Affinity purification methods for adeno associated viruses (AAVS)

Abstract

Provided herein are isolated peptides that can bind an AAV (such as an AAV9). Methods of using the peptides in obtaining an AAV for use in a variety of gene transfer and / or other applications are also provided. The disclosed methods separate the AAV from all or a portion of other components of an AAV starting preparation

Description

AFFINITY PURIFICATION METHODS FOR ADENO ASSOCIATED VIRUSES (AAVS)FIELD OF THE INVENTION

[0001] The present disclosure provides isolated peptides and methods of using the peptides related to obtaining an adeno-associated virus (AAV), such as methods of separating an AAV from an AAV starting preparation comprising the AAV, as well as associated compositions and kits. In some embodiments, the peptides are immobilized on a solid support.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to United States Provisional Application No. 63 / 470,104, filed May 31, 2023, which is incorporated by reference in its entirety.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 28, 2024, is named 01245-0039-00PCT.xml and is 41,745 bytes in size.INTRODUCTION

[0004] Adeno-associated viruses (AAV) vectors are among the leading candidates for in vivo virus-based gene therapy because of their broad tissue tropism, non-pathogenic nature and low immunogenicity. Currently, 12 AAV serotypes and over 100 variants have been identified in human and nonhuman primate populations. Gene therapy vectors using AAV in vivo can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell, although in the native virus some integration of virally carried genes into the host genome can occur. These features make AAV an attractive candidate for creating viral vectors for gene therapy, and for the creation of isogenic human disease models. See, S. Pillay, Nature, 530(7588): 108-112 (2016). The use of adeno-associated viruses (AAVs), such as recombinant adeno-associated viruses (rAAVs) for a variety of gene therapy and vaccine approaches has been described. However, efficient, scalable methods for purification of AAVs are lacking.

[0005] Adeno-associated virus (AAV), a member of the Parvovirus family, is a small, nonenveloped virus. AAV particles comprise an AAV capsid composed of 60 capsid protein subunits that are each made up of VP1, VP2, and VP3 proteins. The VP1, VP2, and VP3 proteinsare present in a predicted ratio of about 1 : 1 : 10 and have icosahedral symmetry. The AAV capsid encloses a small, single-stranded DNA (ssDNA) genome of about 4.8 kilobases (kb). The ssDNA AAV genome includes two open reading frames, Rep and Cap, flanked by two 145-base inverted terminal repeats (ITRs). These ITRs base pair to allow for synthesis of the complementary DNA strand. Rep and Cap are translated to produce multiple distinct proteins (Rep78, Rep68, Rep52, Rep40, which play important roles in the AAV life cycle; and the VP1, VP2, and VP3 capsid proteins).

[0006] Generally, individual AAV particles package only one DNA molecule strand (either the plus or minus strand). The AAV life cycle includes a latent phase and an infectious phase, and particles containing either strand can be infectious. Replication occurs by conversion of the linear single stranded DNA genome to a duplex form followed by amplification. Progeny single strands are subsequently rescued, replicated, and packaged into capsids in the presence of helper molecules. The properties of non-pathogenicity, broad host range of infectivity (including in non-dividing cells), and integration make AAV an attractive delivery vehicle, such as for therapeutic applications.

[0007] Recombinant AAV particles are generally produced in permissive (packaging) cell cultures. Replication and packaging of the recombinant genome into the viral particle requires co-expression of helper virus AAV replication (Rep) and AAV capsid (Cap) genes, such as from a transfer plasmid. When constructing an AAV transfer plasmid, a transgene may be placed between the two ITRs, and Rep and Cap can be supplied in trans. Genes necessary for genome replication, capsid formation, and genome packaging can be expressed from transfected plasmids, integrated into the packaging cell genome, or introduced to the packaging cells using recombinant viruses. For example, a transfer plasmid comprising Rep / Cap, and a helper plasmid comprising E4, E2a, and VA, may be transfected into packaging cells containing the adenovirus gene E1+, to produce infectious AAV particles.

[0008] Packaging cells may be lysed to release infectious AAV particles, such as infectious rAAV particles, and to maximize recovery yield. In such cases, the cell lysate contains various cellular components, such as packaging cell DNA, packaging cell proteins, media components, and in some instances, helper virus or helper virus plasmid DNA, which must be separated from the AAV vector before it is suitable for use, such as for delivery of a therapeutic nucleic acid of interest in vivo. Advances in AAV production include the use of non-adherent cell suspension processes in stirred tank bioreactors and production conditions whereby AAV particles (such as rAAVs) are released into the media or supernatant, reducing the concentration of packaging cellular components present in the production material but still resulting in appreciable amountsof in-process impurities. Therefore, AAV particles (such as rAAVs) may be collected from the media and / or cell lysate and further purified.However, industry standard purification methods, such as standard chromatography purification techniques, rarely achieve efficient separation of AAV particles from impurities present in the media and / or cell lysate, and frequently require harsh conditions, such as pH levels that may damage an AAV or require inefficient buffer exchange steps. Accordingly, the present disclosure presents improved methods and tools for obtaining an AAV particle, such as an AAV particle comprising a therapeutic nucleic acid of interest, from an AAV starting preparation comprising the AAV particle and various impurities.SUMMARY

[0009] Isolated peptides are provided herein that selectively bind an AAV, such as an AAV9, within a particular pH range. In some embodiments, a method of obtaining an AAV (such as an AAV9) from an AAV starting preparation comprising the AAV and various impurities is provided, wherein the AAV is contacted with the isolated peptide at a first pH that is within a pH range within which the AAV binds the peptide. An optional wash step may be performed, such as at a pH within the pH range within which the AAV binds the peptide, to remove all or a portion of the components of the AAV starting preparation that are not bound to the peptide. In the disclosed methods, the AAV can be dissociated from the peptide by changing the pH to a second pH that is within a pH range within which the peptide does not bind the AAV as tightly. The following exemplary embodiments are provided.

[0010] Embodiment 1 is an isolated peptide comprising or consisting of: a) at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-28; b) at least 12 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 2-14 or 16-28; c) at least 13 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 4-14 or 18-28; d) at least 14 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 9-14 or 23-28; or e) an amino acid sequence selected from any one of SEQ ID NOs: 1-28.

[0011] Embodiment 2 is the peptide of embodiment 1, wherein the amino acid sequence of the peptide consists of an amino acid sequence selected from any one of SEQ ID NOs: 1-28.

[0012] Embodiment 3 is the peptide of embodiment 1 or embodiment 2, wherein the C- or N- terminal amino acid of the peptide is acetylated.

[0013] Embodiment 4 is the peptide of any one of embodiments 1-3, wherein the C- or N- terminal amino acid of the peptide is iso-aspartic acid.

[0014] Embodiment 5 is the peptide of any one of embodiments 1-3, wherein the peptide further comprises 2,3 diaminopropionic acid (Dap).

[0015] Embodiment 6 is the peptide of embodiment 5, wherein the C- or N-terminal amino acid comprises 2,3 diaminopropionic acid (Dap).

[0016] Embodiment 7 is the peptide of any one of embodiments 1-6, wherein the C- or N- terminal amino acid of the peptide is amidated.

[0017] Embodiment 8 is the peptide of any one of embodiments 1-7, further comprising a linker.

[0018] Embodiment 9 is the peptide of embodiment 8, wherein the linker is a peptide linker or a polyol linker.

[0019] Embodiment 10 is the peptide of embodiment 9, wherein the polyol linker is a PEG linker.

[0020] Embodiment 11 is the peptide of embodiment 9, wherein the peptide linker comprises serine, glycine, and / or threonine.

[0021] Embodiment 12 is the peptide of embodiment 11, wherein the linker is a glycine-serine linker.

[0022] Embodiment 13 is the peptide of embodiment 12, wherein the glycine-serine linker is GGGSGGGS (SEQ ID NO: 29).

[0023] Embodiment 14 is the peptide of embodiment 10, wherein the PEG linker is PEG2.

[0024] Embodiment 15 is the peptide of any one of embodiments 8-14, wherein the linker is attached to the N- or C-terminal amino acid of the peptide.

[0025] Embodiment 16 is the peptide of any one of embodiments 1-15, further comprising a binding moiety.

[0026] Embodiment 17 is the peptide of embodiment 16, wherein the binding moiety is a biotin or a modified biotin.

[0027] Embodiment 18 is the peptide of embodiment 16 or embodiment 17, wherein the binding moiety is attached to the peptide through the linker, optionally wherein the linker links the N- or C-terminal amino acid of the peptide to the binding moiety, and optionally wherein a lysine links the linker to the binding moiety.

[0028] Embodiment 19 is the peptide of any one of embodiments 1-18, wherein the peptide is immobilized on a solid support.

[0029] Embodiment 20 is the peptide of embodiment 19, wherein the solid support comprises sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose,a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, a flow cell, a hydrogel, a silica gel, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, an alumina gel, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead.

[0030] Embodiment 21 is the peptide of embodiment 19 or embodiment 20, wherein the solid support has a binding affinity for the binding moiety.

[0031] Embodiment 22 is the peptide of any one of embodiments 19-21, wherein the binding moiety is a biotin or a modified biotin, and the solid support comprises streptavidin, avidin, and / or neutravidin.

[0032] Embodiment 23 is the peptide of embodiment 22, wherein the biotin or the modified biotin of the peptide binds to the streptavidin, avidin, and / or neutravidin of the solid support.

[0033] Embodiment 24 is an isolated peptide comprising or consisting of: (a) at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-5; (b) at least 12 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 2-5; (c) at least 13 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 3-5; (d) at least 14 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 4 or 5; or (e) an amino acid sequence selected from any one of SEQ ID NOs: 1-5; and i) a PEG or a glycine-serine linker conjugated to the N- or C-terminal amino acid of the peptide; and ii) a binding moiety conjugated to the linker; wherein the C-terminal amino acid of the peptide is amidated, and optionally wherein the N- terminal amino acid of the peptide is acetylated and / or the N-terminal amino acid of the peptide further comprises 2,3 diaminopropionic acid (Dap).

[0034] Embodiment 25 is the peptide of embodiment 24, wherein the binding moiety comprises biotin or a modified biotin; the peptide is immobilized on a solid support comprising streptavidin, avidin, and / or neutravidin; and the biotin or the modified biotin conjugated to the linker of the peptide binds to the streptavidin, avidin, and / or neutravidin of the solid support.

[0035] Embodiment 26 is the peptide of any one of embodiments 1-25, wherein the peptide binds an adeno-associated virus (AAV).

[0036] Embodiment 27 is the peptide of embodiment 26, wherein the AAV is AAV9.

[0037] Embodiment 28 is the peptide of embodiment 26 or embodiment 27, wherein the peptide binds the AAV between a first pH and a second pH, inclusive.

[0038] Embodiment 29 is the peptide of embodiment 28, wherein the AAV is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or atleast 90% more likely to dissociate from the peptide at a pH that is lower than the first pH than it is to dissociate from the peptide at a pH that is between the first pH and the second pH, inclusive.

[0039] Embodiment 30 is the peptide of embodiment 28 or embodiment 29, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is higher than the second pH than it is to dissociate from the peptide at a pH that is between the first pH and the second pH, inclusive.

[0040] Embodiment 31 is the peptide of any one of embodiments 28-30, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is lower than the first pH or higher than the second pH than it is to dissociate from the peptide at a pH that is between the first pH and the second pH, inclusive.

[0041] Embodiment 32 is the peptide of any one of embodiments 28-31, wherein the second pH is between 0.05 and 2 pH units, between 0.05 and 1.75 pH units, between 0.05 and 1.5 pH units, between 0.05 and 1.25 pH units, between 0.05 and 1 pH units, between 0.05 and 0.75 pH units, between 0.05 and 0.5 pH units, between 0.05 and 0.4 pH units, between 0.05 and 0.3 pH units, between 0.05 and 0.2 pH units, or between 0.05 and 0.1 pH units higher than the first pH.

[0042] Embodiment 33 is the peptide of any one of embodiments 28-32, wherein the AAV dissociates from the peptide at any pH that is lower than the first pH or higher than the second pH.

[0043] Embodiment 34 is the peptide of any one of embodiments 28-33, wherein the AAV dissociates from the peptide at any pH that is lower than a first pH or higher than a second pH, and the second pH is between 0.05 and 2 pH units, between 0.05 and 1.75 pH units, between 0.05 and 1.5 pH units, between 0.05 and 1.25 pH units, between 0.05 and 1 pH units, between 0.05 and 0.75 pH units, between 0.05 and 0.5 pH units, between 0.05 and 0.4 pH units, between 0.05 and 0.3 pH units, between 0.05 and 0.2 pH units, or between 0.05 and 0.1 pH units higher than the first pH.

[0044] Embodiment 35 is the peptide of any one of embodiments 28-34, wherein the peptide binds the AAV only at a pH that is between the first pH and the second pH, inclusive.

[0045] Embodiment 36 is the peptide of any one of embodiments 28-35, wherein a) the first pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0; and / or b) the second pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

[0046] Embodiment 37 is the peptide of any one of embodiments 28-36, wherein the first pH is 7.0 and the second pH is 8.0.

[0047] Embodiment 38 is the peptide of any one of embodiments 28-37, wherein the peptide binds the AAV at pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0048] Embodiment 39 is the peptide of any one of embodiments 28-38, wherein the peptide binds the AAV at pH 7.4.

[0049] Embodiment 40 is a method of obtaining an AAV from an AAV starting preparation comprising the AAV, the method comprising: a) contacting an AAV with the peptide of any one of embodiments 1-39, wherein the contacting occurs at a first pH that is within a pH range within which the AAV binds the peptide; b) optionally performing a wash step at a pH within the pH range within which the AAV binds the peptide to remove all or a portion of the components of the AAV starting preparation that are not bound to the peptide; and c) dissociating the AAV from the peptide by changing the pH to a second pH that is within a pH range within which the AAV does not bind the peptide.

[0050] Embodiment 41 is the method of embodiment 40, further comprising, following step (c), subjecting the AAV to one or more of:(i) a clarifying treatment;(ii) a step of treating the obtained AAV with a nuclease and / or a protease;(iii) a chromatography process; and(iv) a concentrating process.

[0051] Embodiment 42 is the method of embodiment 40 or embodiment 41, wherein the AAV comprises an AAV full capsid and one or more AAV intermediaries.

[0052] Embodiment 43 is the method of embodiment 42, further comprising a step of separating the AAV full capsid from the one or more AAV intermediaries.

[0053] Embodiment 44 is the method of embodiment 43, wherein the step of separating the AAV full capsid from the one or more AAV intermediaries is performed following step (c).

[0054] Embodiment 45 is the method of any one of embodiments 40-44, wherein the first pH is within a pH range of 6.5 to 8.5.

[0055] Embodiment 46 is the method of any one of embodiments 40-45, wherein the second pH is within a pH range of 0.5 to 7.0 or 8.0 to 14.0.

[0056] Embodiment 47 is the method of any one of embodiments 40-46, wherein the first pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

[0057] Embodiment 48 is the method of any one of embodiments 40-47, wherein the first pH is 7.4.

[0058] Embodiment 49 is the method of any one of embodiments 40-48, wherein the pH range within which the AAV binds the peptide is defined by a lower bound pH and an upper bound pH, inclusive.

[0059] Embodiment 50 is the method of embodiment 49, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is lower than the lower bound pH than it is to dissociate from the peptide at a pH that is between the lower bound pH and the upper bound pH, inclusive.

[0060] Embodiment 51 is the method of embodiment 49 or embodiment 50, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is higher than the upper bound pH than it is to dissociate from the peptide at a pH that is between the lower bound pH and the upper bound pH, inclusive.

[0061] Embodiment 52 is the method of any one of embodiments 49-51, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is lower than the lower bound pH or higher than the upper bound pH, than it is to dissociate from the peptide at a pH that is between the lower bound pH and the upper bound pH, inclusive.

[0062] Embodiment 53 is the method of any one of embodiments 49-52, wherein the upper bound pH is between 0.05 and 2 pH units, between 0.05 and 1.75 pH units, between 0.05 and 1.5 pH units, between 0.05 and 1.25 pH units, between 0.05 and 1 pH units, between 0.05 and 0.75 pH units, between 0.05 and 0.5 pH units, between 0.05 and 0.4 pH units, between 0.05 and 0.3 pH units, between 0.05 and 0.2 pH units, or between 0.05 and 0.1 pH units higher than the lower bound pH.

[0063] Embodiment 54 is the method of any one of embodiments 49-53, wherein a) the lower bound pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0; and / or b) the upper bound pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

[0064] Embodiment 55 is the method of any one of embodiments 40-54, comprising the wash step.

[0065] Embodiment 56 is the method of any one of embodiments 40-55, wherein the wash step is carried out within a pH range of 6.5 to 8.5.

[0066] Embodiment 57 is the method of any one of embodiments 40-56, wherein the wash step comprises a wash buffer, optionally wherein the wash buffer is phosphate buffered saline solution.

[0067] Embodiment 58 is the method of any one of embodiments 40-57, wherein step (b), if present, and step (c) occur simultaneously.

[0068] Embodiment 59 is the method of any one of embodiments 40-58, wherein step (b), if present, and step (c) occur sequentially.

[0069] Embodiment 60 is the method of any one of embodiments 40-59, wherein the dissociating step comprises a dissociation buffer, optionally wherein the dissociation buffer comprises any one or more of Citric acid (e.g., at pH 3-4), Sodium acetate (e.g., at pH 4-5), Sodium citrate (e.g., at pH 5-6), Sodium cacodylate (e.g., at pH 6-7), HEPES sodium (e.g., pH 7- 8), TRIS hydrochloride (e.g., at pH 8-9), CAPSO (e.g., at pH 9-10), and / or CAPS (e.g., at pH 10- H).

[0070] Embodiment 61 is the method of any one of embodiments 40-60, wherein the AAV is separated from at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of other components of the AAV starting preparation.

[0071] Embodiment 62 is the method of embodiment 61, wherein the other components of the AAV starting preparation comprise one or more of a cell culture media, whole cells, cellular materials, and cell lysate.

[0072] Embodiment 63 is the method of any one of embodiments 40-62, wherein a yield of the AAV is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the AAV initially present in the AAV starting preparation.

[0073] Embodiment 64 is the method of any one of embodiments 40-63, wherein the peptide is immobilized on a solid support.

[0074] Embodiment 65 is the method of embodiment 64, wherein the solid support comprises sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, a flow cell, a hydrogel, a silica gel, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, an alumina gel, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead.

[0075] Embodiment 66 is the method of any one of embodiments 40-65, wherein the AAV is removed from the solid support by means of one or more elution buffers.

[0076] Embodiment 67 is the method of embodiment 66, wherein the elution buffer has a pH between 2-4, 2.5-3.5, 2.8-3.2, 2.9-3.1, 2.9-3.4, 2.9-3.3, or 2.9-3.2.

[0077] Embodiment 68 is the method of embodiment 66 or embodiment 67, wherein the elution buffer comprises Tris, NaCl, Pluronic, and / or Glycine.

[0078] Embodiment 69 is the method of any one of embodiments 40-68, wherein the AAV is AAV9.

[0079] Embodiment 70 is the method of any one of embodiments 40-69, wherein the AAV is a recombinant AAV.

[0080] Embodiment 71 is the method of any one of embodiments 40-70, wherein the AAV is a self-complementary AAV.

[0081] Embodiment 72 is the method of any one of embodiments 40-71, wherein the AAV comprises a nucleic acid molecule of interest.

[0082] Embodiment 73 is an AAV obtained using the method of any one of embodiments 40-72.

[0083] Embodiment 74 is the AAV of embodiment 73, wherein the AAV is an AAV9.

[0084] Embodiment 75 is a kit comprising the peptide of any one of embodiments 1-39.

[0085] Embodiment 76 is the kit of embodiment 75, further comprising one or more of: a) a wash buffer having a pH within the pH range within which the AAV binds the peptide; b) a dissociation buffer having a pH outside the pH range within which the AAV binds the peptide; c) a solid support, optionally wherein the solid support comprises Sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, a flow cell, a hydrogel, a silica gel, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, an alumina gel, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead; d) a nucleic acid molecule encoding an AAV capsid protein; e) a nucleic acid molecule encoding an AAV rep protein; f) one or more nucleic acids encoding one or more helper functions required for packaging a nucleic acid molecule of interest into an AAV capsid; g) an AAV packaging cell; and h) a control peptide that does not bind the AAV at a pH within which the AAV binds the peptide.BRIEF DESCRIPTION OF THE DRAWINGS

[0086] FIGS. 1A-1E illustrate binding and dissociation of exemplary peptides (LP3-1 and LP3- 2) with an AAV9. FIG. 1A is a diagram depicting the workflow for a biolayer interferometry (BLI)-based binding assay. FIG. IB shows a target peptide displayed with a C-terminal linker followed by a C-terminal biotin (AEGEFNKSTMLAHSDGGGSGGGSL, SEQ ID NO: 30). FIGS. 1C-1E are binding assays showing the association of AAV9 capsids with the depicted peptides (AEGFNKSTMLAHS, SEQ ID NO: 31; AEGFNKSTHLAFY, SEQ ID NO: 32; GSGSGSG, SEQ ID NO: 33) followed by a dissociation step in buffer alone. The AAV9 capsid concentration starts at 30ug / mL with a two-fold dilution between each subsequent measurement.

[0087] FIGS. 2A-2B illustrate epitope mapping experiments used to determine the region of the AAV9 capsid that binds the LP3-1 peptide. FIG. 2A is a diagram depicting the loading and association steps of an exemplary competition assay. FIG. 2B shows the association and dissociation steps of an exemplary competition assay. During the association step, octet sensors loaded with LP3-1 are dipped in wells containing 12.5 ug / mL of AAV9 alone, 12.5 ug / mL AAV9 with lOOug / mL AAVX ligand, or 12.5ug / mL AAV9 with lOOug / mL POROS ligand.

[0088] FIGS. 3A-3C illustrate binding and dissociation of AAV9 with the POROS ligand or with the LP3-1 peptide (as shown in FIG. IB). FIG. 3A is a diagram depicting a BLI-based pH sensitivity assay. FIG. 3B shows POROS ligand binding to AAV9 (12.5 ug / mL) and dissociation of the AAV9 from the ligand at pH 3.0 and pH 10.4. FIG. 3C shows LP3-1 peptide binding to AAV9 (12.5 ug / mL) and dissociation of the AAV9 from the peptide in buffers at the indicated pH.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0089] Reference will now be made in detail to certain embodiments of the disclosure. While the disclosed methods will be described in conjunction with such embodiments, it will be understood that they are not intended to limit the disclosed methods to those embodiments. On the contrary, the disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the disclosure as defined by the appended claims.

[0090] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a bead” includes a plurality of beads, reference to “a cell” includes a plurality of cells, and the like.

[0091] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0092] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as encompassing embodiments “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as being among embodiments “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as encompassing embodiments “consisting of’ or “comprising” the recited components.

[0093] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference contradicts any explicit content of this specification, including definitions, this specification controls.I. Definitions

[0094] As used herein, “or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise.

[0095] As used herein, “adeno-associated virus” or “AAV” refers to an adeno-associated virus vector. As used herein, “AAV” refers to any AAV serotype and variant, including but not limited to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhlO (see, e.g, SEQ ID NO: 81 of US 9,790,472, which is incorporated by reference herein in its entirety), AAVrh74 (see, e.g., SEQ ID NO: 1 of US 2015 / 0111955, which is incorporated by reference herein in its entirety), AAV9, AAV9P also known as AAVMYO (see, Weinmann et al., 2020, Nature Communications, 11 :5432), AAV10, AAV11, AAV12, and Myo- AAV as described in Tabebordbar et al., 2021, Cell, 184: 1-20 (e.g., MyoAAV 1 A, 2A, 3A, 4A, 4C, or 4E), wherein the number following AAV indicates the AAV serotype. The term “AAV” can also refer to any known AAV (vector) system. In some embodiments, the AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the AAV vector is a double-stranded AAV (dsAAV). Any variant of an AAV vector or serotype thereof, such as a self-complementary AAV (scAAV) vector, is encompassed within the general terms AAV vector, AAV1 vector, etc. See, e.g.,McCarty et aL, Gene Ther. 2001; 8: 1248-54, Naso et aL, BioDrugs. 2017; 31 :317-334, and references cited therein for detailed discussion of various AAV vectors. Structurally, AAVs are small (25 nm), single-stranded DNA, non-enveloped viruses with an icosahedral capsid. As used herein, “AAV” can refer to naturally occurring or engineered AAV serotypes and recombinant AAVs (rAAVs) and variants that can differ in the composition and structure of their capsid protein and that may have varying tropism, z.e., ability to transduce different cell types.

[0096] As used herein, “recombinant AAV” refers to an AAV with a capsid having packaged therein a heterologous nucleic acid molecule comprising an expression cassette for a desired product, such as a gene product. Such an expression cassette may contain an AAV 5’ and / or 3’ inverted terminal repeat sequence flanking a nucleic acid of interest, such as a gene sequence, in which the nucleic acid of interest is operably linked to expression control sequences. A “pharmacologically active” rAAV is capable of delivering the transgene to a host cell that is capable of expressing the nucleic acid of interest carried by the expression cassette.

[0097] As used herein, “adeno-associated virus starting preparation” or “AAV starting preparation” refers to a mixture comprising an AAV and other components, such as media collected from a packaging cell culture and / or components of a packaging cell lysate. An AAV starting preparation can comprise impurities present in the packaging cell media and / or packaging cell lysate, such as various cellular components, such as packaging cell DNA, packaging cell proteins, media components, and in some instances, helper virus or helper virus plasmid DNA. In some embodiments, following AAV harvest (such as from an AAV packaging cell and / or from an AAV packaging cell culture medium), an AAV starting preparation is processed prior to contacting the AAV of the AAV starting preparation with the disclosed peptides. Thus, an AAV starting preparation of use herein may comprise impurities that remain following a processing step, such as a processing step comprising one or more filtration and / or centrifugation steps, and / or treatment of the AAV starting preparation with cell lysis agents, enzymes such as endonucleases (such as a DNase), and other such reagents known in the art.

[0098] In some embodiments, an AAV is endonuclease resistant (e.g., DNase resistant). Hence, an AAV starting preparation may in some cases include one or more endonucleases. In addition to endonucleases (DNase), exonucleases may also be added to an AAV starting preparation described herein, to remove contaminating nucleic acids. Such nucleases may be selected to degrade single stranded DNA and / or double-stranded DNA, and RNA. Such steps may contain a single nuclease, or mixtures of nucleases directed to different targets, and may be endonucleases or exonucleases. An AAV is “nuclease resistant” when the AAV capsid has fully assembled around the expression cassette which is designed to deliver a nucleic acid of interest (e.g., a transgene) to a host cell and protects these packaged genomic sequences from degradation(digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.

[0099] As used herein, “bind” or “binding” can refer to a covalent or non-covalent interaction between macromolecules (e.g., a non-covalent interaction between a peptide and an AAV, or a non-covalent interaction between a biotin or a modified biotin and a biotin binding agent, such as streptavidin). While in a state of non-covalent interaction, the macromolecules can be said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). “Affinity” or “binding affinity” refers to the total strength of all non-covalent interactions between a binding site of a molecule (e.g., a biotin) and its binding partner (e.g., biotin binding agent, such as avidin, streptavidin, or neutravidin). Unless otherwise indicated, “binding affinity” as used herein refers to the intrinsic binding affinity that reflects a 1 : 1 interaction between members of a binding pair (e.g., biotin and biotin binding agent). The affinity of a molecule X for its partner Y can usually be determined by the dissociation constant (Kd), with increased binding affinity being correlated with a lower Kd. Affinity can be measured by common methods known in the art. Binding interactions are generally characterized by a dissociation constant (Kd) of less than 10'6M, less than 10'7M, less than 10'8M, less than 10'9M, less than IO'10M, less than 10'11M, less than 10'12M, less than 10'13M, less than 10'14M, or less than 10'15M. By “binding domain” it is meant a protein domain that is able to bind non-covalently to another molecule. A binding domain of an AAV can bind to, for example, a disclosed peptide (a proteinbinding protein). As used herein, a molecule that “binds” or “has an affinity for” or “has a binding affinity for” another molecule refers to a molecule that interacts with the other molecule with a dissociation constant (Kd) indicative of a specific interaction between the molecules (i.e., with a Kd less than that of random, nonspecific interactions between molecules in the solution, or characterized by a Kd of less than 10'6M, less than 10'7M, less than 10'8M, less than 10'9M, less than IO'10M, less than 10'11M, less than 10'12M, less than 10'13M, less than 10'14M, or less than 10’15M).

[0100] The term “conservative amino acid substitution” refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate;and a group of amino acids having sulfur containing side chains consists of cysteine and methionine. A “conservative amino acid substitution” herein thus refers to the replacement of one or more amino acids of a peptide with an alternative amino acid with similar properties and which does not substantially alter the physical-chemical properties and / or structure or function of the respective comparator peptide, e.g., a substitution of one amino acid within one of the above groups for another of the amino acids within that same group (e.g., a substitution of one basic amino acid for another basic amino acid or of one aromatic amino acid for another aromatic amino acid). Such exemplary conservative amino acid substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and / or asparagine-glutamine.

[0101] As used herein, “binding moiety” refers to any moiety that binds (such as non-covalently) to a target “affinity moiety” as described herein. Exemplary binding moieties include, but are not limited to biotin, modified biotin, and antibodies, or any of the peptides disclosed herein. Exemplary affinity moieties can thus include, but are not limited to, molecules having an affinity for a given binding moiety, such as biotin binding agents (such as avidin, streptavidin, neutravidin, anti-biotin antibodies, biotin receptors, and / or biotin-binding enzymes).

[0102] As used herein, “contact” or “contacting” refers to bringing together physically, such as bringing two molecules (such as a peptide and an AAV) into close proximity with one another such that an association (such as non-covalent bonding) may occur, so long as the two molecules have an affinity for one another. For example, in some embodiments, an AAV starting preparation comprising an AAV (such as an AAV9) is introduced to (such as mixed with) a solution comprising a peptide that has an affinity for the AAV. In such embodiments, when the AAV starting preparation is introduced to the solution comprising the peptide (or when the peptide is introduced to the AAV starting preparation) the AAV and the peptide can contact one another and binding (such as a non-covalent association) can occur.

[0103] As used herein, “dissociate” or “dissociation” refers to physically separating two molecules (such as a peptide and an AAV). Dissociation may be induced, for example, by altering optimal or favorable binding conditions between two molecules, such as by changing the pH that is optimal or favorable for binding conditions between two molecules in a buffer comprising the two molecules. Molecules that are dissociated from one another are no longer bound (such as non-covalently) to one another. In some embodiments, the dissociation is not permanent. For example, if optimal / favorable conditions for binding between two molecules are changed to facilitate dissociation, the optimal / favorable conditions may be returned to facilitate binding between the two molecules.

[0104] As used herein, “immobilized” refers to direct or indirect, covalent or non-covalent attachment of a molecule (e.g., a peptide) to a substrate, such as a solid support. When referringto immobilization of molecules (e.g., peptides) to a solid support, the terms “immobilized” and “attached” are used interchangeably herein and both terms are intended to encompass direct or indirect, covalent or non-covalent attachment, unless indicated otherwise, either explicitly or by context. In certain embodiments of the invention covalent attachment may be preferred, but generally all that is required is that the molecules (e.g., peptides) remain immobilized or attached to the support under the conditions in which it is intended to use the support, for example in applications requiring buffer modification or exchange (e.g., flow-through exchange). In particular embodiments, immobilization of a molecule is not permanent (e.g., immobilization only occurs under specific conditions).

[0105] As used herein, “isolated” refers to a biological component (such as an AAV) that has been separated from some or all other components of a mixture in which it was previously present (such as an AAV starting preparation comprising the AAV, cell culture media, whole cells, cellular materials, and / or cell lysate), or that is not in its native biological context (such as a naturally occurring peptide that has been chemically synthesized or recombinantly produced). A biological component (such as an AAV, such as an AAV9) need not be completely separated from other components of the mixture to be “isolated,” but may be separated from at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of other components of the mixture (such as other components of the AAV starting preparation). A peptide or polynucleotide molecule may be “isolated,” for example, if it has been produced synthetically or recombinantly, or if it has been at least partially separated from other components of a biological mixture, such as whole cells or a cell lysate, and thus, is not present in any naturally occurring form.

[0106] As used herein, “peptide” refers to a polymer of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Most commonly, “peptide” refers to an amino acid sequence that is less than or equal to 50 amino acids long, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acids long. In some embodiments, a peptide may be more than 50 amino acids long, such as 75, 100, 125, 150, 175, or 200 amino acids long. Exemplary peptides are described herein.

[0107] As used herein, “solid support” and other grammatical equivalents herein refer to any material that is appropriate for or can be modified to be appropriate for the attachment of the disclosed peptides. In some embodiments, the solid support comprises particles (such as beads), a patterned surface, or wells. Exemplary solid supports include a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, a flow cell, a hydrogel, a silica gel, polymericmembranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, an alumina gel, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead. Exemplary solid support substrates include sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, Additional exemplary substrates include glass and modified or functionalized glass, plastics (including, for example, acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon, ceramics, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, optical fiber bundles, and a variety of other polymers. Certain useful solid supports and solid surfaces for some embodiments are located within a flow cell apparatus.

[0108] Additional definitions are provided elsewhere in the disclosure, including in the sections that follow.II. Isolated PeptidesA. Overview

[0109] In some embodiments, an isolated peptide that can bind an AAV (such as an AAV9) is provided. In certain embodiments, such isolated peptides comprise at least 8 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-28 (Table 1).

[0110] Table 1: Exemplary Peptides

[0111] In some embodiments, the isolated peptide comprises at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-28. In other embodiments, the isolated peptide comprises at least 12 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 2-14 or 16-28. In other embodiments, the isolated peptide comprises at least 13 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 4-14 or 18-28. In other embodiments, the isolated peptide comprises at least 14 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 9-14 or 23-28. In other embodiments, the isolated peptide comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 28. In some embodiments, the isolated peptide consists of an amino acid sequence selected from any one of SEQ ID NOs: 1-28. In some embodiments, the isolated peptide comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 28, wherein any 1, 2, 3, or 4 of the first four amino acids of SEQ ID NO: 14 or SEQ ID NO: 28 are deleted.

[0112] In particular embodiments, the isolated peptide comprises at least 8 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-14. In another particular embodiment, the isolated peptide comprises at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-14. In another particular embodiment, the isolated peptide comprises at least 12 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 2-14. In another particular embodiment, the isolated peptide comprises at least 13 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 4-14. In another particular embodiment, the isolated peptide comprises at least 14 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 9-14. In another particular embodiment, the isolated peptide comprises the amino acid sequence of SEQ ID NO: 14. In other particular embodiments, the isolated peptide consists of an amino acid sequence selected from any one of SEQ ID NOs: 1-14. In another particular embodiment, the isolated peptide comprises the aminoacid sequence of SEQ ID NO: 14, wherein any 1, 2, 3, or 4 of the first four amino acids of SEQ ID NO: 14 are deleted.

[0113] In some embodiments, a disclosed peptide comprising at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-28 further comprises one or more additional amino acids at the N-terminus and / or at the C-terminus of the peptide. In some embodiments, the peptide comprising at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-28 further comprises 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, 30, 35, 40, 45, 50, 75, or 100 or more additional amino acids at the N- terminus of the peptide. In some embodiments, the peptide comprising at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-28 further comprises 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, 30, 35, 40, 45, 50, 75, or 100 or more additional amino acids at the C- terminus of the peptide.

[0114] In some embodiments, a disclosed peptide comprising at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-28 is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more amino acids in length. In particular embodiments, a disclosed peptide is 8-200, such as 8-175, 8-150, 8-125, 8-100, 8-75, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15, 9-200, 9-175, 9-150, 9-125, 9-100, 9-75, 9-50, 9-45, 9-40, 9- 35, 9-30, 9-25, 9-20, 9-15, 10-200, 10-175, 10-150, 10-125, 10-100, 10-75, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 11-200, 11-175, 11-150, 11-125, 11-100, 11-75, 11-50, 11- 45, 11-40, 11-35, 11-30, 11-25, 11-20, 11-15, 12-200, 12-175, 12-150, 12-125, 12-100, 12-75, 12-50, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 12-15, 13-200, 13-175, 13-150, 13-125, 13- 100, 13-75, 13-50, 13-45, 13-40, 13-35, 13-30, 13-25, 13-20, 13-15, 14-200, 14-175, 14-150, 14- 125, 14-100, 14-75, 14-50, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 14-15, 15-200, 15-175, 15- 150, 15-125, 15-100, 15-75, 15-50, 15-45, 15-40, 15-35, 15-30, 15-25, or 15-20 amino acids in length. In particular embodiments, the peptide is 8-50 amino acids in length. In some embodiments, the peptide is 8-25 amino acids in length. In some embodiments, the peptide is 8- 15 amino acids in length.

[0115] Peptides of the present disclosure may be produced using any suitable means known in the art, such as chemical synthesis. Peptide synthesis most frequently occurs by coupling the carboxyl group of the incoming amino acid to the N-terminus of the growing peptide chain, such as through a condensation reaction. This C-to-N synthesis is opposite from protein biosynthesis, during which the N-terminus of the incoming amino acid is linked to the C-terminus of theprotein chain (N-to-C). The addition of amino acids to a growing peptide chain typically occurs in a precise, stepwise, and cyclic manner. Strategies such as the use of protecting groups can help to prevent undesired side reactions. While common methods of peptide synthesis may differ, they commonly follow the same stepwise addition of amino acids one at a time to the growing peptide chain.

[0116] Peptide isolation and purification strategies may be based on one or more separation methods that utilize the physiochemical characteristics of the particular peptides, including size, charge, and hydrophobicity. Purification techniques are known in the art and include sizeexclusion chromatography (SEC), ion exchange chromatography (IEC), partition chromatography, and high-performance liquid chromatography (HPLC). For example, reversephase chromatography (RPC) is a versatile and widely used method of peptide purification. In RPC, hydrophobic molecules from aqueous solutions are captured by a stationary phase using hydrophobic (e.g., C4, C8, or Cl 8 n-alkyl hydrocarbon) ligands, and their retention time is a function of the hydrophobicity of the molecule and of the mobile phase. RPC can separate target peptides from impurities from the synthesis steps, such as isomers, deletion sequences, peptide products from side reactions with free coupling and protecting groups, or peptides that have undergone side-chain reactions. Peptide purity can be measured as a percentage of the target peptide to impurities that absorb at the peptide bond absorption wavelength (210-220 nm).

[0117] In certain embodiments, a disclosed peptide may comprise one or more amino acid substitutions, insertions, or deletions. In a substitution, at least one amino acid is exchanged for another amino acid in the sequence in comparison to the respective comparator sequence (such as an amino acid sequence selected from any one of SEQ ID NOs: 1-28). In an insertion, at least one amino acid or nucleotide is inserted into the sequence in comparison to the respective comparator sequence. In a deletion, at least one amino acid or nucleotide is omitted in the sequence in comparison to the respective comparator sequence.

[0118] In some embodiments, a substitution may be a conservative amino acid substitution in the primary sequence (such as an amino acid sequence selected from any one of SEQ ID NOs: 1-28). In some embodiments, a disclosed peptide comprising at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1- 28 comprises 1, 2, 3, 4, or 5 conservative amino acid substitutions. Analogues of this type are also encompassed within the scope of this disclosure. In one embodiment, substitute amino acids may be selected from other members of the class to which the amino acid belongs. Examples of conservative substitutions include Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free OH is maintained; and Gin for Asn to maintain a free NH2.

[0119] In some embodiments, the amino acid corresponding to the first and / or twelfth amino acid of SEQ ID NO: 14 or 28 is substituted with a glycine, valine, leucine, or isoleucine. In some embodiments, the amino acid corresponding to the second and / or fourth amino acid of SEQ ID NO: 14 or 28 is substituted with an aspartate. In some embodiments, the amino acid corresponding to the third amino acid of SEQ ID NO: 14 or 28 is substituted with alanine, valine, leucine, or isoleucine. In some embodiments, the amino acid corresponding to the fifth amino acid of SEQ ID NO: 14 or 28 is substituted with a tyrosine or tryptophan. In some embodiments, the amino acid corresponding to the sixth amino acid of SEQ ID NO: 14 or is substituted with a glutamine. In some embodiments, the amino acid corresponding to the seventh amino acid of SEQ ID NO: 14 or 28 is substituted with an arginine or histidine. In some embodiments, the amino acid corresponding to the eighth and / or fourteenth amino acid of SEQ ID NO: 14 or 28 is substituted with threonine. In some embodiments, the amino acid corresponding to the ninth amino acid of SEQ ID NO: 14 is substituted with a serine. In some embodiments, the amino acid corresponding to the tenth amino acid of SEQ ID NO: 14 is substituted with a cysteine. In some embodiments, the amino acid corresponding to the eleventh amino acid of SEQ ID NO: 14 is substituted with a glycine, alanine, valine, or isoleucine. In some embodiments, the amino acid corresponding to the thirteenth amino acid of SEQ ID NO: 14 is substituted with a lysine or arginine. In some embodiments, the amino acid corresponding to the fifteenth amino acid of SEQ ID NO: 14 or 28 is substituted with a glutamate. In some embodiments, the amino acid corresponding to the tenth amino acid of SEQ ID NO: 28 is substituted with a lysine or arginine. In some embodiments, the amino acid corresponding to the eleventh amino acid of SEQ ID NO: 28 is substituted with a glycine, alanine, valine or isoleucine. In some embodiments, the amino acid corresponding to the twelfth amino acid of SEQ ID NO: 28 is substituted with a glycine, valine, leucine, or isoleucine. In some embodiments, the amino acid corresponding to the thirteenth amino acid of SEQ ID NO: 28 is substituted with a tyrosine or tryptophan. In some embodiments, the amino acid corresponding to the fourteenth amino acid of SEQ ID NO: 28 is substituted with a tryptophan or phenylalanine. In some embodiments, any of the referenced amino acids of SEQ ID NO: 14 in this paragraph are the corresponding amino acids of any one of SEQ ID Nos: 1-13. In some embodiments, any of the referenced amino acids of SEQ ID NO: 28 in this paragraph are the corresponding amino acids of any one of SEQ ID Nos: 15-27.

[0120] Peptides of the present disclosure can selectively bind an AAV, such as an AAV9. In some embodiments, the peptide binds the AAV (such as the AAV9) between a first pH and a second pH, inclusive, wherein the second pH is between 0.05 and 2.00 pH units higher than the first pH, such as between 0.05 and 1.75 pH units, between 0.05 and 1.50 pH units, between 0.05 and 1.25 pH units, between 0.05 and 1 pH units, between 0.05 and 0.75 pH units, between 0.05and 0.50 pH units, between 0.05 and 0.40 pH units, between 0.05 and 0.30 pH units, between 0.05 and 0.20 pH units, or between 0.05 and 0.10 pH units higher than the first pH. In some embodiments, the AAV dissociates from the peptide at any pH that is lower than the first pH or higher than the second pH. In some embodiments, the AAV is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more likely to dissociate from the peptide at any pH that is lower than the first pH. In some embodiments, the AAV is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more likely to dissociate from the peptide at any pH that is higher than the second pH. The pH values of the first pH and the second pH can depend on components of the particular buffer being used. “Between a first and second pH, inclusive” means the provided pH range including the end points. Accordingly, pH ranges “between a first and second pH, inclusive” are intended to include each end pH unless stated otherwise. One of ordinary skill will understand that pH values are approximate, and may be rounded up or down accordingly to the nearest significant figure and may vary somewhat depending upon the instrumentation used for the measurement and its calibration. In some instances, a provided pH value is an average, such as if pH measurements are taken more than once and then averaged. In some instances, a pH value is rounded to the nearest significant figure.

[0121] In some embodiments, the AAV (such as the AAV9) dissociates from the peptide at any pH that is lower than a first pH or higher than a second pH, and the second pH is between 0.05 and 2 pH units higher than the first pH, such as between 0.05 and 1.75 pH units, between 0.05 and 1.5 pH units, between 0.05 and 1.25 pH units, between 0.05 and 1 pH units, between 0.05 and 0.75 pH units, between 0.05 and 0.5 pH units, between 0.05 and 0.4 pH units, between 0.05 and 0.3 pH units, between 0.05 and 0.2 pH units, or between 0.05 and 0.1 pH units higher than the first pH. In particular embodiments, the peptide binds the AAV only at a pH that is between the first pH and the second pH, inclusive. In some embodiments, the AAV is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more likely to dissociate from the peptide at any pH that is lower than a first pH or higher than a second pH, and the second pH is between 0.05 and 2 pH units higher than the first pH, such as between 0.05 and 1.75 pH units, between 0.05 and 1.5 pH units, between 0.05 and 1.25 pH units, between 0.05 and 1 pH units, between 0.05 and 0.75 pH units, between 0.05 and 0.5 pH units, between 0.05 and 0.4 pH units, between 0.05 and 0.3 pH units, between 0.05 and 0.2 pH units, or between 0.05 and 0.1 pH units higher than the first pH.

[0122] In certain embodiments, the first pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8, 7.9, or 8.0. In certain embodiments, the second pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In particular embodiments, the first pH is 7.0 and the second pH is 8.0. In other particular embodiments, the peptide binds the AAV at pH 7.0, 7.1, 7.2,7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In a specific, non-limiting embodiment, the peptide binds the AAV at pH 7.4.B. Modifications

[0123] In some embodiments of the present disclosure, isolated peptides comprise one or more modifications, such as one or more modified amino acids. For example, chemically synthesized peptides may comprise free (e.g., unmodified) N- and C-termini that may be modified during or following completion of synthesis. In some embodiments, one or more amino acids (such as the N- and / or C-terminal amino acids) of the disclosed peptides may be modified by amidation, acetylation, and / or other modifications disclosed herein. In some embodiments, one or more amino acids are acetylated. In some embodiments, one or more amino acids are amidated. Any amino acid of a disclosed peptide may be modified, such as, but not limited to, at the N-terminal amino group, the amino group of Lys, the thiol group of Cys, the hydroxyl groups of Ser, Thr, and / or Tyr, the C=NH group of Arg, and / or the C-terminal carboxyl group.

[0124] For example, a peptide of the present disclosure can comprise one or more acetylated amino acids acetylated under acetylating conditions known in the art (e.g., using an acetyltransferase). In some embodiments, an amino acid comprises an acetyl group (COCH3). In some embodiments, peptides of the present disclosure are acetylated at the N-terminus and / or the C-terminus. In some embodiments, the N-terminal amino acid of the peptide is acetylated. In some embodiments, the C-terminal amino acid of the peptide is acetylated. In particular embodiments, the stability of a disclosed peptide comprising one or more acetylated amino acids is increased compared to that of an otherwise identical peptide not comprising the one or more acetylated amino acids. In some embodiments, enhanced stability is a feature that is of use, such as when the disclosed peptides are employed under conditions where they will be subject to pH changes or proteolytic degradation (e.g., hydrolysis by naturally occurring enzymes, such as enzymes present in an AAV starting preparation), before the peptides perform their desired function.

[0125] In some embodiments, a disclosed peptide comprises one or more amidated amino acids amidated under amidation conditions known in the art. In some embodiments, an amino acid comprises an amide (R-C=O(N)R’R”, e.g., R-C=O(NH2)). The peptides of the present invention may be amidated at the N-terminus and / or the C-terminus. In some embodiments, the N-terminal amino acid of the peptide is amidated. In some embodiments, the C-terminal amino acid of the peptide is amidated. In particular embodiments, the stability of a disclosed peptide comprising one or more amidated amino acids is increased compared to that of an otherwise identical peptide not comprising the one or more amidated amino acids. For example, a peptidecomprising one or more amidated amino acids may be less sensitive to proteolytic degradation and / or changes in pH than the same peptide that does not comprise the one or more amidated amino acids.

[0126] In some embodiments, the peptides of the present invention are acetylated at the N- terminus, amidated at the C-terminus, or both acetylated at the N-terminus and amidated at the C-terminus. In some embodiments, the peptides of the present invention may have both an acetylated N-terminus and a carboxy terminal amide. In some embodiments, acetylation and / or amidation of a disclosed peptide can reduce the overall charge of the peptide. In the present application, when a peptide is acetylated on an N or C terminus, the letters Ac are indicated, and when a peptide is amidated on an N or C terminus, the designation NH2 is employed.

[0127] In certain embodiments, a peptide of the present disclosure comprises one or more isoaspartic acid residues, such as at the N- or C-terminus of the peptide. Iso-aspartic acid (also known as isoaspartate, isoaspartyl, and P-aspartate) is an aspartic acid residue isomeric to the typical a peptide linkage. Iso-aspartic acid is a P-amino acid, with the side chain carboxyl moved to the backbone.

[0128] In some embodiments, a disclosed peptide comprises one or more 2,3 diaminopropionic acid (Dap; C3H8N2O2) residues. Dap is a diamino acid that is alanine in which one of the hydrogens of the methyl group is replaced by an amino group. In some embodiments, the C- or N-terminal amino acid of the peptide comprises Dap. In particular embodiments, the N-terminal amino acid of the peptide comprises Dap. In some embodiments, the Dap is acetylated.C. Linkers

[0129] In some embodiments, a peptide of the present disclosure comprises one or more linkers, such as one or more linkers attached (e.g., covalently bound) to the N- and / or C-terminal amino acid of the peptide. Any suitable linker may be used. For example, the linker can comprise a flexible linker and / or a cleavable linker. In certain embodiments, the linker includes at least a linear group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether (-S-) and hydroxylamino (-O-N(H)-) groups. In certain embodiments, the linear group comprises groups selected from alkyl, amide, and ether groups. In certain embodiments, the linear group comprises one or more alkyl groups.

[0130] In some embodiments, a disclosed peptide is covalently linked to a linker (such as a peptide linker or a non-peptide linker) through an amine group of a lysine amino acid. In particular embodiments, a binding moiety (such as described herein) is attached (such as covalently bound) to the peptide through the linker. For example, in some embodiments, the linker links the N- or C-terminal amino acid of the peptide to the binding moiety. In certainembodiments, the linker is covalently bound to the peptide and a lysine links the linker to the binding moiety. In particular embodiments, the binding moiety is a biotin or a modified biotin, the linker is covalently bound to the C-terminal amino acid of the peptide, and a lysine links the linker to the biotin or the modified biotin.

[0131] In some embodiments, the one or more linkers comprise one or more amino acids (e.g., a peptide linker). Such linkers can be 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, 30, 35, 40 or more amino acids in length. An illustrative peptide linker has a length from 3 amino acids (AA) to 90 AA, from 3 AA to 80 AA, from 3 AA to 70 AA, from 3 AA to 60 AA, from 3 AA to 50 A A, from 3 AA to 40 AA, from 3 AA to 30 AA, from 3 AA to 20 AA, from 3 AA to 10 AA. For example, the linker can have a length from 3 AA to 5 AA, from 5 AA to 10 AA, from 10 AA to 15 AA, from 15 AA to 20 AA, from 20 AA to 25 AA, from 25 AA to 30 AA, from 30 AA to 35 AA, from 35 AA to 40 AA, from 40 AA to 50 AA, from 50 AA to 60 AA, from 60 AA to 70 AA, from 70 AA to 80 AA, from 80 AA to 90 AA, or from 90 AA to 100 AA. In some embodiments, the peptide linker is 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, or at least 15 amino acids in length.

[0132] A peptide linker can comprise any of a variety of sequences. For example, a peptide linker can comprise one or more serine (S), glycine (G), and / or threonine (T) residues. Glycine may impart flexibility in a given linker, whereas serine and / or threonine may improve solubility. In some embodiments, the linker is a glycine-serine linker. In particular embodiments, the glycine-serine linker comprises (Glym-Ser)n, where m is 1 to 10 (such as 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 7, 3 to 6, 3 to 5, or 3 to 4) and n is 1 to 10 (such as 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3). In a particular embodiment, the glycine-serine linker is GGGSGGGS (SEQ ID NO: 29). In some embodiments, the linker comprises one or more alanine residues. In some embodiments, the linker comprises one or more proline residues. Mechanisms by which a peptide linker (such as a glycine-serine linker) can be attached to an amino acid of a disclosed AAV-binding peptide are known in the art, and site-specific linker attachment can be accomplished at a variety of sites on the disclosed peptide.

[0133] In some embodiments, the one or more linker is a polyol linker, such as a polyol linker comprising ethylene glycol, glycerol, glucose, or trehalose. In some embodiments, the linker comprises a polyethylene glycol, e.g., a PEG linker. In some embodiments, the polyethylene glycol (e.g., PEG) linker is between 2 and 18 PEGs long, e.g., PEG2 (di ethylene glycol), PEG3 (tri ethylene glycol), PEG4 (tetraethyl ene glycol), PEG5, PEG6, PEG7, PEG8, PEG9, PEG10, PEGU, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, or PEG18. In some embodiments, thePEG linker is a modified PEG linker, such as mini-PEG2 (8-amino-3,6-dioxaoctanoic acid), mini-PEG3 (amino-3,6,9-trioxaundecanoic acid), or NH2-PEG2-acid (3-(2-(2- aminoethoxy)ethoxy)-propanoic acid), or a branched PEG linker (also known as a multi-arm PEG linker). In some embodiments, the linker comprises the structure ofH (O CH2Ci hin OH, wherein n is 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, n is 2. In some embodiments, n is 3.

[0134] Mechanisms by which a PEG linker can be attached to an amino acid of a peptide are known in the art, and site-specific PEGylation can be accomplished at a variety of sites on the peptide. For example, N-terminal PEGylation can be accomplished by direct PEG carboxylic acid coupling or native chemical ligation with PEG thioester and a cysteine residue. C-terminal PEGylation can be achieved, for example, through a thiocarboxylic acid modification and sulfone-azide PEG reagent. Hydrazide modifications combined with a pyruvoyl PEG reagent is also a useful approach to C-terminal PEGylation. In addition to the N- and C-terminal, PEGylation is also possible at virtually any amino acid side chain bearing the appropriate functional group. Three methodologies are commonly used for site-specific PEGylations: (1) click chemistry, e.g., between an azide group of the PEG reagent and an alkyne group of the peptide, or vice versa; (2) Suzuki-Miyaura coupling between the iodophenyl group of the PEG reagent and an aryl boronic acid group of peptide, or vice versa; or Sonogashira coupling between an iodophenyl group of the PEG reagent and an alkyne group of the peptide, or vice versa.

[0135] In some embodiments, a disclosed peptide does not comprise a linker. In some embodiments, a peptide comprises one linker. In some embodiments, a peptide comprises two or more than two linkers.D. Binding Moieties

[0136] Some embodiments of the disclosed peptides comprise one or more binding moieties. In certain embodiments, a binding moiety is attached (such as covalently) to the peptide, such as to the C-terminal amino acid of the peptide, such as through a linker described herein. In certain embodiments, the binding moiety has an affinity for an affinity moiety (such as an affinity moiety of a solid support described herein) and can bind (such as noncovalently) the affinity moiety under conditions that allow for such binding. In some embodiments, the binding moiety is biotin or a modified biotin (z.e., the peptide is biotinylated). In such embodiments, the affinity moiety is a biotin binding agent. Biotin binding agents include, but are not limited to, avidin, streptavidin, neutravidin, anti-biotin antibodies, biotin receptors, and biotin-binding enzymes. In some embodiments, the biotin-binding enzyme is biotinidase or biotin holocarboxylasesynthetase. As used herein, the term “biotinylated” refers to the process of covalently attaching biotin or a modified biotin to another molecule, such as on the C-terminal amino acid of a disclosed peptide.

[0137] In other embodiments, the binding moiety is a biotin binding agent, such as avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor, and / or a biotin-binding enzyme. In such embodiments, the affinity moiety is a biotin or a modified biotin.

[0138] In other embodiments, the binding moiety is another peptide or an antibody. As used herein, “antibody” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, and antibody or antigen-binding fragments so long as they exhibit the desired binding activity. A range of suitable binding moieties that can bind to suitable affinity moieties are known in the art, and a user may choose any suitable pair of a binding moiety and affinity moiety.

[0139] In some embodiments, the binding moiety (such as biotin or a modified biotin) binds to an affinity moiety (such as an avidin, streptavidin, or neutravidin) on a surface of a solid support as described herein. In such embodiments, binding of the binding moiety to the affinity moiety immobilizes the peptide on the solid support, such that it will not be released from the support during a fluid exchange, e.g., a flow-through buffer exchange, such as in an exemplary embodiment wherein the solid support is a flow cell channel, or wherein the solid support is a bead comprised in a column.

[0140] In some embodiments, the binding moiety is a biotin molecule connected to a linker, e.g., a PEG2 linker. In some embodiments, the biotin is part of an amine-PEG2 -biotin or an amine- PEG3-biotin molecule. In some embodiments, the biotin comprises the structure of Formula I:Formula ICertain Exemplary Peptides

[0141] Certain exemplary peptides of the present disclosure are provided in Table 2 below.Table 2. Exemplary peptides (AEGEFNKSTMLAHSD is SEQ ID NO: 14).Acetyl-{Dap}AEGEFNKSTMLAHSD{PEG2}{Lys(Biotin)}-Amide

[0142] In some embodiments, a disclosed peptide is covalently linked to a glycine-serine linker (such as the glycine-serine linker of SEQ ID NO: 29) or a PEG linker (such as a PEG2 linker). In particular embodiments, the glycine-serine linker of SEQ ID NO: 29 or a PEG2 linker is covalently linked to the C-terminal amino acid of the peptide. In particular embodiments, a binding moiety (such as a biotin or a modified biotin) is covalently bound to the peptide through the linker. For example, in some embodiments, the linker links the C-terminal amino acid of the peptide to the binding moiety. In certain embodiments, the linker is covalently bound to the peptide and a lysine links the linker to the binding moiety. In particular embodiments, the binding moiety is a biotin or a modified biotin, the linker is the glycine-serine linker of SEQ ID NO: 29 or a PEG2 linker, the linker is covalently bound to the C-terminal amino acid of the peptide, and a lysine links the linker to the biotin or the modified biotin.

[0143] In certain embodiments of a peptide comprising at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1- 28, (a) the N-terminal amino acid of the peptide is acetylated, (b) the C-terminal amino acid of the peptide is amidated, (c) the N-terminal amino acid comprises Dap, or (d) any combination of (a)-(c). In some embodiments of a peptide comprising at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1- 28, the N-terminal amino acid of the peptide is acetylated. In some embodiments of a peptide comprising at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-28, the C-terminal amino acid of the peptide is amidated. In some embodiments of a peptide comprising at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-28, the N-terminal amino acid comprises Dap. In particular embodiments, the peptide of any of the above embodiments further comprises a linker, such as a glycine-serine linker (such as the linker of SEQ ID NO: 29) or a PEG linker (such as a PEG2 linker). In other particular embodiments, the peptide of any of the above embodiments further comprises a binding moiety, such as a biotin or a modified biotin. In particular embodiments, the linker is covalently bound to the C-terminal amino acid of the peptide, and a lysine links the linker to the biotin or the modified biotin. In specific, non-limiting embodiments, the peptide does not comprise a linker. In other specific, non-limiting embodiments, the peptide does not comprise a binding moiety.

[0144] Exemplary structures depicting a biotin bound to a PEG linker (comprising an optional N-terminal amine) through a lysine are shown below. In embodiments comprising suchstructures, the N-terminus of the PEG linker is bound to an amino acid of the peptide, such as the C-terminal amino acid of the peptide.Formula II

[0145] In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10. In particular embodiments, n is 2 (PEG2) as depicted below.Formula I

[0146] In particular embodiments, a disclosed peptide comprises at least 8 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-14. In another particular embodiment, the peptide comprises at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-14. In another particular embodiment, the peptide comprises at least 12 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 2-14. In another particular embodiment, the peptide comprises at least 13 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 4-14. In another particular embodiment, the peptide comprises at least 14 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 9-14. In another particular embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 14. In other particular embodiments, the isolated peptide consists of an amino acid sequence selected from any one of SEQ ID NOs: 1-14. In another particular embodiment, the isolated peptide comprises the amino acid sequence of SEQ ID NO: 14, wherein any 1, 2, 3, or 4 of the first four amino acids of SEQ ID NO: 14 are deleted.

[0147] In particular embodiments, a PEG linker or a glycine-serine linker is attached to the N- or C-terminal amino acid of the peptide, a binding moiety is attached to the linker, and the C- terminal amino acid of the peptide is amidated. In particular embodiments, (a) the N-terminal amino acid of the peptide is acetylated, (b) the N-terminal amino acid of the peptide comprisesDap, or (c) both (a) and (b). In other particular embodiments, the binding moiety is biotin or a modified biotin; the peptide is immobilized on a solid support comprising streptavidin, avidin, and / or neutravidin; and the biotin or the modified biotin attached to the linker of the peptide binds to the streptavidin, avidin, and / or neutravidin of the solid support, thereby immobilizing the peptide to the solid support.III. Methods of Obtaining an AAV from an AAV Starting PreparationA. Overview

[0148] Methods of obtaining an AAV (such as an AAV9) for use in a variety of gene transfer and / or other applications are provided. The disclosed methods separate the AAV from all or a portion of other components of an AAV starting preparation, such as production culture components, such as helper virus, helper virus proteins, plasmids, cellular materials (such as proteins and nucleic acids), media components, serum proteins, AAV rep proteins, unassembled AAV VP1, VP2, and VP3 proteins, and similar. In some embodiments of the disclosed methods, the AAV is an AAV9, such as an rAAV9.B. Exemplary Methods

[0149] In some embodiments, a method for obtaining an AAV from an AAV starting preparation as described herein comprises contacting the AAV with a peptide disclosed herein. In some embodiments, the contacting occurs at a first pH that is within a pH range within which the AAV binds the peptide. In some embodiments, a wash step is performed at a pH within the pH range within which the AAV binds the peptide to remove all or a portion of the components of the AAV starting preparation that are not bound to the peptide. In some embodiments, all or a portion of the AAV is dissociated from the peptide by changing the pH to a second pH that is within a pH range within which the AAV does not bind the peptide as tightly as when in the first pH. In the disclosed embodiments, a wash step can be optional. In some embodiments, a wash step is performed at the same time (simultaneously) as a dissociating step. In other embodiments, a wash step is performed prior to a dissociating step. In particular embodiments, the AAV is an AAV9. In some embodiments, the AAV (such as the AAV9) comprises a nucleic acid of interest, such as described elsewhere herein.

[0150] In certain embodiments, the first pH is within a pH range of 6.5 to 8.5. In certain embodiments, the second pH is within a pH range of 0.5 to 7.0 or 8.0 to 14.0. In particular embodiments, the first pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In a specific, non-limiting embodiment, the first pH is 7.4.

[0151] In some embodiments, the pH range within which the AAV binds the peptide is defined by a lower bound pH and an upper bound pH, inclusive. In some embodiments, the AAV is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more likely to dissociate from the peptide at a pH that is lower than the lower bound pH than it is to dissociate from the peptide at a pH that is between the lower bound pH and the upper bound pH, inclusive. In some embodiments, the AAV is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more likely to dissociate from the peptide at a pH that is higher than the upper bound pH than it is to dissociate from the peptide at a pH that is between the lower bound pH and the upper bound pH, inclusive. In some embodiments, the AAV is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more likely to dissociate from the peptide at a pH that is lower than the lower bound pH or higher than the upper bound pH than it is to dissociate from the peptide at a pH that is between the lower bound pH and the upper bound pH, inclusive.

[0152] In some embodiments, the upper bound pH is between 0.05 and 2 pH units, between 0.05 and 1.75 pH units, between 0.05 and 1.5 pH units, between 0.05 and 1.25 pH units, between 0.05 and 1 pH units, between 0.05 and 0.75 pH units, between 0.05 and 0.5 pH units, between 0.05 and 0.4 pH units, between 0.05 and 0.3 pH units, between 0.05 and 0.2 pH units, or between 0.05 and 0.1 pH units higher than the lower bound pH. In certain embodiments, the lower bound pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In certain embodiments, the upper bound pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

[0153] In embodiments comprising a wash step, the method comprises a wash buffer. In certain embodiments, the wash step can be carried out within a pH range of 6.5 to 8.5. In some embodiments, the wash buffer comprises 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM buffering agent. Suitable wash buffers may include ions contributed from a variety of buffering agents, such as, e.g., N-methylpiperazine; piperazine; Bis-Tris; BisTris propane; CHES (N-cyclohexyl-2-aminoethanesulfonic acid); AMPSO (N-( 1,1 -dimethylshydroxy ethyl)-3-amino-2-hydroxypropanesulfonic acid); MES, Hepes, BTP or a phosphate buffer N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acid, phosphate buffered solution, and the like. In some embodiments, the wash buffer is phosphate buffered solution (PBS). Such buffers may be used, for example, at a neutral pH e.g., pH 6.5 to pH 8, pH 7 to pH 7.5, or pH 7.5).

[0154] In some embodiments, the dissociating step comprises a dissociation buffer. In some embodiments, the dissociation buffer comprises 5-100 mM, 10-100 mM, 10-50 mM, 10-40 mM, 10-30 mM, 20-30 mM, or 25 mM buffering agent. Suitable dissociation buffers may include ions contributed from a variety of buffering agents, such as, e.g., N-methylpiperazine; piperazine; Bis-Tris; Bis-Tris propane; CHES (N-cyclohexyl-2-aminoethanesulfonic acid); AMPSO (N-(l,l-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid); MES, Hepes, BTP or a phosphate buffer N-methyldiethanolamine; 1,3-diaminopropane; ethanolamine; acetic acid, and the like. In some embodiments, the dissociation buffer comprises any one or more of the following: Citric acid (e.g., at pH 3-4), Sodium acetate (e.g., at pH 4-5) Sodium citrate (e.g., at pH 5-6), Sodium cacodylate (e.g., at pH 6-7), HEPES sodium (e.g., pH 7-8), TRIS hydrochloride (e.g., at pH 8-9), CAPSO (e.g., at pH 9-10) and / or CAPS (e.g., at pH 10-11). In some embodiments, the dissociation buffer comprises any one or more of the following: Citric acid (e.g., at pH 3.4), Sodium acetate (e.g., pH 4.4), Sodium citrate (e.g., pH 5.4), Sodium cacodylate (e.g., pH 6.4), HEPES sodium (e.g., pH 7.4), TRIS hydrochloride (e.g., pH 8.4), CAPSO (e.g., pH 9.4) and / or CAPS (e.g., pH 10.4).

[0155] In embodiments of the disclosed methods, an AAV of an AAV starting preparation (such as an AAV9 of an AAV9 starting preparation) is separated from at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the other components of the AAV starting preparation. As described elsewhere herein, the other components of the AAV starting preparation can include impurities present in the packaging cell media and / or packaging cell lysate, such as one or more of empty capsids, packaging cell materials (such as packaging cell DNA, packaging cell proteins, etc.), media components, whole cells, and in some instances, helper virus or helper virus plasmid DNA. In some embodiments, following AAV harvest (such as from an AAV packaging cell and / or from an AAV packaging cell culture medium), an AAV starting preparation is processed (such as described herein) prior to contacting the AAV of the AAV starting preparation with the disclosed peptides. Thus, the AAV starting preparation can comprise impurities initially present in the packaging cell media and / or packaging cell lysate, and that remain following a processing step, such as a processing step comprising one or more clarification steps (such as one or more filtration and / or centrifugation steps), and / or treatment of the AAV starting preparation with cell lysis agents, enzymes such as endonucleases and / or exonucleases, and other such reagents described elsewhere herein and as known in the art.

[0156] In some embodiments, a yield of the AAV (such as the AAV9) is at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the AAV initially present in the AAV starting preparation.

[0157] In some embodiments, the physical titer of the AAV (such as the AAV9) in a solution obtained using the disclosed methods is at least IxlO10to IxlO13viral genomes (vg) / mL. AAVphysical titer can be calculated using any suitable method known in the art, such as quantitative PCR or digital droplet PCR. In such embodiments, copy number of an ITR region may be used to quantify AAVs in a solution obtained using the disclosed methods.

[0158] In one embodiment, the concentration of AAV viral genomes (vg) can be measured by qPCR. Samples can be diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA, and can optionally be further treated with proteinase K (or another suitable proteinase). After inactivation of the nuclease, samples can be further diluted and amplified using primers and a probe (e.g., a TagMan™ fluorogenic probe) specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample using a suitable detection system. Plasmid DNA containing identical sequences to that contained in the AAV is employed to generate a standard curve in the qPCR reaction. The Ct values obtained from the samples are used to determine vector genome titer by normalizing them to the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.

[0159] Additionally, or alternatively, droplet digital PCR (ddPCR) may be used. For example, methods for determining single-stranded and self-complementary AAV genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, 2014, 25(2): 15-25.

[0160] In some embodiments of the disclosed methods, the peptide is immobilized on a solid support, such as a solid support disclosed herein. In some embodiments, the solid support comprises sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, a flow cell, a hydrogel, a silica gel, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, an alumina gel, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead. Because the methods provided herein allow an AAV to be bound to a peptide, such as a peptide immobilized on a solid support, any unbound impurities in an AAV starting preparation can be removed, such as by washing the solid support after AAV-peptide binding has occurred. For example, an AAV starting preparation comprising cell culture materials, a crude cell lysate, and / or or whole cells that is applied to a solid support in a method set forth herein need not have been subjected to one or more processing steps that can be used to separate the AAV from a portion of or all of the cell culture materials, crude cell lysate, and / or or whole cells. Alternatively, an AAV starting preparation comprising cell culture materials, a crude cell lysate, and / or or whole cells may be subjected to one or more processing steps (suchas described herein and as known in the art) prior to applying the AAV starting preparation to a solid support in a method set forth herein.C. AAV Starting Preparations

[0161] In some embodiments, obtaining an AAV starting preparation comprises culturing an AAV packaging cell in media that allows production of the AAV. Accordingly, in some embodiments an AAV starting preparation comprises an AAV (such as an AAV9) and other components, such as media collected from a packaging cell culture and / or components of a packaging cell lysate. An AAV starting preparation can comprise impurities present in the packaging cell media and / or packaging cell lysate, such as various cellular components, such as packaging cell DNA, packaging cell proteins, media components, and in some instances, helper virus or helper virus plasmid DNA.

[0162] A variety of suitable cells and cell lines have been described for use in production of AAV. A packaging cell useful in the present disclosure can be a eukaryotic cell, a fungal cell, an insect cell, a prokaryotic cell (e.g., bacterial or archaeal cell), or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, and include the progeny of the original cell if such cell has been transformed by the nucleic acid. In some embodiments, the cell is a eukaryotic cell, such as a 293T cell (such as a HEK293T cell). Exemplary mammalian cells include without limitation, A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, HEK 293 (which express functional adenoviral El), Saos, C2C12, L cells, HT1080, HepG2 cells, and primary fibroblast, hepatocyte, and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster. In certain embodiments, the cells are suspension-adapted cells. The selection of the mammalian species providing the cells is not a limitation of this invention; nor is the type of mammalian cell, z.e., fibroblast, hepatocyte, tumor cell, etc.

[0163] In other embodiments, the cell is a fungal cell, such as a yeast cell, such as a yeast cell of species Saccharomyces (such as Saccharomyces cerevisiae). In other embodiments, the cell is an insect cell (e.g., for use in baculovirus-based AAV production systems), such as an Sf9 cell. A “recombinant packaging cell” (also referred to as a “genetically modified packaging cell”) is a packaging cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector. For example, a bacterial packaging cell is a genetically modified bacterial packaging cell by virtue of introduction of an exogenous nucleic acid (e.g., a plasmid or recombinant expression vector) into a suitable bacterial packaging cell, and a eukaryotic packaging cell is a genetically modified eukaryotic packaging cell (e.g., a mammalian cell), by virtue of introduction of an exogenous nucleic acid into a suitable eukaryotic packaging cell.

[0164] In certain embodiments, the disclosed methods comprise culturing an AAV packaging cell in media that allows production of the AAV. Methods of culturing an AAV packaging cell to produce an AAV are known in the art, and exemplary methods are discussed herein. Suitable media known in the art may be used for AAV production including, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), custom formulations such as those described in U.S. Pat. No. 6,566,118, and Sf-900 II SFM media as described in U.S. Pat. No. 6,723,551.

[0165] AAV production culture media may be supplemented with serum or serum-derived recombinant proteins, e.g., at a level of 0.5%-20% (v / v or w / v). Alternatively, as is known in the art, AAV vectors may be produced in serum-free conditions which may also be referred to as media with no animal-derived products. Commercial or custom media designed to support production of AAVs may also be supplemented with one or more cell culture components know in the art, including without limitation, glucose, vitamins, amino acids, and / or growth factors, in order to increase the titer or yield of AAV in production cultures.

[0166] AAV production cultures can be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized. As is known in the art, AAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. AAV vector production cultures may also include suspension-adapted host cells such as HeLa, 293, and SF-9 cells that can be cultured in a variety of attachment-independent vessels including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system.

[0167] In some embodiments, the AAV is harvested from the AAV packaging cell and / or from the media, thereby providing an AAV starting preparation. The harvesting may comprise cell disruption (such as cell lysis) or may substantially not comprise cell disruption (such as in embodiments wherein the AAV is harvested from cell culture media (e.g., supernatant)). Thus, AAVs of the invention may be harvested from AAV production cultures by lysis of the packaging cells of the production culture or by harvest of the media (“spent” media) from the production culture, provided the cells are cultured under conditions known in the art to cause release of AAV particles into the media from intact cells (such as described more fully in U.S. Pat. No. 6,566,118). Suitable methods of lysing cells are also known in the art and include for example multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and / or proteases.

[0168] On its own, AAV does not possess the ability to efficiently replicate its genetic material, and thus typically requires the presence of a helper virus. Three helper viruses are commonly used for AAV production: adenovirus (Ad), herpes simplex virus (HSV), and baculovirus (Bac). Ad and HSV helper AAV production methods use mammalian cell lines while the Bac system uses insect cells. Unlike the HSV and Bac systems, the Ad-based system genes required for helper function have been identified and can be expressed via a plasmid, thus eliminating the need for actual Ad to be produced. An exemplary “helper free” system can use three plasmids: one containing the Ad E2A, E4, and VA RNA helper genes, a second expressing the AAV rep and cap genes, and third, a nucleic acid of interest (such as described elsewhere herein) flanked by AAV inverted terminal repeats (ITRs). Accordingly, an AAV production system useful in the disclosed methods may be helper free.

[0169] In some embodiments of the disclosed peptides and methods, the peptide may selectively bind an AAV (such as an AAV9) comprising an AAV capsid protein and a nucleic acid molecule that comprises an AAV 5’ inverted terminal repeat (ITR), a nucleic acid molecule of interest to be packaged into the AAV capsid, and a 3’ ITR. In such embodiments, the packaging cell can thus comprise (i) the at least one nucleic acid of interest to be packaged into the AAV capsid, (ii) a nucleic acid molecule encoding the AAV capsid protein under control of one or more sequences that direct its expression in the packaging cell, (iii) a nucleic acid molecule encoding an AAV Rep protein that expresses the AAV Rep protein in the cell to permit packaging of the nucleic acid of interest into the AAV capsid, and / or (iv) one or more helper functions required for packaging the nucleic acid molecule of interest into the AAV capsid.

[0170] In some embodiments, the preparation of a packaging cell suitable for use in the disclosed methods involves techniques such as assembly of selected DNA sequences. This assembly may be accomplished utilizing conventional techniques. Such techniques include cDNA and genomic cloning, which are well known and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., including polymerase chain reaction, synthetic methods, and any other suitable methods which provide the desired nucleotide sequence.

[0171] In some embodiments, an AAV starting preparation useful in the disclosed methods can be an AAV production culture harvest without further treatment, or can be such a harvest treated using one or more of a clarifying treatment (such as filtration and / or centrifugation), one or more nucleases and / or proteases (to digest contaminating nucleic acids and / or proteins), an additional chromatography step (such as affinity chromatography), and / or a concentrating step. At harvest, an AAV starting preparation of the present disclosure may contain one or more of the following: packaging cell materials (such as packaging cell proteins and / or DNA); plasmid DNA; helpervirus; helper virus proteins; helper virus DNA; and media components including, for example, serum proteins, amino acids, transferrins, and other low molecular weight proteins. In some embodiments, prior to separating the AAV from the other components of the AAV starting preparation, the AAV starting preparation is clarified to remove packaging cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters including, for example, a grade DOHC Millipore Millistak+HC Pod Filter, a grade A1HC Millipore Millistak+HC Pod Filter, and / or a 0.2 pm Filter Opticap XL 10 Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as, centrifugation or filtration through any suitable filter (such as any suitable cellulose acetate filter of 0.2 pm or greater pore size) known in the art. Still other suitable depth filters, e.g., in the range of 0.045 pm to 0.2 pm, or other filtration techniques may be used.

[0172] In some embodiments, the AAV starting preparation can be treated with a nuclease, or a combination of nucleases, to digest any contaminating high molecular weight nucleic acid present in the production culture. Suitable nucleases include but are not limited to a DNAse, e.g., Benzonase® digestion performed under standard conditions known in the art. For example, a final concentration of 1 unit / mL to 2.5 units / mL of Benzonase® is used at a temperature ranging from ambient temperature to 37°C for a period of 30 minutes to several hours, such as 2 hours. In another example, a turbonuclease is used. However, one of skill in the art may utilize another suitable nuclease, or a mixture of nucleases. Exonucleases may also be used to remove contaminating nucleic acids. Such nucleases may be selected to degrade single stranded DNA and / or double-stranded DNA, and RNA. Such steps may contain a single nuclease, or mixtures of nucleases directed to different targets, and may be endonucleases or exonucleases.

[0173] In some embodiments, AAVs of the AAV starting preparation may be isolated or purified prior to subjecting the AAVs to a method of the present disclosure, using one or more of the following steps: tangential flow filtration (TFF) for concentrating the AAV particles, heat inactivation of helper virus, AAV capture by hydrophobic interaction chromatography, affinity capture chromatography to remove production system contaminants, buffer exchange by size exclusion chromatography (SEC), and / or nanofiltration. These steps may be used alone, in various combinations, or in different orders. In some embodiments, the method comprises all the steps in the order.

[0174] In some embodiments, a nuclease (e.g., Benzonase®)-treated mixture is concentrated via TFF. Large scale concentration of viruses using TFF ultrafiltration has been described by R. Paul et al., Human Gene Therapy, 4:609-615 (1993). TFF concentration of the AAV starting preparation enables a technically manageable volume of the preparation to be subjected to themethods of the present disclosure and allows for more reasonable sizing of a solid support (such as beads, such as in a column). In some embodiments, the AAV starting preparation is concentrated between at least two-fold or at least ten-fold. In some embodiments, the AAV starting preparation is concentrated between at least ten-fold and at least twenty-fold, such as at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold. In some embodiments, the AAV starting preparation is concentrated between at least twenty-fold and at least fifty-fold. One of ordinary skill in the art will also recognize that TFF can also be used at any step in the disclosed methods where it is desirable to exchange buffers before performing the next step in the method.

[0175] In one embodiment, AAVs of the AAV starting preparation have been separated from contaminants (e.g., packaging cell, viral, and other nucleic acid or proteinaceous materials which are present in the production culture or are by-products thereof) present from the production system. In some embodiments, the AAV starting preparation comprising the AAV contains less than 10% contamination from non- AAV viral and cellular proteinaceous and nucleic acid materials, or less than 5% contaminants, or less than 1% contaminating viral and cellular proteinaceous and nucleic acid materials. Thus, in some embodiments, the AAV starting preparation contacted with the disclosed peptides is 95% to 99% free of contaminants.

[0176] In some embodiments, affinity capture chromatography may be used to separate AAVs of an AAV starting preparation from production system contaminants. This affinity capture can be performed using an antibody-capture affinity resin. In one embodiment, the solid support is a cross-linked poly(styrene-divinylbenzene) having an average particle size of 50 pm and having an AAV-specific antibody. An example of one such commercially available affinity resin is POROS™ high performance affinity resin commercially available from Thermo Fisher Scientific. The resin contains ligands created by a proprietary technology based on camelid- derived single-domain antibody fragments coupled to the resin via carbonyldiimidazole (CDI). The ligand is a 13-kDa single-domain fragment that comprises the 3 CDRs that form the antigen binding domain and is efficiently produced by the yeast Saccharomyces cerevisiae in a production process free of animal components. Other suitable affinity resins may be selected or designed which contain an AAV-specific antibody, AAV9 specific antibody, or other immunoglobulin construct which is an AAV-specific ligand. Such solid supports may be any suitable polymeric matrix material, e.g., agarose, Sepharose®, Sephadex®, amongst others.

[0062] In some embodiments of the present disclosure, an AAV starting preparation comprises at least 1%, 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 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, or at least 50% AAV (such as AAV9). In particular embodiments, the AAV starting preparation comprises 1-50% AAV, such as 1-40%, 1-30%, 1- 20%, 1-19%, 1-18%, 1-17%, 1-16%, 1-15%, 1-14%, 1-13%, 1-12%, 2-40%, 3-40%, 4-40%, 5- 40%, 5-30%, 5-20%, 5-15%, 10-40%, 10-30%, 10-20%, or 10-15% AAV (such as AAV9).

[0177] Alternatively or additionally to treating an AAV starting preparation prior to subjecting the AAV starting preparation to a disclosed method to obtain an AAV, an AAV obtained using a method of the present disclosure can be subjected to one or more additional processes, such as treatment using one or more of a clarifying treatment (such as filtration and / or centrifugation, such as ultracentrifugation), one or more nucleases and / or proteases (e.g., to digest contaminating nucleic acids and / or proteins), an additional chromatography step (such as affinity chromatography and / or ion exchange chromatography (such as anion exchange chromatography)), a concentrating step (such as filtration (such as ultrafiltration) and / or centrifugation), and / or another treatment, such as one or more additional isolation or purification processes known in the art. Such one or more additional processes may be beneficial for certain downstream applications of an AAV obtained using the disclosed methods.

[0178] In some embodiments, an AAV obtained using a disclosed method comprises an AAV full capsid AAV full capsid and one or more AAV intermediaries, such as an AAV empty capsid and / or an AAV partially empty capsid. In particular embodiments, the disclosed methods further comprise a step of separating the AAV full capsid from the one or more AAV intermediaries, such as an AAV empty capsid and / or an AAV partially empty capsid. Such a separating step can be performed prior to, simultaneously, or after subjecting the AAV to one or more additional processes as described above. An “AAV full capsid” refers to a nuclease-resistant particle comprising an AAV capsid (such as an AAV9 capsid), the capsid having packaged therein a nucleic acid molecule comprising an expression cassette that comprises a nucleic acid molecule of interest (e.g., a nucleic acid molecule encoding a desired gene product, such as a Cas9 protein and one or more gRNA molecules). Methods of separating an AAV full capsid from one or more AAV intermediaries are known in the art. An AAV full capsid is “pharmacologically active” when it can deliver the nucleic molecule of interest to a host cell that is capable of expressing the desired gene product. “AAV empty capsid” refers to an assembled AAV capsid (such as an AAV9 capsid) that lacks genomic sequences packaged therein. Such a capsid contains no detectable genomic sequences of an expression cassette. An “AAV partially empty capsid” as referred to herein contains only partial genomic sequences that are insufficient to express the complete gene product. AAV empty capsids and AAV partially empty capsids are non-functional to transfer the complete gene of interest to a host cell. AAV empty capsids and AAV partially empty capsids are also referred to herein as “AAV intermediaries.”IV. Additional Features of Certain Disclosed Isolated Peptides and MethodsA. Adeno-associated Viruses (AAVs)

[0179] Peptides of the present disclosure can selectively bind an AAV. In some embodiments, the AAV is a recombinant or engineered AAV. In some embodiments, the AAV is a pseudotyped AAV. Use of a recombinant or engineered AAV enables insertion, deletion, or substitution of target DNA sequences into the genomes of mammalian cells. AAV comprises a protein capsid surrounding and protecting a single-stranded DNA genome of approximately 4.8 kilobases (kb). Naso et al., BioDrugs. 2017; 31(4): 317-334. AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Its single-stranded genome contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly). These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging, while Cap expression gives rise to the viral capsid proteins (VP; VP1 / VP2 / VP3), which form the outer capsid shell that protects the viral genome, and is involved in cell binding and internalization. The viral capsid is comprised of approximately 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of 1 : 1 : 10 (VP1 :VP2:VP3). The aap gene encodes the assemblyactivating protein (AAP) in an alternate reading frame overlapping the cap gene. This nuclear protein is thought to provide a scaffolding function for capsid assembly, but may be nonessential in certain AAV serotypes.

[0180] Recombinant AAV (rAAV), which lacks viral DNA, is a protein-based nanoparticle engineered to traverse the cell membrane, where it can ultimately traffic and deliver a DNA cargo (comprised within the viral capsid) into the nucleus of a cell. Naso et al., BioDrugs. 2017; 31(4): 317-334. In some embodiments, in the absence of Rep proteins, ITR-flanked transgenes encoded within rAAV can form circular concatemers that persist as episomes in the nucleus of a transduced cell. In some embodiments, because recombinant episomal DNA does not integrate into host genomes, it will eventually be diluted over time as the cell undergoes repeated rounds of replication in mitotic cells. This will eventually result in the loss of the transgene and transgene expression, with the rate of transgene loss dependent on the turnover rate of the transduced cell. These characteristics make rAAV appealing for certain gene therapy applications.

[0181] In some embodiments, the AAV that is selectively bound by a disclosed peptide is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhlO, AAVrh74, AAV9, AAV9P, AAV10, AAV11, AAV12, or Myo- AAV. In particular embodiments, the AAV is AAV9. AAV9 has been shown to have enhanced capsid-associated tropism in tissues, such as liver, heart, and skeletal muscle, such as following intravascular delivery (Zincarelli et al., Mol. Ther. 2008; 16: 1073-1080). Like other AAVs, AAV9 can transduce non-dividing cells (DiMattia et al., J. Virol. 2012; 86(12): 6947-6958). In addition, AAV9 can traverse the blood-brain barrier and transduce cells of the central nervous system (CNS), providing opportunities for CNS gene therapy (Foust et al., Nat. Biotechnol. 2009; 27:59-65; Duque et al., Mol. Ther. 2009; 17: 1187- 1196). In some embodiments, the AAV9 is less than 5 kb from ITR to ITR in size, inclusive of both ITRs. In particular embodiments, the AAV9 is less than 4.9 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 is less than 4.85 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 is less than 4.8 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 is less than 4.75 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV9 is less than 4.7 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the AAV9 is 3.9- 5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the vector is 4.4-4.85 kb from ITR to ITR in size, inclusive of both ITRs.

[0182] Exemplary methods of generating a capsid, coding sequences for a capsid, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al. , Proc. Natl.Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003), U.S. Pat. Nos. 6,759,237, 7,105,345, and 7,186,552, and US Patent Publication 2013 / 0045186A1.

[0183] In some embodiments, the AAV comprises a nucleic acid molecule of interest (i.e., enclosed within the fully assembled AAV capsid), such as a nucleic acid molecule that encodes a therapeutic molecule, such as a therapeutic protein or RNA molecule. A nucleic acid molecule of interest can be operably linked to one or more regulatory elements, such as a promoter, such as a tissue-specific specific promoter. The term “operably linked” means that the nucleic acid molecule of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. An exemplary promoter useful in the present disclosure can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / “ON” state), an inducible promoter (i.e., a promoter whose state, active / “ON” or inactive / “OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein), a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc., e.g., a tissuespecific promoter, a cell type specific promoter, etc.), or a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process).

[0184] Suitable promoters for expression of AAV cargo genes can be derived from viruses (viral promoters), or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a Rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi el al.. Nature Biotechnology . 20: 497-500; 2002), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. Sep 1(31): 17; 2003), a human Hl promoter (Hl), and the like.

[0185] AAVs obtained using the disclosed peptides and methods can include any AAV comprising a tissue-specific promoter in facilitating administration of gene therapy, which can include any known gene editing system in the art. A promoter as described herein can also be “cell specific,” meaning that the particular promoter selected for the AAV can direct expression of the selected transgene / nucleotide sequence of interest in a particular cell or cell type. In some embodiments, for example, the promoter is a muscle-specific promoter, including a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, or an SPc5-12 promoter. In some embodiments, the muscle-specific promoter is a CK8 promoter. In some embodiments, the muscle-specific promoter is a CK8e promoter. Muscle-specific promoters are described in detail, e.g., in US2004 / 0175727 Al; Wang et al., Expert Opin Drug Deliv. (2014) 11, 345-364; Wang et al., Gene Therapy (2008) 15, 1489-1499. In some embodiments, the tissue-specific promoter is a neuron-specific promoter, such as an enolase promoter. See, e.g., Naso et al., BioDrugs 2017; 31 :317-334; Dashkoff et al., Mol Ther Methods Clin Dev. 2016;3: 16081, and references cited therein for detailed discussion of tissue-specific promoters including neuron-specific promoters. Any known promoters may be used in conjunction with the AAVs to administer the gene therapy to the intended target tissues or cells.

[0186] Nucleic acids of interest useful herein can also include other regulatory elements, i.e., transcriptional and translational control sequences, such as enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., site- directed modifying polypeptide, or Cas9 polypeptide) and / or regulate translation of an encoded polypeptide. Exemplary regulatory sequences are well known in the art and are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

[0187] In some embodiments, the AAV (such as the AAV9) includes CRISPR-Cas components, any of which are known in the art. In some embodiments, the nucleic acid of interest encodes a Cas9 protein. Such embodiments include for example, AAVs comprising a nucleic acid encoding Staphylococcus aureus (SaCas9) and / or Staphylococcus lugdunensis (SluCas9) and further comprising a nucleic acid encoding one or more guide RNAs. In some embodiments, in addition to guide RNA and Cas9 sequences, the AAV further comprises nucleic acids that do not encode guide RNAs. Nucleic acids that do not encode guide RNAs and Cas9 include, but are not limited to, regulatory elements described elsewhere herein (e.g., promoters, enhancers, and the like). In some embodiments, the AAV comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA. In some embodiments, the AAV comprises multiple nucleic acids encoding more than one guide RNA. In some embodiments, the AAV comprises two nucleic acids encoding two guide RNA sequences.B. Solid Supports

[0188] In some embodiments of the present disclosure, any of the peptides disclosed herein is immobilized to the surface of a solid support. In certain embodiments, the peptide is physically linked to the surface of the solid support. Thus, some embodiments of the present disclosure include solid supports, such as beads, having peptides immobilized thereon. In certain embodiments, a disclosed peptide immobilized on the solid support comprises a binding moiety (such as a biotin or a modified biotin), and the solid support comprises an affinity moiety (such as an avidin or a streptavidin) that has a binding affinity for the binding moiety. In such embodiments, interaction of the binding moiety of the peptide and the affinity moiety of the solid support can be used to immobilize the peptide to the solid support.

[0189] In particular embodiments, a binding moiety of a disclosed peptide is a biotin or a modified biotin (i.e., the peptide is biotinylated) and an affinity moiety of the solid support is a biotin binding agent, such as avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor, and / or a biotin-binding enzyme. In other particular embodiments, a binding moiety of a disclosed peptide is a biotin binding agent, such as avidin, streptavidin, neutravidin, an antibiotin antibody, a biotin receptor, and / or a biotin-binding enzyme and an affinity moiety of the solid support is a biotin or a modified biotin (i.e., the solid support is biotinylated). In some embodiments, the affinity moiety, such as a biotin binding agent, is immobilized to the surface of the solid support, such as physically linked to the surface of the solid support, and a peptide comprising a biotin or a modified biotin binds to the biotin binding agent immobilized to thesurface of the solid support, thereby physically linking the biotinylated peptide to the solid support. In some embodiments, a peptide is immobilized to the solid support via binding of a binding moiety attached to the C-terminal amino acid of the peptide to an affinity moiety on the surface of the solid support.

[0190] Biotin binding agents include, but are not limited to, avidin, streptavidin, neutravidin, anti-biotin antibodies, biotin receptors, and biotin-binding enzymes. Thus, in some embodiments, the biotin binding agents (such as biotin binding agents of the solid support) comprise one or more of avidin, streptavidin, neutravidin, an anti-biotin antibody, a biotin receptor, and / or a biotin-binding enzyme. In some embodiments, the biotin-binding enzyme is biotinidase or biotin holocarboxylase synthetase.

[0191] In some embodiments, as described herein, contacting a disclosed peptide with an AAV (such as an AAV9 comprised in an AAV9 starting preparation) allows selective binding of the AAV and the peptide at a first pH that is within a pH range within which the AAV binds the peptide. Thus, when a peptide is immobilized to a solid support as disclosed herein (either directly or through association of a binding moiety of the peptide, such as through a biotin-biotin binding agent association), selective binding of the peptide to the AAV (such as the AAV9) immobilizes the AAV to the solid support. In some embodiments, during a buffer exchange step (e.g., flow-through buffer exchange, such as in a flow cell channel), the AAV (such as the AAV9) remains bound to the peptide while a portion of (such as at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) or all components of the AAV starting preparation not bound to the peptide are removed (such as from the flow cell channel). As described herein, dissociating the AAV from the peptide can then be achieved by changing the pH to a second pH that is within a pH range within which the AAV does not bind the peptide or does not bind the peptide as tightly as when in the first pH range.

[0192] The density of biotin binding agents immobilized on the surface of a solid support can vary. For example, in some embodiments, the biotin binding agents are present on the solid support at a density of at least 102, at least 103, at least 104, at least 105, or at least 106biotin binding agents per mm2.

[0193] Certain embodiments of the present disclosure may include solid supports comprised of an inert substrate or matrix (e.g., glass slides, polymer beads, or similar) that has been functionalized, for example by application of a layer or coating of an intermediate material comprising reactive groups which permit covalent attachment to a peptide disclosed herein and / or to a molecule (such as a biotin binding moiety) having an affinity for a binding moiety (such as a biotin or a modified biotin) of the peptide. Examples of such supports include, but arenot limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, such as polyacrylamide hydrogels as described in WO 2005 / 065814 and US 2008 / 0280773, the contents of which are incorporated herein in their entirety by reference. In such embodiments, a peptide may be directly covalently attached to the intermediate material (e.g., the hydrogel), or may be associated with the intermediate material through association of a binding moiety and a molecule having an affinity for the binding moiety, but the intermediate material may itself be non- covalently attached to the substrate or matrix (e.g., the glass substrate).

[0194] In some embodiments, the solid support comprises a patterned surface suitable for immobilization of the disclosed peptides in an ordered pattern. A “patterned surface” refers to an arrangement of different regions in or on an exposed layer of a solid support. For example, one or more of the regions can be features where one or more peptides are present. The features can be separated by interstitial regions where peptides are not present. In some embodiments, the pattern can be an x-y format of features that are in rows and columns. In some embodiments, the pattern can be a repeating arrangement of features and / or interstitial regions. In some embodiments, the pattern can be a random arrangement of features and / or interstitial regions. In some embodiments, the peptides are randomly distributed upon the solid support. In some embodiments, peptides are distributed on a patterned surface. Exemplary patterned surfaces that can be used with the peptides or the methods set forth herein are described in US Pat. App. Publ. Nos. 2012 / 0316086 Al and 2013 / 0116153 Al.

[0195] In some embodiments, the solid support comprises an array of wells or depressions in a surface. This may be fabricated as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques. The technique used will depend on the composition and shape of the array substrate.

[0196] The composition and geometry of the solid support can vary with its use. In some embodiments, the solid support is a planar structure such as a slide, chip, microchip, and / or array. As such, the surface of a substrate can be in the form of a planar layer. In some embodiments, the solid support comprises one or more surfaces of a flow cell. The term “flow cell” as used herein refers to a chamber comprising a solid surface across which one or more fluid reagents can be flowed. Examples of flow cells and related fluidic systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497; US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281, and US 2008 / 0108082.

[0197] In some embodiments, the solid support or its surface is non-planar, such as the inner or outer surface of a tube or vessel. In some embodiments, the solid support comprises microspheres or beads. By “spheres” or “beads” or “particles” or grammatical equivalents herein is meant small discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphite, titanium dioxide, latex or cross-linked dextrans such as sepharose, cellulose, nylon, cross-linked micelles, and Teflon™. Any other materials described herein for solid supports may be used. In certain embodiments, the microspheres are magnetic microspheres or beads.

[0198] Beads used in the present disclosure need not be spherical; irregular particles may be used. Alternately or in addition thereto, the beads may be porous. In some embodiments, bead size ranges from nanometers, such as 100 nm, to millimeters, such as 1 mm, such as 0.2 micron to 200 microns, or from 0.5 to 5 microns, although in some embodiments smaller or larger beads may be used.

[0199] In some embodiments, the solid support may be a sorbent, such as a sorbent of a liquid chromatography column. In such embodiments, a solid support can be a monolithic chromatography medium, which is cast as a single macroscopic entity of the desired (usually rod-like) shape, or can be a compressible column packing made of loose microscopic particles. Monolithic columns can comprise silica or polymeric sorbents such as, for example, polymethacrylates, and their microstructure can contain fibrous capillaries or sintered particle agglomerates. Thus, solid supports of the present embodiment can include a column comprising a sorbent within a tubular containment, wherein the peptides are immobilized on the sorbent. Such a column can be used as stationary phase of a fixed, desired size (length x diameter) in liquid chromatography or solid phase extraction applications.

[0200] In some embodiments, an AAV (such as an AAV9) is immobilized on a solid support through binding of a peptide to the AAV, such as to the AAV capsid. In such embodiments, the AAV remains immobilized or attached to the support under the conditions in which it is intended to use the support, for example in applications requiring buffer modification or exchange (e.g., flow-through exchange), such as during a wash step disclosed herein.

[0201] In some embodiments, the AAV is removed from the solid support by adding one or more elution buffers. In some embodiments, the elution buffer has a pH lower than 4. In some embodiments, the elution buffer has a pH between 2-4, 2.5-3.5, 2.8-3.2, 2.9-3.1, 2.9-3.4, 2.9-3.3, or 2.9-3.2. In some embodiments, the elution buffer has a pH of 2.8-3.2. In some embodiments, the elution buffer comprises Tris, NaCl, Pluronic, and / or Glycine. In some embodiments, the elution buffer comprises 10-50 mM Tris, 15-300 mM NaCl, 0.005-.02% Pluronic, 50-200 mMGlycine. In some embodiments, the elution buffer comprises 20 mM Tris, 250 mM NaCl, 0.01% Pluronic, 100 mM Glycine, and is at pH 3.0.V.Kits

[0202] Also provided are kits comprising the isolated peptides described herein. The kits can be useful in, or for use in, performing the methods as described herein. In some embodiments, the kit comprises one or both of (i) a wash buffer having a pH within the pH range within which the AAV binds the peptide and (ii) a dissociation buffer having a pH outside the pH range within which the AAV binds the peptide. In some embodiments, the kit comprises a solid support, such as a solid support comprising the isolated peptide immobilized on the solid support. In some embodiments, the kit comprises a solid support comprising an affinity moiety that has an affinity for a binding moiety of the peptide. In kits of the disclosed embodiments, the solid support can comprise, e.g., Sepharose®, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, a flow cell, a hydrogel, a silica gel, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, an alumina gel, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, a latex bead, a magnetic bead, a paramagnetic bead, or a supraparamagnetic bead. In some embodiments, the kit comprises one or more of (i) a nucleic acid molecule encoding an AAV capsid protein; (ii) a nucleic acid molecule encoding an AAV Rep protein; (iii) one or more nucleic acids encoding one or more helper functions required for packaging a nucleic acid molecule of interest into an AAV capsid; and (iv) a nucleic acid molecule of interest. In some embodiments, the kit comprises an AAV packaging cell, such as a eukaryotic, fungal, insect, or bacterial AAV packaging cell. In some embodiments, the kit comprises a control, such as a control peptide, such as a control peptide that does not bind the AAV in the pH range within which a disclosed peptide binds the AAV. In some embodiments, the kit comprises instructions for using a disclosed peptide to obtain an AAV, and / or instructions for performing a disclosed method of obtaining the AAV using the peptide.EXAMPLESExample 1: Identification of peptides that bind AAV9

[0203] This example describes identification of peptides that bind to AAV9 for improved isolation of the AAV9 from an AAV9 preparation. A commonly used current manufacturing process uses the POROS™ CaptureSelect™ AAV9 Affinity Resin (ThermoFisher Scientific),which requires a low pH elution (< pH 3.0) and results in retention of a significant amount of AAV9 on the resin post-elution. The present example describes peptides that bind to AAV9 with high affinity and that do not require low pH conditions to release the AAV9 from the peptides.

[0204] Two linear peptides were identified that bound to AAV9 capsids: AEGEFNKSTMLAHSD (LP3-1; SEQ ID NO: 14) and AEGEFNKSTHLAFYD (LP3-2; SEQ ID NO: 28). These peptides were synthesized with a C-terminal linker followed by a biotin, for use in a biolayer interferometry-based binding assay (Figure IB). For this assay, streptavidin coated biosensors were loaded with biotinylated peptides and then bound to AAV9 capsids (Figure 1 A). The LP3-1 peptide bound with high affinity to AAV9 while the LP3-2 peptide did not bind as well at equivalent concentrations of AAV9, indicating that the affinity for AAV9 is lower than that of LP3-1. A control peptide with only the linker sequence and C-terminal biotin did not bind AAV9 (Figures 1C-1E).

[0205] Epitope mapping was then performed to determine the region that LP3-1 binds on the AAV9 capsid. This experiment was performed using a similar BLI-based method as described previously. AAV9 was pre-incubated with 100 ug / mL of the POROS™ AAV9 ligand, which has a defined binding site at the five-fold axis of symmetry of the AAV9 capsid (Figure 2A). The presence of the POROS™ ligand prevents binding of the peptide to the AAV9 capsid, suggesting that they have overlapping epitopes. A separate ligand (AAVX) was tested that binds to a similar site on other AAV serotypes but does not bind well to AAV9 (Figure 2B).

[0206] Because the low pH required for elution is a drawback of commonly used current affinity protocol, the ability of LP3-1 to release AAV9 under different pH conditions was tested. Using the same BLI system, AAV9 was contacted with the LP3-1 peptide in phosphate buffered saline (PBS) pH 7.4 (as in all previous experiments) for the association step. For the dissociation step, the peptide-bound AAV9 was incubated in buffers ranging from pH 3.0 to pH 10.4. As a positive control, the same analysis was performed using the biotinylated POROS™ ligand. Once bound to the AAV9 capsid, dissociation of the AAV9 from the POROS™ ligand was negligible at every pH tested. The LP3-1 peptide dissociated from the AAV9 at every pH tested except pH 7.4 (FIG. 3).Example 2: Separation of an AAV9 from other components of an AAV9 preparation

[0207] This example describes a non-limiting technique for separating an AAV9 from other components of an AAV9 starting preparation using a peptide described herein. A peptide of SEQ ID NOs: 1-28 is selected, synthesized, and modified as follows: (a) the N-terminal amino acid of the peptide is acetylated, (b) the C-terminal amino acid of the peptide is amidated, (c) the N- terminal amino acid comprises Dap, and / or (d) the peptide is biotinylated at the C-terminal,wherein a PEG2 linker links the biotin moiety to the C-terminal amino acid of the peptide. The peptide is immobilized on the surface of a bead via non-covalent binding of the biotin moiety of the peptide to a streptavidin moiety attached to the surface of the bead.

[0208] The AAV9 starting preparation is prepared by harvesting media from an AAV9 production culture of HEK293T packaging cells cultured under conditions known in the art to cause release of the AAV9 particles into the media from intact cells. The AAV9 starting preparation is clarified by centrifugation or filtration through a suitable filter, and is then treated with a nuclease to digest any contaminating high molecular weight nucleic acid molecules present in the production culture. The AAV9 starting preparation is then concentrated approximately 20-fold, such as through tangential flow filtration.

[0209] The peptide-bound beads are then contacted with the AAV9 starting preparation in PBS at pH 7.4. At this pH, the peptides selectively bind AAV9. The AAV9 starting preparation is washed from the beads using a wash buffer that comprises PBS at pH 7.4. The beads are then contacted with a dissociation buffer having a pH at which the AAV9 dissociates from the beads, z.e., a pH higher or lower than 7.4, such as pH 6.5 or pH 8.5. The AAV9 is separated from the peptide-bound beads, and is collected in the dissociation buffer for downstream analyses and / or use.

[0210] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number, if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant, unless otherwise indicated.

[0211] While the present disclosure has been described with reference to one or more embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof. Therefore, it is intended that the disclosure not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this disclosure, but that the disclosure will include all embodiments falling within the scope of the appended claims. In addition, allnumerical values identified in the detailed description shall be interpreted as though the precise and approximate values are both expressly identified.

Claims

What is claimed is:

1. An isolated peptide comprising or consisting of: a) at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-28; b) at least 12 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 2-14 or 16-28; c) at least 13 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 4-14 or 18-28; d) at least 14 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 9-14 or 23-28; or e.) an amino acid sequence selected from any one of SEQ ID NOs: 1-28.

2. The peptide of claim 1, wherein the amino acid sequence of the peptide consists of an amino acid sequence selected from any one of SEQ ID NOs: 1-28.

3. The peptide of claim 1 or claim 2, wherein the C- or N-terminal amino acid of the peptide is acetylated.

4. The peptide of any one of claims 1-3, wherein the C- or N-terminal amino acid of the peptide is iso-aspartic acid.

5. The peptide of any one of claims 1-3, wherein the peptide further comprises 2,3 diaminopropionic acid (Dap).

6. The peptide of claim 5, wherein the C- or N-terminal amino acid comprises 2,3 diaminopropionic acid (Dap).

7. The peptide of any one of claims 1-6, wherein the C- or N-terminal amino acid of the peptide is amidated.

8. The peptide of any one of claims 1-7, further comprising a linker.

9. The peptide of claim 8, wherein the linker is a peptide linker or a polyol linker.

10. The peptide of claim 9, wherein the polyol linker is a PEG linker.

11. The peptide of claim 9, wherein the peptide linker comprises serine, glycine, and / or threonine.

12. The peptide of claim 11, wherein the linker is a glycine-serine linker.

13. The peptide of claim 12, wherein the glycine-serine linker is GGGSGGGS (SEQ ID NO: 29).

14. The peptide of claim 10, wherein the PEG linker is PEG2.

15. The peptide of any one of claims 8-14, wherein the linker is attached to the N- or C- terminal amino acid of the peptide.

16. The peptide of any one of claims 1-15, further comprising a binding moiety.

17. The peptide of claim 16, wherein the binding moiety is a biotin or a modified biotin.

18. The peptide of claim 16 or claim 17, wherein the binding moiety is attached to the peptide through the linker, optionally wherein the linker links the N- or C-terminal amino acid of the peptide to the binding moiety, and optionally wherein a lysine links the linker to the binding moiety.

19. The peptide of any one of claims 1-18, wherein the peptide is immobilized on a solid support.

20. The peptide of claim 19, wherein the solid support comprises sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, a flow cell, a hydrogel, a silica gel, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, an alumina gel, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead.

21. The peptide of claim 19 or claim 20, wherein the solid support has a binding affinity for the binding moiety.

22. The peptide of any one of claims 19-21, wherein the binding moiety is a biotin or a modified biotin, and the solid support comprises streptavidin, avidin, and / or neutravidin.

23. The peptide of claim 22, wherein the biotin or the modified biotin of the peptide binds to the streptavidin, avidin, and / or neutravidin of the solid support.

24. An isolated peptide comprising or consisting of: (a) at least 8, at least 9, at least 10, or at least 11 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 1-5; (b) at least 12 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 2-5; (c) at least 13 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 3-5; (d) at least 14 consecutive amino acids of an amino acid sequence selected from any one of SEQ ID NOs: 4 or 5; or (e) an amino acid sequence selected from any one of SEQ ID NOs: 1-5; and i) a PEG or a glycine-serine linker conjugated to the N- or C-terminal amino acid of the peptide; and ii) a binding moiety conjugated to the linker; wherein the C-terminal amino acid of the peptide is amidated, and optionally wherein the N-terminal amino acid of the peptide is acetylated and / or the N-terminal amino acid of the peptide further comprises 2,3 diaminopropionic acid (Dap).

25. The peptide of claim 24, wherein the binding moiety comprises biotin or a modified biotin; the peptide is immobilized on a solid support comprising streptavidin, avidin, and / or neutravidin; and the biotin or the modified biotin conjugated to the linker of the peptide binds to the streptavidin, avidin, and / or neutravidin of the solid support.

26. The peptide of any one of claims 1-25, wherein the peptide binds an adeno-associated virus (AAV).

27. The peptide of claim 26, wherein the AAV is AAV9.

28. The peptide of claim 26 or claim 27, wherein the peptide binds the AAV between a first pH and a second pH, inclusive.

29. The peptide of claim 28, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is lower than the first pH than it is to dissociate from the peptide at a pH that is between the first pH and the second pH, inclusive.

30. The peptide of claim 28 or claim 29, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is higher than the second pH than it is to dissociate from the peptide at a pH that is between the first pH and the second pH, inclusive.

31. The peptide of any one of claims 28-30, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is lower than the first pH or higher than the second pH than it is to dissociate from the peptide at a pH that is between the first pH and the second pH, inclusive.

32. The peptide of any one of claims 28-31, wherein the second pH is between 0.05 and 2 pH units, between 0.05 and 1.75 pH units, between 0.05 and 1.5 pH units, between 0.05 and 1.25 pH units, between 0.05 and 1 pH units, between 0.05 and 0.75 pH units, between 0.05 and 0.5 pH units, between 0.05 and 0.4 pH units, between 0.05 and 0.3 pH units, between 0.05 and 0.2 pH units, or between 0.05 and 0.1 pH units higher than the first pH.

33. The peptide of any one of claims 28-32, wherein the AAV dissociates from the peptide at any pH that is lower than the first pH or higher than the second pH.

34. The peptide of any one of claims 28-33, wherein the AAV dissociates from the peptide at any pH that is lower than a first pH or higher than a second pH, and the second pH is between 0.05 and 2 pH units, between 0.05 and 1.75 pH units, between 0.05 and 1.5 pH units, between 0.05 and 1.25 pH units, between 0.05 and 1 pH units, between 0.05 and 0.75 pH units, between 0.05 and 0.5 pH units, between 0.05 and 0.4 pH units, between0.05 and 0.3 pH units, between 0.05 and 0.2 pH units, or between 0.05 and 0.1 pH units higher than the first pH.

35. The peptide of any one of claims 28-34, wherein the peptide binds the AAV only at a pH that is between the first pH and the second pH, inclusive.

36. The peptide of any one of claims 28-35, wherein a) the first pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0; and / or b) the second pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

37. The peptide of any one of claims 28-36, wherein the first pH is 7.0 and the second pH is 8.0.

38. The peptide of any one of claims 28-37, wherein the peptide binds the AAV at pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

39. The peptide of any one of claims 28-38, wherein the peptide binds the AAV at pH 7.4.

40. A method of obtaining an AAV from an AAV starting preparation comprising the AAV, the method comprising: a) contacting an AAV with the peptide of any one of claims 1-39, wherein the contacting occurs at a first pH that is within a pH range within which the AAV binds the peptide; b) optionally performing a wash step at a pH within the pH range within which the AAV binds the peptide to remove all or a portion of the components of the AAV starting preparation that are not bound to the peptide; and c) dissociating the AAV from the peptide by changing the pH to a second pH that is within a pH range within which the AAV does not bind the peptide.

41. The method of claim 40, further comprising, following step (c), subjecting the AAV to one or more of(i) a clarifying treatment;(ii) a step of treating the obtained AAV with a nuclease and / or a protease;(iii) a chromatography process; and(iv) a concentrating process.

42. The method of claim 40 or claim 41, wherein the AAV comprises an AAV full capsid and one or more AAV intermediaries.

43. The method of claim 42, further comprising a step of separating the AAV full capsid from the one or more AAV intermediaries.

44. The method of claim 43, wherein the step of separating the AAV full capsid from the one or more AAV intermediaries is performed following step (c).

45. The method of any one of claims 40-44, wherein the first pH is within a pH range of 6.5 to 8.5.

46. The method of any one of claims 40-45, wherein the second pH is within a pH range of 0.5 to 7.0 or 8.0 to 14.0.

47. The method of any one of claims 40-46, wherein the first pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

48. The method of any one of claims 40-47, wherein the first pH is 7.4.

49. The method of any one of claims 40-48, wherein the pH range within which the AAV binds the peptide is defined by a lower bound pH and an upper bound pH, inclusive.

50. The method of claim 49, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is lower than the lower bound pH than it is to dissociate from the peptide at a pH that is between the lower bound pH and the upper bound pH, inclusive.

51. The method of claim 49 or claim 50, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is higher than the upperbound pH than it is to dissociate from the peptide at a pH that is between the lower bound pH and the upper bound pH, inclusive.

52. The method of any one of claims 49-51, wherein the AAV is at least 10%, 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% more likely to dissociate from the peptide at a pH that is lower than the lower bound pH or higher than the upper bound pH, than it is to dissociate from the peptide at a pH that is between the lower bound pH and the upper bound pH, inclusive.

53. The method of any one of claims 49-52, wherein the upper bound pH is between 0.05 and 2 pH units, between 0.05 and 1.75 pH units, between 0.05 and 1.5 pH units, between 0.05 and 1.25 pH units, between 0.05 and 1 pH units, between 0.05 and 0.75 pH units, between 0.05 and 0.5 pH units, between 0.05 and 0.4 pH units, between 0.05 and 0.3 pH units, between 0.05 and 0.2 pH units, or between 0.05 and 0.1 pH units higher than the lower bound pH.

54. The method of any one of claims 49-53, wherein a) the lower bound pH is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0; and / or b) the upper bound pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5.

55. The method of any one of claims 40-54, comprising the wash step.

56. The method of any one of claims 40-55, wherein the wash step is carried out within a pH range of 6.5 to 8.5.

57. The method of any one of claims 40-56, wherein the wash step comprises a wash buffer, optionally wherein the wash buffer is phosphate buffered saline solution.

58. The method of any one of claims 40-57, wherein step (b), if present, and step (c) occur simultaneously.

59. The method of any one of claims 40-58, wherein step (b), if present, and step (c) occur sequentially.

60. The method of any one of claims 40-59, wherein the dissociating step comprises a dissociation buffer, optionally wherein the dissociation buffer comprises any one or more of Citric acid (e.g., at pH 3-4), Sodium acetate (e.g., at pH 4-5), Sodium citrate (e.g., at pH 5-6), Sodium cacodylate (e.g., at pH 6-7), HEPES sodium (e.g., pH 7-8), TRIS hydrochloride (e.g., at pH 8-9), CAPSO (e.g., at pH 9-10), and / or CAPS (e.g., at pH 10-11).

61. The method of any one of claims 40-60, wherein the AAV is separated from at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of other components of the AAV starting preparation.

62. The method of claim 61, wherein the other components of the AAV starting preparation comprise one or more of a cell culture media, whole cells, cellular materials, and cell lysate.

63. The method of any one of claims 40-62, wherein a yield of the AAV is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the AAV initially present in the AAV starting preparation.

64. The method of any one of claims 40-63, wherein the peptide is immobilized on a solid support.

65. The method of claim 64, wherein the solid support comprises sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, a flow cell, a hydrogel, a silica gel, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, an alumina gel, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead.

66. The method of any one of claims 40-65, wherein the AAV is removed from the solid support by means of one or more elution buffers.

67. The method of claim 66, wherein the elution buffer has a pH between 2-4, 2.5-3.5, 2.8- 3.2, 2.9-3.1, 2.9-3.4, 2.9-3.3, or 2.9-3.2.

68. The method of claim 66 or claim 67, wherein the elution buffer comprises Tris, NaCl, Pluronic, and / or Glycine.

69. The method of any one of claims 40-68, wherein the AAV is AAV9.

70. The method of any one of claims 40-69, wherein the AAV is a recombinant AAV.

71. The method of any one of claims 40-70, wherein the AAV is a self-complementary AAV.

72. The method of any one of claims 40-71, wherein the AAV comprises a nucleic acid molecule of interest.

73. An AAV obtained using the method of any one of claims 40-72.

74. The AAV of claim 73, wherein the AAV is an AAV9.

75. A kit comprising the peptide of any one of claims 1-39.

76. The kit of claim 75, further comprising one or more of: a) a wash buffer having a pH within the pH range within which the AAV binds the peptide; b) a dissociation buffer having a pH outside the pH range within which the AAV binds the peptide; c) a solid support, optionally wherein the solid support comprises Sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, a flow cell, a hydrogel, a silica gel, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, an alumina gel, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead; d) a nucleic acid molecule encoding an AAV capsid protein;e) a nucleic acid molecule encoding an AAV rep protein; f) one or more nucleic acids encoding one or more helper functions required for packaging a nucleic acid molecule of interest into an AAV capsid; g) an AAV packaging cell; and h) a control peptide that does not bind the AAV at a pH within which the AAV binds the peptide.

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