ to 1 × 10⁷ virus particles per microliter (vp / μL). The ability of the purified matrix from nuclease-treated cell lysates to capture AAV8 particles was also evaluated. Nuclease-treated cell lysates were incubated with 50 U / mL benzonase (Millipore®) at 34°C for 1 hour.

[0295]

[0275] The ability of the purified matrix from clarified cell lysates or culture media to capture AAV8 particles was also evaluated. Cell lysates and / or culture media were clarified by centrifugation at 13,200 rpm for 10 minutes. This supernatant was used for the subsequent isolation of AAV8 particles at 13,200 rpm for 10 minutes.

[0296]

[0276] From each sample, the sample was mixed with a 10 μM purified matrix and 0.6 M NaCl (i.e., the first environmental factor), and the sample was centrifuged at 13,200 rpm for 10 minutes to isolate the AAV8 particles.

[0297]

[0277] The pellet containing the AAV-purified matrix complex was resuspended on ice in an elution buffer containing 100 mM glycine at pH 3 (i.e., the second environmental factor), warmed to room temperature, transferred with 0.6 M salt, and then centrifuged a second time. The amount of eluted AAV8 was compared to the starting material (i.e., culture medium or cell lysate containing AAV8 particles) using reverse-end repeat (ITR) quantitative polymerase chain reaction (qPCR). This technique quantifies the number of AAV particles by measuring the number of ITRs using PCR. The AAV capture efficiency for each sample was calculated using the following formula: 100 × (number of AAV8 particles captured by the purified matrix / number of AAV8 particles in the composition before purification).

[0298]

[0278] The purified matrix robustly captured over 98% of AAV particles, regardless of titer (1 × 10⁷ to 4 × 10¹⁰), clarification, nuclease treatment, or whether lysate or culture medium was used as the starting material (Figure 10). Evaluation of lysate samples with and without nuclease treatment showed that nuclease treatment did not affect the purity of the ultimately eluted AAV8 particles when compared by silver-stained SDS-PAGE (Figure 11) and Quant-iT® picogreen assay for dsDNA (Figure 12).

[0299] Example 7. Effect of centrifugal velocity on AAV8 capture

[0279] AAV-purified matrix complex was formed by mixing the culture medium recovered from AAV8 HEK293 suspension cell culture with 10 μM purified matrix and 0.6 M NaCl. This sample was centrifuged for 10 minutes at relative centrifugal force (RCF) ranging from 100 to 16,000. For each RCF, the amount of uncaptured AAV8 remaining in the supernatant was quantified using ITR qPCR and calculated as a percentage of the amount measured in the initial recovered material. AAV capture efficiency was measured by subtracting the percentage remaining uncaptured in the supernatant from 100%, and the results showed that extremely efficient AAV8 capture occurred at rates of 500 RCF or higher (Figure 13).

[0300] Example 8. Stabilization of AAV2 by a purified matrix.

[0280] Adhered HEK293 cells were transfected using a standard triple transfection method, and recombinant AAV2 particles carrying the luciferase trans gene were produced using these cells. These cells were cultured for 6 days in or without purified matrix. Cells cultured in the presence of 10 μM of the purified matrix from Example 4 were triplicately compared with control cells for each treatment or control group. On day 4, the culture medium was collected and replenished with equal volumes of medium with or without purified matrix additive. On day 6, the medium was collected again, and the cells were recovered by rinsing with PBS and detaching. The total amount of vector genome collected in all fractions was quantified for comparison using qPCR with primers for ITR2. Inclusion of purified matrix in the culture medium increased the vector genome (vg) titer by at least 8% (Figure 14).

[0301] Example 9. Stabilization of AAV8 with a purified matrix.

[0281] Culture medium was collected from HEK293 cells grown in suspension, where the cells produced GFP-AAV8. The medium was divided into aliquots and stored at -20°C with or without the addition of 100 μM purified matrix from Example 4. As an accelerated stability study, the samples were subjected to a freeze-thaw cycle (-20°C to room temperature), and then assayed using AAV8 ELISA (Progen) to quantify the total amount of intact AAV particles. The quantified particles of the control and purified matrix-treated samples were normalized with starting materials without freeze-thaw cycles. This data indicates that the resistance of AAV particles to degradation and aggregation via freeze-thaw cycles can be enhanced by the purified matrix (Figure 15).

[0302] Example 10. Capture of AAV8 particles by a purified matrix.

[0282] Culture medium was collected from HEK293 suspension cells producing AAV8 particles carrying the luciferase (luc) trans gene. This medium was contacted with 0 μM, 0.1 μM, 0.5 μM, 1 μM, 10 μM, and 50 μM purified matrix from Example 4, and complexes were formed between the AAV8 particles and the purified matrix. The size of the complexes was increased by applying the first environmental factor (i.e., 0.6 M NaCl). Subsequently, the medium containing the complexes was centrifuged at 13,200 revolutions per minute (rpm) for 10 minutes (min). According to this protocol, it was possible to separate the complexes from impurities based on size. The amount of AAV particles captured from the medium using the purified matrix was evaluated using reverse-end repeat (ITR) quantitative polymerase chain reaction (qPCR) compared to the amount of AAV particles in the starting material. This technique quantifies the number of AAV particles by measuring the number of ITRs using PCR. The capture efficiency of the purified matrix at each concentration was calculated using the following formula: 100 × (number of AAV8 particles captured by the purified matrix / number of AAV8 particles in the composition before purification). Figure 16 shows that purified matrix concentrations of 10 μM or higher are sufficient for robust capture of over 98% of viruses.

[0303] Numbered Embodiments of the Present Disclosure

[0283] Notwithstanding the attached claims, this disclosure illustrates the following numbered embodiments: 1. A purified matrix comprising an AAV-binding polypeptide coupled to a support, wherein the AAV-binding polypeptide comprises the ectodomain of the AAV receptor (AAVR), or an AAV-binding fragment or derivative thereof. 2. The purified matrix according to Embodiment 1, wherein AAVR is human AAVR. 3. The purified matrix according to Embodiment 1, wherein AAVR is monkey AAVR. 4. The purification matrix according to Embodiment 1, wherein AAVR is orangutan AAVR. 5. The purified matrix according to Embodiment 1, wherein AAVR is mouse AAVR. 6. A purified matrix according to any one of Embodiments 1 to 5, wherein AAVR is wild-type AAVR. 7. A purified matrix according to any one of Embodiments 1 to 5, wherein AAVR is mutant AAVR. 8. The purified matrix according to Embodiment 1, wherein the AAV-conjugated polypeptide contains the sequence of SEQ ID NO: 33. 9. The purified matrix according to Embodiment 1, wherein the AAV-binding polypeptide comprises the sequence of SEQ ID NO: 33 having up to 25 amino acid mutations. 10. The purified matrix according to Embodiment 1, wherein the AAV binding domain comprises amino acids 411-499 of Sequence ID No. 35 having at least one, at least two, at least three, at least four, or at least five mutations, and each mutation is individually selected from the group consisting of V440H, S431H, Q432H, T434H, Y442H, I462H, D435H, D436H, K438H, and I439H. 11. The purified matrix according to Embodiment 1, wherein the AAV-conjugated polypeptide comprises one of SEQ ID NOs. 28-32, 37-41, 43-47, and 52-86. 12. The purification matrix according to Embodiment 1, wherein the AAV-binding polypeptide comprises at least two, at least three, at least four, or at least five of the sequence numbers 28-32, 37-41, 43-47, and 52-86. 13. The purified matrix according to Embodiment 1, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 1 (PKD1) domain or a fragment thereof. 14. The purified matrix according to Embodiment 1, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 2 (PKD2) domain or a fragment thereof. 15. The purified matrix according to Embodiment 1, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 3 (PKD3) domain or a fragment thereof. 16. The purified matrix according to Embodiment 1, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 4 (PKD4) domain or a fragment thereof. 17. The purified matrix according to Embodiment 1, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 5 (PKD5) domain or a fragment thereof. 18. The purified matrix according to Embodiment 1, wherein the AAV-conjugated polypeptide contains the sequence of SEQ ID NO: 29. 19. The purified matrix according to Embodiment 18, wherein the AAV-binding polypeptide comprises the sequence of SEQ ID NO: 29, in which at least one amino acid is mutated to histidine. 20. A purification matrix according to any one of Embodiments 1 to 19, wherein the support is a bead, resin, membrane, fiber, polymer, plate, or chip. 21. The purified matrix according to Embodiment 20, wherein the support is beads comprising Sepharose, agarose, cellulose, polystyrene, polymethacrylate, and / or polyacrylamide. 22. The purification matrix according to Embodiment 20, wherein the support is magnetic beads. 23. The purification matrix according to Embodiment 20, wherein the support is a polymer. 24. The purified matrix according to Embodiment 23, wherein the support is a synthetic polymer. 25. A purification matrix according to any one of Embodiments 1 to 24, wherein an AAV-conjugated polypeptide is reversibly coupled to a support. 26. A purified matrix according to any one of Embodiments 1 to 24, wherein an AAV-conjugated polypeptide is covalently coupled to a support. 27. A purified matrix according to any one of Embodiments 1 to 24, wherein an AAV-conjugated polypeptide is non-covalently coupled to a support. 28. A purified matrix according to any one of Embodiments 1 to 27, wherein an AAV-conjugated polypeptide is coupled to a support via a linker. 29. The purification matrix according to Embodiment 28, wherein the linker is a peptide linker. 30. The purified matrix according to Embodiment 29, wherein the peptide linker contains a protease cleavage site. 31. The purification matrix according to Embodiment 28, wherein the linker is a chemical linker. 32. A reusable purification matrix according to any one of Embodiments 1 to 31. 33. A purified matrix according to any one of Embodiments 1 to 31, comprising an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 9. 34. A purified matrix according to any one of Embodiments 1 to 31, comprising an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 52. 35. A purified matrix according to any one of Embodiments 1 to 31, comprising an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 64. 36. A purified matrix comprising an AAV-binding polypeptide coupled to a polypeptide having phase behavior, wherein the AAV-binding polypeptide comprises the ectodomain of the AAV receptor (AAVR), or an AAV-binding fragment or derivative thereof. 37. The purified matrix according to Embodiment 36, wherein AAVR is human AAVR. 38. The purified matrix according to Embodiment 36, wherein AAVR is monkey AAVR. 39. The purification matrix according to Embodiment 36, wherein AAVR is orangutan AAVR. 40. The purified matrix according to Embodiment 36, wherein AAVR is mouse AAVR. 41. A purified matrix according to any one of embodiments 36 to 40, wherein AAVR is wild-type AAVR. 42. A purified matrix according to any one of embodiments 36 to 40, wherein AAVR is mutant AAVR. 43. The purification matrix according to Embodiment 36, wherein the AAV-binding polypeptide comprises the sequence of SEQ ID NO: 33. 44. The purified matrix according to Embodiment 36, wherein the AAV-binding polypeptide comprises the sequence of SEQ ID NO: 33 having up to 25 amino acid mutations. 45. The purified matrix according to Embodiment 36, wherein the AAV binding domain comprises amino acids 411-499 of Sequence ID No. 35 having at least one, at least two, at least three, at least four, or at least five mutations, and each mutation is individually selected from the group consisting of V440H, S431H, Q432H, T434H, Y442H, I462H, D435H, D436H, K438H, and I439H. 46. ​​The purification matrix according to Embodiment 36, wherein the AAV-conjugated polypeptide comprises one of SEQ ID NOs. 28-32, 37-41, 43-47, and 52-86. 47. The purification matrix according to Embodiment 36, wherein the AAV-binding polypeptide comprises at least two, at least three, at least four, or at least five of the sequence numbers 28-32, 37-41, 43-47, and 52-86. 48. The purified matrix according to Embodiment 36, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 1 (PKD1) domain or a fragment thereof. 49. The purified matrix according to Embodiment 36, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 2 (PKD2) domain or a fragment thereof. 50. The purified matrix according to Embodiment 36, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 3 (PKD3) domain or a fragment thereof. 51. The purified matrix according to Embodiment 36, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 4 (PKD4) domain or a fragment thereof. 52. The purified matrix according to Embodiment 36, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 5 (PKD5) domain or a fragment thereof. 53. The purified matrix according to Embodiment 36, wherein the AAV-conjugated polypeptide contains the sequence of SEQ ID NO: 29. 54. The purified matrix according to Embodiment 53, wherein the AAV-binding polypeptide comprises the sequence of SEQ ID NO: 29, in which at least one amino acid is mutated to histidine. 55. A purification matrix according to any one of embodiments 36 to 54, wherein an AAV-conjugated polypeptide is reversibly coupled to a polypeptide having phase behavior. 56. A purification matrix according to any one of embodiments 36 to 54, wherein an AAV-conjugated polypeptide is covalently coupled to a polypeptide having phase behavior. 57. A purification matrix according to any one of embodiments 36 to 54, wherein an AAV-conjugated polypeptide is non-covalently coupled to a polypeptide having phase behavior. 58. A purified matrix according to any one of embodiments 36 to 57, wherein an AAV-conjugated polypeptide is coupled to a polypeptide having phase behavior via a linker. 59. The purification matrix according to Embodiment 58, wherein the linker is a peptide linker. 60. The purified matrix according to Embodiment 58, wherein the peptide linker contains a protease cleavage site. 61. The purification matrix according to Embodiment 58, wherein the linker is a chemical linker. 62. A purified matrix according to any one of embodiments 36 to 61, wherein the fusion protein comprises an AAV-binding polypeptide and a polypeptide having phase behavior. 63. The purified matrix according to any one of Embodiments 36 to 62, wherein the polypeptide exhibiting phase behavior is an elastin-like polypeptide. 64. The purified matrix according to any one of embodiments 36 to 62, wherein the polypeptide exhibiting phase behavior is a resilin-like polypeptide. 65. The purified matrix according to Embodiment 63, wherein the polypeptide exhibiting phase behavior is a polymer comprising a pentapeptide repeat having the sequence (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 10) or a randomized scrambled analog thereof; where Xaa may be any amino acid other than proline. The purification matrix according to embodiment 65, wherein 66.n is an integer between 1 and 360. 67. Polypeptides exhibiting phase behavior a.(GRGDSPY)n(Sequence ID 1) b.(GRGDSPH)n(Sequence ID 2) c.(GRGDSPV)n(Sequence ID 3) d.(GRGDSPYG)n(Sequence ID 4) e.(RPLGYDS)n(Sequence ID 5) f.(RPAGYDS)n(Sequence ID 6) g.(GRGDSYP)n(Sequence ID 7) h.(GRGDSPYQ)n(Sequence ID 8) i.(GRGNSPYG)n(Sequence ID 9) j.(GVGVP)n(Sequence ID 11); k.(GVGVPGLGVPGVGVPGLGVPGVGVP)m(Sequence ID 12); l.(GVGVPGVGVPGAGVPGVGVPGVGVP)m(Sequence No. 13); m.(GVGVPGWGVPGVGVPGWGVPGVGVP)m(Sequence ID 14); n.(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP)m(SEQ ID NO: 15); o.(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP)m(Sequence ID 16); and p.(GAGVPGVGVPGAGVPGVGVPGAGVP)m(Sequence ID 17) Selected amino acid sequence; or its randomized scrambled analogue (In the formula: n is an integer in the range of 20 to 360; and A purification matrix according to any one of embodiments 1 to 63, wherein m is an integer in the range of 4 to 25. 68. Polypeptides exhibiting phase behavior (a)(GVGVP)m(Sequence ID 143); (b)(ZZPXXXXGZ)m(Sequence ID 148); (c)(ZZPXGZ)m(Sequence ID 149); (d)(ZZPXXGZ)m(sequence number 150); or (e)(ZZPXXXGZ)m(Sequence ID 151) A purified matrix according to any one of Embodiments 1 to 63, comprising an amino acid sequence selected from (wherein m is an integer between 10 and 160, X is any amino acid other than proline or glycine if present, and Z is any amino acid if present). 69. Polypeptides exhibiting phase behavior (a)(GVGVPGVGVPGAGVPGVGVPGVGVP)m(Sequence ID 144); or (b)(GVGVPGVGVPGLGVPGVGVPGVGVP)m(Sequence ID 146); A purified matrix according to any one of embodiments 1 to 63, comprising an amino acid sequence selected from (wherein m is an integer between 2 and 32). 70. Polypeptides exhibiting phase behavior (a)(GVGVPGVGVPGAGVPGVGVPGVGVP)m(Sequence ID 144)(wherein m is 8 or 16); (b)(GVGVPGAGVP)m(sequence code 145)(wherein m is an integer between 5 and 80); or A purified matrix according to any one of Embodiments 1 to 63, comprising an amino acid sequence selected from (c)(GXGVP)m(SEQ ID NO: 147) (wherein m is an integer between 10 and 160, and in each repeat, X is independently selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine). 71. A reusable purification matrix according to any one of embodiments 36 to 70. 72. A purified matrix according to any one of embodiments 36 to 70, comprising an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 29. 73. A purified matrix according to any one of embodiments 36 to 70, comprising an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 52. 74. A purified matrix according to any one of embodiments 36 to 70, comprising an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 64. 75. A purified matrix according to any one of Embodiments 36 to 70, comprising an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 29 and a polypeptide exhibiting phase behavior having the amino acid sequence of SEQ ID NO: 88 or (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16). 76. The purified matrix according to any one of embodiments 36 to 70, wherein the purified matrix comprises an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 52 and a polypeptide exhibiting phase behavior having the amino acid sequence of SEQ ID NO: 88 or (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16). 77. The purified matrix according to any one of embodiments 36 to 70, wherein the purified matrix comprises an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 64 and a polypeptide exhibiting phase behavior having the amino acid sequence of SEQ ID NO: 88 or (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12) (wherein m is 16). 78. A purified matrix according to any one of embodiments 36 to 70, comprising one amino acid sequence from sequence number 87 and 171 to 174. 79. A method for purifying AAV, comprising contacting AAV with a purification matrix described in any one of Embodiments 1 to 35. 80. The method according to Embodiment 79, wherein AAV comprises a wild-type AAV capsid. 81. The method according to Embodiment 79, wherein AAV comprises a mutant AAV capsid. 82. The method according to Embodiment 79, wherein the AAV comprises a capsid of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, or AAVrh74. 83. The method according to any one of embodiments 79 to 82, wherein AAV reversibly binds to the purified matrix to form an AAV-purified matrix complex. 84. The method according to Embodiment 83, comprising separating the AAV-purified matrix complex from one or more impurities. 85. The method according to Embodiment 84, wherein the AAV-purified matrix complex is separated from one or more impurities by washing the AAV-purified matrix complex. 86. The method according to any one of embodiments 83 to 85, comprising eluting AAV from the AAV-purified matrix complex. 87. The method according to Embodiment 86, wherein AAV is eluted from the AAV-purified matrix complex by changing the pH of the composition containing the AAV-purified matrix complex. 88. The method according to Embodiment 86, wherein AAV is eluted from the AAV-purified matrix complex by changing the temperature of the composition containing the AAV-purified matrix complex. 89. The method according to Embodiment 86, wherein AAV is eluted from the AAV-purified matrix complex by changing the ionic strength of the composition containing the AAV-purified matrix complex. 90. The method according to Embodiment 86, wherein AAV is eluted from the AAV-purified matrix complex by adding a reducing agent to a composition containing the AAV-purified matrix complex. 91. A method for purifying AAV, comprising contacting AAV with a purification matrix described in any one of embodiments 36 to 78. 92. The method according to Embodiment 91, wherein AAV comprises a wild-type AAV capsid. 93. The method according to Embodiment 91, wherein AAV comprises a mutant AAV capsid. 94. The method according to Embodiment 91, wherein the AAV comprises a capsid of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32,33, AAVrh74, avian AAV, or bovine AAV. 95. The method according to any one of embodiments 91 to 94, wherein AAV reversibly binds to the purified matrix to form an AAV-purified matrix complex. 96. The method according to embodiment 95, comprising increasing the size of the AAV-purified matrix complex using a first environmental factor. 97. The first environmental factor is, a. A change in one or more of the following: temperature, pH, salt concentration, or pressure; b. Addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, enzymes, or denaturants; or c. Application of electromagnetic waves or sound waves The method according to embodiment 96, comprising one or more of the following. 98. The method according to Embodiment 96 or 97, comprising separating the AAV-purified matrix complex from at least one impurity based on size. 99. The method according to Embodiment 98, wherein the AAV-purified matrix complex is separated from at least one impurity based on diameter. 100. The method according to Embodiment 98, wherein the AAV-purified matrix complex is separated from at least one impurity based on mass. 101. The method according to any one of Embodiments 98 to 100, wherein size-based separation is performed using tangential flow filtration, analytical ultracentrifugation, membrane chromatography, high-performance liquid chromatography, normal flow filtration, ultrasonic separation, centrifugation, countercurrent centrifugation, and high-performance protein liquid chromatography. 102. The method according to any one of Embodiments 91 to 101, wherein a second environmental factor is used to elute AAV from the AAV-purified matrix complex. 103. The second environmental factor is, a. A change in one or more of the following: temperature, pH, salt concentration, or pressure; b. Addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, denaturants, or enzymes; or c. Application of electromagnetic waves or sound waves The method according to Embodiment 102, comprising one or more of the following. 104. The method according to any one of embodiments 79 to 103, comprising regenerating the purified matrix and reusing the purified matrix for the purification of a second AAV. 105. The method according to any one of embodiments 79 to 103, wherein the purified AAV has an infectivity level higher than 95% of the infectivity level before purification. 106. The method according to any one of embodiments 79 to 103, wherein AAV retains at least 96% of its infectivity after purification. 107. The method according to any one of embodiments 79 to 103, wherein AAV retains at least 97% of its infectivity after purification. 108. The method according to any one of embodiments 79 to 103, wherein AAV retains at least 98% of its infectivity after purification. 109. The method according to any one of embodiments 79 to 103, wherein AAV retains at least 99% of its infectivity after purification. 110. The method according to any one of embodiments 79 to 103, wherein AAV retains at least 100% of its infectivity after purification. 111. The method according to any one of Embodiments 79 to 103, wherein the encapsulated capsid is concentrated in purified AAV. 112. The method according to Embodiment 111, wherein the purified AAV exhibits an increased qPCR:ELISA ratio compared to the qPCR:ELISA ratio before purification. 113. The method according to any one of embodiments 79 to 112, which can be completed in approximately 2 to 10 hours. 114. The method according to embodiment 113, which can be completed in approximately 4 to 8 hours. 115. A composition comprising AAV purified by any one of the methods described in Embodiments 79 to 114. 116. The composition according to Embodiment 115, wherein at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% are free of impurities. 117. An AAV-binding polypeptide containing one of the sequences from sequence numbers 28-47 having at least one mutation. 118. A nucleic acid encoding the AAV-binding polypeptide described in Embodiment 117. 119. A vector comprising the nucleic acid described in Embodiment 118. 120. A composition comprising the AAV-conjugated polypeptide described in Embodiment 117, the nucleic acid described in Embodiment 118, or the vector described in Embodiment 119. 121. A kit comprising the AAV-conjugated polypeptide described in Embodiment 117, the nucleic acid described in Embodiment 118, or the vector described in Embodiment 119. 122. A method for increasing the yield of AAV particles during their production, comprising culturing AAV-producing cells in the presence of a purified matrix. 123. A method for stabilizing AAV particles during their production, comprising culturing AAV-producing cells in the presence of a purified matrix. 124. A method for stabilizing AAV particles during their purification, comprising contacting the AAV particles with a purification matrix during their purification. 125. A method for stabilizing AAV particles during storage, comprising storing the AAV particles in the presence of a purification matrix. 126. A method for increasing the storage life of AAV particles, comprising storing the AAV particles in the presence of a purification matrix. 127. The method according to any one of embodiments 122 to 126, wherein the purification matrix is ​​the purification matrix described in any one of embodiments 1 to 78. 128. The method according to any one of Embodiments 122 to 127, wherein the AAV particle is a wild-type AAV particle of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32,33, AAVrh74, avian AAV, or bovine AAV. 129. The method according to any one of embodiments 122 to 127, wherein the AAV particle is a mutant AAV particle. 130. The method according to any one of embodiments 122 to 127, wherein AAV particles reversibly bind to a purified matrix to form an AAV-purified matrix complex. 131. The method according to any one of embodiments 122 to 127, wherein the purified matrix is ​​present at a concentration of at least about 10 μM.

[0304] Reference

[0284] All references, articles, publications, patents, patent gazettes, and patent applications cited herein are incorporated by reference in their entirety for any purpose. However, no reference made herein to any reference, article, publication, patent, patent gazette, or patent application is, and should not be construed as, an endorsement or in any way of suggestion that it constitutes prior art or is part of common technical knowledge in any country in the world.

Claims

1. A purified matrix comprising an AAV-binding polypeptide coupled to a polypeptide having phase behavior, wherein the AAV-binding polypeptide comprises the ectodomain of the AAV receptor (AAVR), or an AAV-binding fragment or derivative thereof.

2. The purified matrix according to claim 1, wherein the AAVR is human, monkey, orangutan, or mouse AAVR, or a derivative thereof.

3. The purification matrix according to claim 1, wherein the AAV-binding polypeptide comprises one of SEQ ID NOs: 28-32, 37-41, 43-47, and 52-86.

4. The purified matrix according to claim 1, wherein the AAV-binding polypeptide comprises any one of the sequences of SEQ ID NOs: 29, 33, 52, and 64, or a sequence having up to 25 amino acid mutations compared to any one of SEQ ID NOs: 29, 33, 52, or 64.

5. The purified matrix according to claim 1, wherein the AAV-binding polypeptide comprises a polycystic kidney disease 2 (PKD2) domain or a fragment thereof.

6. The purification matrix according to any one of claims 1 to 5, wherein the AAV-conjugated polypeptide is reversibly coupled to the polypeptide having the phase behavior described above.

7. The purification matrix according to any one of claims 1 to 5, wherein the AAV-conjugated polypeptide is covalently coupled to the polypeptide having the phase behavior.

8. The purified matrix according to any one of claims 1 to 7, wherein the polypeptide exhibiting the aforementioned phase behavior is an elastin-like polypeptide.

9. The polypeptide exhibiting the aforementioned phase behavior has the sequence (Val-Pro-Gly-Xaa-Gly) ｎ A purified matrix according to any one of claims 1 to 7, comprising a pentapeptide repeat having (SEQ ID NO: 10) or a randomized scrambled analog thereof; wherein Xaa may be any amino acid other than proline, and n is an integer between 1 and 360.

10. The polypeptide having the aforementioned phase behavior a. (GRGDSPY) ｎ (Sequence No. 1) b. (GRGDSPH) ｎ (Sequence No. 2) c. (GRGDSPV) ｎ (Sequence No. 3) d. (GRGDSPYG) ｎ (Sequence No. 4) e. (RPLGYDS) ｎ (Sequence No. 5) f. (RPAGYDS) ｎ (Sequence No. 6) g. (ZZPXXXXGZ) ｍ (Sequence ID 148); h. (GRGDSPY) ｎ (SEQ ID NO: 7) i. (GRGDSPYQ) ｎ (Sequence No. 8) j. (GRGNSPYG) ｎ (Sequence No. 9) k. (GVGVP) ｎ (Sequence No. 11); l. (GVGVPGLGVPGVGVPGLGVPGVGVP) ｍ (Sequence No. 12); m. (GVGVPGVGVPGAGVPGVGVPGVGVP) ｍ (Sequence No. 13); n. (GVGVPGWGVPGVGVPGWGVPGVGVP) ｍ (Sequence ID 14); o. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGFGVPGVGVPGVGVPGVP) ｍ (SEQ ID NO: 15); p. (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP) ｍ (Sequence ID 16); and q. (GAGVPGVGVPGAGVPGVGVPGAGVP) ｍ (Sequence No. 17) amino acid sequence selected from; or its randomized scrambled analogue (In the formula: n is an integer in the range of 20 to 360; and A purification matrix according to any one of claims 1 to 8, comprising m being an integer in the range of 4 to 25.

11. The polypeptide having the aforementioned phase behavior (a) (GVGVP) ｍ (Sequence ID 143); (b) (ZZPXGZ) ｍ (Sequence ID 149); (c) (ZZPXXGZ) ｍ (Sequence ID 150); or (d) (ZZPXXXGZ) ｍ (Sequence No. 151) A purified matrix according to any one of claims 1 to 8, comprising an amino acid sequence selected from (wherein m is an integer between 10 and 160, X is any amino acid other than proline or glycine if present, and Z is any amino acid if present).

12. The polypeptide having the aforementioned phase behavior (a) (GVGVPGVGVPGAGVPGVGVPGVGVP) ｍ (Sequence ID 144); or (b) (GVGVPGVGVPGLGVPGVGVPGVGVP) ｍ (Sequence ID 146); A purified matrix according to any one of claims 1 to 8, comprising an amino acid sequence selected from (wherein m is an integer between 2 and 32).

13. The polypeptide having the aforementioned phase behavior (a) (GVGVPGVGVPGAGVPGVGVPGVGVP) ｍ (Sequence ID 144) (wherein m is 8 or 16); (b) (GVGVPGAGVP) ｍ (Sequence ID 145) (wherein m is an integer between 5 and 80); or (c) (GXGVP) ｍ A purified matrix according to any one of claims 1 to 8, comprising an amino acid sequence selected from (Sequence ID 147) (wherein m is an integer between 10 and 160, and in each repeat, X is independently selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine).

14. The purified matrix comprises an AAV-binding polypeptide having the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 52, or SEQ ID NO: 64, and the amino acids of SEQ ID NO: 88 or (GVGVPGLGVPPGVGVPGLGVPPGVGVP) ｍ A purification matrix according to any one of claims 1 to 8, comprising a polypeptide having phase behavior including the sequence (SEQ ID NO: 12) (wherein m is 16).

15. The purified matrix according to any one of claims 1 to 8, wherein the purified matrix comprises one amino acid sequence from sequence number 87 and 171 to 174.

16. The purified matrix according to claim 15, wherein the purified matrix comprises the amino acid sequence of sequence number 171.

17. The purification matrix according to claim 15, wherein the purification matrix comprises a polypeptide having phase behavior having the amino acid sequence of SEQ ID NO: 170 and an AAV-binding polypeptide of SEQ ID NO:

169.

18. A method for purifying AAV particles, comprising contacting the AAV particles with a purification matrix according to any one of claims 1 to 17.

19. The method according to claim 18, wherein the AAV particles contain a capsid protein from any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32,33, AAVrh74, avian AAV, or bovine AAV.

20. The method according to claim 18 or 19, wherein the AAV particles reversibly bind to the purified matrix to form an AAV-purified matrix complex.

21. A method according to any one of claims 18 to 20, comprising increasing the size of the AAV-purified matrix complex using a first environmental factor, The first environmental factor is, a. A change in one or more of the following: temperature, pH, salt concentration, or pressure; b. Addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, enzymes, or denaturants; or c. Application of electromagnetic waves or sound waves A method that includes one or more of the following.

22. The method according to any one of claims 18 to 20, comprising separating the AAV-purified matrix complex from at least one impurity on a size basis using a technique selected from the group consisting of tangential flow filtration, analytical ultracentrifugation, membrane chromatography, high-performance liquid chromatography, normal flow filtration, ultrasonic separation, centrifugation, countercurrent centrifugation, and high-performance protein liquid chromatography.

23. The AAV particles are eluted from the AAV-purified matrix complex using a second environmental factor, and the second environmental factor is a. A change in one or more of the following: temperature, pH, salt concentration, or pressure; b. Addition of one or more surfactants, cofactors, vitamins, molecular crowding agents, denaturants, or enzymes; or c. Application of electromagnetic waves or sound waves The method according to any one of claims 18 to 22, comprising one or more of the above.

24. AAV-binding polypeptide comprising one sequence from sequence numbers 28-47, 52, 64, or 169, or a sequence having at least one mutation compared to one of sequence numbers 28-47, 52, 64, or 169.

25. A nucleic acid encoding an AAV-binding polypeptide as described in claim 24.

26. A vector comprising the nucleic acid described in claim 25.

27. A composition comprising the AAV-conjugated polypeptide described in claim 24, the nucleic acid described in claim 25, the vector described in claim 26, or a combination thereof.

28. A method for increasing the yield of AAV particles during their production, comprising culturing AAV-producing cells in the presence of a purified matrix.

29. A method for stabilizing AAV particles during their production, comprising culturing AAV-producing cells in the presence of a purified matrix.

30. A method for stabilizing AAV particles during their purification, comprising contacting the AAV particles with a purification matrix during their purification.

31. A method for stabilizing AAV particles during storage, comprising storing the AAV particles in the presence of a purification matrix.

32. A method for increasing the storage life of AAV particles, comprising storing the AAV particles in the presence of a purification matrix.

33. The method according to any one of claims 28 to 32, wherein the purification matrix is ​​the purification matrix according to any one of claims 1 to 17.

34. The method according to claim 33, wherein at least about 10 μM of purified matrix is ​​present.

35. The method according to any one of claims 28 to 34, wherein the AAV particle is a wild-type AAV particle of any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32,33, AAVrh74, avian AAV, or bovine AAV.

36. The method according to any one of claims 28 to 34, wherein the AAV particle is a mutant AAV particle.

37. The method according to any one of claims 28 to 34, wherein the AAV particles reversibly bind to the purified matrix to form an AAV-purified matrix complex.

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