Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14151—Methods of production or purification of viral material

Definitions

• Viral vectors are poised to become fundamental tools in modern medicine and biotechnology owing to their role as delivery agents of gene therapies targeting rare diseases, oncolytic agents to fight aggressive forms of cancer, vaccine platforms to counter infectious diseases, and a gateway to engineer plants and animals for a sustainable agriculture.
• the landscape of viral vector technology is rich of promises as much as challenges: novel vector designs are constantly being introduced with improved tissue targeting and gene delivery activity as well as lower genotoxicity, hepatotoxicity, and immunogenicity.
• the bioprocess technology utilized in viral vector manufacturing draws heavily upon a decades-old platform established decades ago for producing monoclonal antibodies (mAbs).
• AAVs adeno-associated viruses
• Embodiments of the present disclosure include a composition for purifying an adeno- associated virus (AAV) from a biological fluid.
• AAV adeno- associated virus
• the composition comprises at least one peptide ligand that is at least ten amino acids in length and comprises an AAV-binding motif having at least 80% identity to one of the following: (a) YIHFSGYT (SEQ ID NO: 18); (b) STDDD (SEQ ID NO: 19); (c) CYHFS (SEQ ID NO: 20); and/or (d) LITHPRDYS (SEQ ID NO: 21).
• the AAV is a recombinant AAV (rAAV).
• the at least one peptide ligand is cyclic.
• the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrh10.
• the AAV is selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9.
• the at least one peptide ligand comprises an AAV-binding motif having at least 80% identity with SEQ ID NO: 18, and further comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 1, 2, 6, 7, and 14-16.
• the at least one peptide ligand comprises an AAV-binding motif having at least 80% identity with SEQ ID NO: 19, and further comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 10-13, and 33-35.
• the at least one peptide ligand comprises an AAV-binding motif having at least 80% identity with SEQ ID NO: 20, and further comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 3-5, 8, and 9.
• the at least one peptide ligand comprises an AAV-binding motif having at least 80% identity with SEQ ID NO: 21, and further comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 17.
• the at least one peptide ligand binds AAV1 by interacting with at least one of the following amino acids located on the solvent-accessible surface of the conserved regions of its capsid virion proteins (VP1, VP2, and VP3): THR504, ASN500, SER499, TRP503, ASN269, ASP270, SER268, ASN271, ALA267, GLY266, HIS272, SER262, SER385, ALA263, GLN386, GLY384, ASN383, ASN512, GLY513, and/or LYS508.
• the at least one peptide ligand binds AAV2 by interacting with at least one of the following amino acids located on the solvent-accessible surface of the conserved regions of its capsid virion proteins (VP1, VP2, and VP3): GLN385, THR503, GLU499, TRP502, LYS507, ASN268, ASP269, SER267, ALA266, GLY265, HIS271, SER384, SER264, GLN263, SER262, GLY383, ASN382, and/or ASN511.
• VP1, VP2, and VP3 conserved regions of its capsid virion proteins
• the at least one peptide ligand binds AAV5 by interacting with at least one of the following amino acids located on the solvent-accessible surface of the conserved regions of its capsid virion proteins (VP1, VP2, and VP3): SER319, GLU708, GLN532, PRO533, ASN535, TYR542, ALA534, ASN530, ASN546, ASP704, SER531, GLY545, ARG710, PHE698, MET547, THR711, GLU544, THR712, LEU548, ARG713, GLN697, LEU543, THR541, ALA540, THR538, and/or GLY478.
• the at least one peptide ligand binds AAV9 by interacting with at least one of the following amino acids located on the solvent-accessible surface of the conserved regions of its capsid virion proteins (VP1, VP2, and VP3): GLY266, ASN262, THR264, SER263, GLY267, GLU500, SER499, ASN498, PRO504, TRP503, SER269, ASN270, ASP271, SER268, SER386, ALA273, GLN387, ASP384, and/or GLY385.
• the at least one peptide ligand binds AAV2 by interacting with at least one of the following amino acids located on the solvent-accessible surface of the conserved regions of its capsid virion proteins (VP1, VP2, and VP3): THR716, ASN717, VAL719, VAL708, LYS706, ASN709, GLU548, LYS556, and/or ASP553.
• the at least one peptide ligand comprises more than one of the AAV-binding motif of SEQ ID NOs: 18-21.
• the at least one peptide ligand comprises any combination of the AAV-binding motifs of SEQ ID NOs: 18-21.
• the at least one peptide ligand is no more than 25 amino acids in length.
• the at least one peptide ligand comprises an isoelectric point from about 3.5 to about 9.5.
• the at least one peptide ligand comprises a polarity value from about -1.2 to about 1.2.
• the at least one peptide ligand exhibits a disassociation constant (KD) less than or equal to about 10 -5 M at a pH that is higher than or equal to 7.0.
• KD disassociation constant
• the at least one peptide ligand exhibits a disassociation constant (KD) higher than or equal to about 10 -4 M at a pH that is lower than or equal to 6.5. [025] In some embodiments, the at least one peptide ligand from (a) and/or (b) exhibits a dynamic binding capacity (DBC10%) of at least 10 13 vp/mL of resin.
• KD disassociation constant
• DRC10% dynamic binding capacity
• the at least one peptide ligand from (a) comprises: (i) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1; (ii) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2; (iii) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 3; (iv) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 4; (v) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 5; (vi) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 6; (vii) an amino acid sequence having at least 80% sequence identity NCSU-2023-105-02 NCSU-41892.601 3199.0019WO with SEQ ID NO: 7; (viii) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8; (ix) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9; (x) an amino acid sequence having at least 80% sequence identity with SEQ
• the composition comprises: (i) at least two peptide ligands from SEQ ID NOs: 1-17 and 33-35; (ii) at least three peptide ligands from SEQ ID NOs: 1-17 and 33- 35; (iii) at least four peptide ligands from SEQ ID NOs: 1-17 and 33-35; (iv) at least five peptide ligands from SEQ ID NOs: 1-17 and 33-35; (v) at least six peptide ligands from SEQ ID NOs: 1- 17 and 33-35; (vi) at least seven peptide ligands from SEQ ID NOs: 1-17 and 33-35; (vii) at least eight peptide ligands from SEQ ID NOs: 1-17 and 33-35; (viii) at least nine peptide ligands from SEQ ID NOs: 1-17 and 33-35; (ix) at least ten peptide ligands from SEQ ID NOs:
• the at least one peptide ligand comprises a linker. In some embodiments, the at least one peptide ligand is bound to a solid support. In some embodiments, the solid support comprises a non-porous or porous particle, a membrane, a plastic surface, a fiber NCSU-2023-105-02 NCSU-41892.601 3199.0019WO or a woven or non-woven fibermat, a hydrogel, a microplate, and/or a microfluidic device.
• the solid support comprises polymethacrylate and derivatives, polyolefin and derivatives, polyesters and derivatives, polyethers and derivatives, polystyrene, crosslinked polysaccharides, iron oxide, silica, titania, and/or zirconia.
• the biological fluid is a cell culture fluid.
• the biological fluid comprises a supernatant and/or a cellular lysate.
• the biological fluid is derived from a virus production cell line.
• the virus production cell line is selected from the group consisting of CHO cells, HEK293 cells, MDCK-S, MDCK-A, Vero cells, LLC-MK2D, PER.C6, EB66, AGE1.CR cells, Spodoptera frugiperda (Sf9) cells, and HeLa cells, or any derivatives or variants thereof.
• Embodiments of the present disclosure also include an adsorbent comprising any of the compositions described herein.
• Embodiments of the present disclosure also include a method of purifying an adeno- associated virus (AAV) from a biological fluid.
• AAV adeno- associated virus
• the method comprising contacting the composition comprising any of the peptide ligands described herein (or any of the adsorbents described herein) with a biological fluid comprising the AAV, wherein the peptide ligand binds the AAV; and eluting the AAV from the peptide ligand.
• the elution is performed at pH from about 6.0 to about 7.5.
• a composition comprising at least one peptide ligand described herein, or an adsorbent comprising at least one peptide ligand described herein is substantially resistant to NaOH, or a similar alkaline agent, at a concentration from about 0.1M to about 0.5M.
• a composition comprising at least one peptide ligand described herein, or an adsorbent comprising at least one peptide ligand described herein is substantially reusable for up to about 10 cycles.
• the method further comprises a washing step before eluting the AAV from the at least one peptide ligand.
• the method results in at least a 50% yield for the AAV. [037] In some embodiments, the method produces at least an 80-fold reduction in host cell proteins. NCSU-2023-105-02 NCSU-41892.601 3199.0019WO [038]
• Embodiments of the present disclosure also include an adeno-associated virus (AAV) purified using an of the methods described herein, wherein the AAV exhibits at least 25% transduction efficiency.
• AAV adeno-associated virus
• FIGS 1A-1F Representative complexes formed by A20-mimetic peptides CYIHFSGYTNYNPSLKSC (A1, red; SEQ ID NO: 1), CYGHFSGYGNYGPC (A4, green; SEQ ID NO: 4), CYIHFSGYTNYNPC (A6, blue; SEQ ID NO: 6), CVIDGSQSTDDDKIC (A10, yellow; SEQ ID NO: 10), CDGSQSTDDDKIC (A11, magenta), and LITHPRDYSPKLTPGLYEFG (A17, orange) with the capsids of (A) AAV1 (PDB IDs: 6JCQ, 6JCR, 7RK9, and 8FQ4), (B) AAV2 (5IPI, 6IH9, 6IHB, and 6U0V); (C) AAV5 (6JCS, 6JCT, 7KP3, and 7KPN); (D) AAV5 (6JCS, 6JCT, 7KP3, and 7KPN
• FIGS.2A-2F Values of loss (orange, calculated as the ratio of the AAV titer in the flow-through and wash fractions vs. load) and yield (green, calculated as the ratio of the AAV titer in the elution fraction vs.
• FIGS.3A-3B Values of loss (orange, calculated as the ratio of the AAV titer in the flow-through and wash fractions vs. load) and yield (green, calculated as the ratio of the AAV titer in the elution fraction vs.
• HCP LRV logarithmic reduction of HCPs
• HCP LRV logarithmic reduction of HCPs
• B a clarified Sf9 cell lysate NCSU-2023-105-02 NCSU-41892.601 3199.0019WO
• FIG.4 Values of relative transduction efficiency of AAV2 purified from a clarified HEK293 cell lysate using peptide-based adsorbents (A1; SEQ ID NO: 1) CYIHFSGYTNYNPSLKS-, (A4; SEQ ID NO: 4) CYGHFSGYGNYGPC-, and (A10; SEQ ID NO: 10) CVIDGSQSTDDDKIC-Toyopearl resins together with control adsorbent POROSTM CaptureSelectTM AAVX resin (note: AAVX and AAVX* denote the eluate that was neutralized respectively immediately and 48 hrs after collection).
• the transduction efficiency (TU/vp) of eluted AAV2 was measured on human epithelial (HT1080; 10 7 vp per cell) by performing a green fluorescence assay using a CytoFLEX Flow Cytometer.
• the values of relative transduction efficiency were calculated as the ratio of transduction efficiency of eluted AAV2 vs. AAV2 in the feedstock. [043] FIGS.
• 5A-5D Contacts formed by (A) AAV1 in complex with AAVR (PDB ID: 6JCQ and 7TI5), (B) AAV2 in complex with AAVR (6IHB and 6NZ0) and monoclonal antibody A20 (3J1S), (C) AAV5 in complex with AAVR (6JCS), and (D) AAV9 in complex with AAVR (7WJX); the full capsid in is gray cartoon, the target VP1 is in light blue cartoon, the AAVR is in light pink cartoon, and the A20 is in light green cartoon; the interacting residues on VP1 are in blue sticks, on AAVR are in magenta sticks, and on A20 are in green sticks. [044] FIGS.
• 6A-6C Examples of complexes formed by peptides (A) CYIHFSGYTNYNPSLKSC (A1, blue cartoon; SEQ ID NO: 1), (B) CYGHFSGYGNYGPC (A4, green cartoon; SEQ ID NO: 4), and (C) CVIDGSQSTDDDKIC (A10, yellow cartoon; SEQ ID NO: 10) with the solvent accessible peptide segments displayed on the convex side of the VP1 protein of AAV1 (PDB ID: 6JCR), AAV2 (6IH9), AAV5 (7KP3), AAV6 (5EGC), AAV8 (2QA0), and AAV9 (7WJX).
• the segments of the VP that are not solvent accessible or whose homology among AAV serotypes is lower than 95% are in grey cartoon, the homologous segments of VP that are solvent accessible and displayed on the concave side of the capsid are in pink cartoon, and the homologous segments of VP that are solvent accessible and displayed on the convex side of the capsid are in red cartoon.
• Binding was conducted in 20 mM NaCl in 10 mM Bis-Tris buffer at pH 7.0 (RT: 3 min); elution from the peptide-functionalized resins was conducted using 1 M MgCl2 in 10 mM Bis-Tris buffer at pH 6.0 (RT: 1 min); elution from POROSTM CaptureSelectTM AAVX affinity resin (AAVX) and AVB Sepharose HP resin (AVB) was conducted using 0.2 M MgCl2 in 200 mM citrate buffer at pH 2.2 and PBS at pH 2.0, respectively (RT: 2 min).
• SEC Size Exclusion Chromatography analysis of (A) a clarified HEK293 cell lysate (AAV2 titer: ⁇ 2.51 ⁇ 10 12 vp/mL; HCP titer: ⁇ 0.3 mg/mL) and the elution fractions obtained from the purification of AAV2 from the clarified lysate using peptide-based adsorbents (B) (A1; SEQ ID NO: 1) CYIHFSGYTNYNPSLKSC-, (C) (A4; SEQ ID NO: 4) CYGHFSGYGNYGPC-, (D) (A10; SEQ ID NO: 10) CVIDGSQSTDDDKIC-Toyopearl resins, and control adsorbents (E) (AAVX) POROSTM CaptureSelectTM AAVX and (F) (AVB) AVB Sepharose HP resins.
• AAVX a clarified HEK293 cell lysate
• HCP LRV logarithmic reduction of HCPs
• HCP LRV logarithmic reduction of HCPs
• Serotypes AAV1, AAV5, AAV7, and AAV8 were bound in 2mM MgCl2 in 50 mM Sodium Acetate buffer at pH 5.0 (RT: 3 min) and eluted using 20mM NaCl in 10 mM Bis-Tris buffer at pH 7.0 (RT: 1 min); and 0.4 M MgCl 2 in 10 mM Bis-Tris buffer at pH 6.0 (RT: 1 min).
• Elution from POROSTM CaptureSelectTM AAVX affinity resin and AVB Sepharose HP resin was conducted using 0.2 M MgCl2 in 200 mM citrate buffer at pH 2.2 and PBS at pH 2.0, respectively (RT: 2 min).
• A20-mimetic YIHFSGYTNYNPSLKS (SEQ ID NO: 1) and AAVR-mimetic VIDGSQSTDDDKI (SEQ ID NO: 10) demonstrated excellent capture of serotypes belonging to distinct clones/clades – AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 – corroborating the in silico models documenting their ability to target regions of the virion proteins that are conserved across all serotypes.
• VIDGSQSTDDDKI-Toyopearl resin features values of binding capacity ( ⁇ 10 14 vp per mL) and product yields ( ⁇ 60-80%) on par with commercial adsorbents, and purified AAV2 from a HEK293 cell lysate affording high recovery (70-80%), a 700-fold reduction of host cell proteins (HCPs), and high transduction activity (up to 65%) of the purified viruses.
• the present disclosure provides compositions and methods for purifying an adeno-associated virus (AAV) from a biological fluid.
• AAV adeno-associated virus
• cyclic NCSU-2023-105-02 NCSU-41892.601 3199.0019WO peptides or polypeptides are molecules that are primarily composed of a peptide segment modified via head-to-tail, head-to-side-chain, side-chain-to-tail, or side-chain-to-side-chain cyclization.
• the peptide ligands of the present disclosure can be cyclized by any method available to one of skill in the art. For example, the N-terminal and C-terminal ends can be condensed to form a peptide bond by known procedures.
• Functional groups present on the side chains of amino acids in the peptides can also be joined to cyclize the peptides of the present disclosure.
• functional groups that can form covalent bonds include -COOH and -OH; -COOH and -NH2; and -COOH and -SH.
• Pairs of amino acids that can be used to cyclize a peptide include, Asp and Lys; Glu and Lys; Asp and Arg; Glu and Arg; Asp and Ser; Glu and Ser; Asp and Thr; Glu and Thr; Asp and Cys; and Glu and Cys.
• amino acid residues that are capable of forming covalent linkages cV ⁇ U [ZR NZ[ ⁇ UR ⁇ VZPXaQR Pe_ ⁇ RVZR&XVWR NYVZ[ NPVQ_ _aPU 9e_% U9e_% t&YR ⁇ UeX&9e_ NZQ Penicillamine (Pen), a non-proteinogenic an alpha-amino acid having the structure of valine substituted at the beta position with a sulfanyl group, which can form disulfide bridges with one another.
• Preferred cysteine-like amino acid residues include Cys and Pen.
• Other pairs of amino acids that can be used for cyclization of the peptide will be apparent to those skilled in the art.
• sequences of the peptide ligands of the present disclosure can be represented as “C-[sequence]-C” or a version thereof (e.g., “cycloCC[SEQUENCE]”), in which the primary sequence of the peptide ligand is flanked by Cys residues.
• the groups used to cyclize a peptide need not be amino acids. Examples of functional groups capable of forming a covalent linkage with the amino terminus of a peptide include carboxylic acids and esters.
• Examples of functional groups capable of forming a covalent linkage with the carboxyl terminus of a peptide include -OH, -SH, -NH 2 and -NHR where R is (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkenyl and (C 1 -C 6 ) alkynyl.
• the N-terminal amino acid is covalently linked to the C-terminal amino acid in the cyclic peptide.
• the covalent linkage is head to tail between the free C-terminal carboxyl and the free N-terminal amine.
• the covalent linkage involves a side chain of the N-terminal amino acid, a side chain of the C-terminal amino acid, or both.
• the N-terminal amino acid is covalently linked to the C-terminal amino acid with a thioether bond.
• the reaction conditions used to cyclize the peptides are sufficiently mild so as not to degrade or otherwise damage the peptide.
• Suitable groups for protecting the various functionalities as necessary are well known in the art (see, e.g., Greene & Wuts, 1991, 2nd ed., NCSU-2023-105-02 NCSU-41892.601 3199.0019WO John Wiley & Sons, NY), as are various reaction schemes for preparing such protected molecules.
• the cyclic peptides of the present invention may be synthesized by solid-phase synthesis and purified according to methods known in the art. Any of a number of well-known procedures utilizing a variety of resins and reagents may be used to prepare the cyclic peptides of the present invention.
• the peptides are modified to stabilize them, to facilitate their uptake and/or absorption, or to improve any other characteristic or property of the peptides that is known to one of skill in art.
• charges on the peptides can be neutralized and the peptides can be linked to other chemical moieties.
• the peptides may also be modified by the addition of: radioactive atoms; detectable labels (e.g., radioactive labels, dyes, fluorescent moieties, chemiluminescent moieties, quantum dots); affinity tags (e.g., His tag, biotin); PEG moieties; carbohydrates (e.g., glycosylation, hesylation); and organic molecules (e.g., alkylation, acetylation, acylation).
• detectable labels e.g., radioactive labels, dyes, fluorescent moieties, chemiluminescent moieties, quantum dots
• affinity tags e.g., His tag, biotin
• PEG moieties e.g., carbohydrates
• carbohydrates e.g., glycosylation, hesylation
• organic molecules e.g., alkylation, acetylation, acylation
• the AAV is a recombinant AAV (rAAV).
• the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrh10.
• the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 85% identity with SEQ ID NO: 18, and further comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NOs: 1, 2, 6, 7, and 14-16.
• the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 90% identity with SEQ ID NO: 18, and further comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 1, 2, 6, 7, and 14-16.
• the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 95% identity with SEQ ID NO: 18, and further comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NOs: 1, 2, 6, 7, and 14-16. [086] In some embodiments, the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 80% identity with SEQ ID NO: 19, and further comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 10- 13, and 33-35.
• the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 85% identity with SEQ ID NO: 19, and further comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NOs: 10- 13, and 33-35. In some embodiments, the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 90% identity with SEQ ID NO: 19, and further comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 10- 13, and 33-35.
• the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 85% identity with SEQ ID NO: 20, and further comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NOs: 3- 5, 8, and 9.
• the at least one peptide ligand of the present disclosure NCSU-2023-105-02 NCSU-41892.601 3199.0019WO comprises an AAV-binding motif having at least 90% identity with SEQ ID NO: 20, and further comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 3- 5, 8, and 9.
• the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 95% identity with SEQ ID NO: 20, and further comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NOs: 3- 5, 8, and 9. [088] In some embodiments, the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 80% identity with SEQ ID NO: 21, and further comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 17.
• the at least one peptide ligand of the present disclosure comprises an AAV- binding motif having at least 85% identity with SEQ ID NO: 21, and further comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NO: 17. In some embodiments, the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 90% identity with SEQ ID NO: 21, and further comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 17. In some embodiments, the at least one peptide ligand of the present disclosure comprises an AAV-binding motif having at least 95% identity with SEQ ID NO: 21, and further comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO: 17.
• the at least one peptide ligand binds AAV1 by interacting with at least one of the following amino acids located on the solvent-accessible surface of the conserved regions of its capsid virion proteins (VP1, VP2, and VP3): THR504, ASN500, SER499, TRP503, ASN269, ASP270, SER268, ASN271, ALA267, GLY266, HIS272, SER262, SER385, ALA263, GLN386, GLY384, ASN383, ASN512, GLY513, and/or LYS508 (see, e.g., Table 8).
• THR504, ASN500, SER499, TRP503, ASN269, ASP270, SER268, ASN271, ALA267, GLY266, HIS272, SER262, SER385, ALA263, GLN386, GLY384, ASN383, ASN512, GLY513, and/or LYS508 see, e
• the at least one peptide ligand binds AAV2 by interacting with at least one of the following amino acids: GLN385, THR503, GLU499, TRP502, LYS507, ASN268, ASP269, SER267, ALA266, GLY265, HIS271, SER384, SER264, GLN263, SER262, GLY383, ASN382, and/or ASN511 (see, e.g., Table 8).
• the at least one peptide ligand binds AAV5 by interacting with at least one of the following amino acids located on the solvent- accessible surface of the conserved regions of its capsid virion proteins (VP1, VP2, and VP3): SER319, GLU708, GLN532, PRO533, ASN535, TYR542, ALA534, ASN530, ASN546, ASP704, SER531, GLY545, ARG710, PHE698, MET547, THR711, GLU544, THR712, LEU548, NCSU-2023-105-02 NCSU-41892.601 3199.0019WO ARG713, GLN697, LEU543, THR541, ALA540, THR538, and/or GLY478 (see, e.g., Table 8).
• the at least one peptide ligand binds AAV9 by interacting with at least one of the following amino acids located on the solvent-accessible surface of the conserved regions of its capsid virion proteins (VP1, VP2, and VP3): GLY266, ASN262, THR264, SER263, GLY267, GLU500, SER499, ASN498, PRO504, TRP503, SER269, ASN270, ASP271, SER268, SER386, ALA273, GLN387, ASP384, and/or GLY385 (see, e.g., Table 8).
• GLY266, ASN262, THR264, SER263, GLY267, GLU500, SER499, ASN498, PRO504, TRP503, SER269, ASN270, ASP271, SER268, SER386, ALA273, GLN387, ASP384, and/or GLY385 see, e.g., Table 8).
• the at least one peptide ligand binds AAV2 by interacting with at least one of the following amino acids located on the solvent-accessible surface of the conserved regions of its capsid virion proteins (VP1, VP2, and VP3): THR716, ASN717, VAL719, VAL708, LYS706, ASN709, GLU548, LYS556, and/or ASP553 (see, e.g., Table 8).
• the at least one peptide ligand comprises more than one of the AAV-binding motif of SEQ ID NOs: 18-21.
• the at least one peptide ligand comprises any combination of the AAV-binding motifs of SEQ ID NOs: 18-21. [091] In some embodiments, the at least one peptide ligand is no more than 20 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 25 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 30 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 35 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 40 amino acids in length. In some embodiments, the at least one peptide ligand is no more than 45 amino acids in length.
• the at least one peptide ligand is no more than 50 amino acids in length. [092] In some embodiments, the at least one peptide ligand is from about 10 amino acids to about 100 amino acids in length. In some embodiments, the at least one peptide ligand is from about 10 amino acids to about 90 amino acids in length. In some embodiments, the at least one peptide ligand is from about 10 amino acids to about 80 amino acids in length. In some embodiments, the at least one peptide ligand is from about 10 amino acids to about 70 amino acids in length. In some embodiments, the at least one peptide ligand is from about 10 amino acids to about 60 amino acids in length.
• the at least one peptide ligand is from about 10 amino acids to about 50 amino acids in length. In some embodiments, the at least one peptide ligand is from about 10 amino acids to about 40 amino acids in length. In some embodiments, the at least one peptide ligand is from about 10 amino acids to about 30 amino acids in length. In some NCSU-2023-105-02 NCSU-41892.601 3199.0019WO embodiments, the at least one peptide ligand is from about 20 amino acids to about 100 amino acids in length. In some embodiments, the at least one peptide ligand is from about 30 amino acids to about 100 amino acids in length. In some embodiments, the at least one peptide ligand is from about 40 amino acids to about 100 amino acids in length.
• the at least one peptide ligand is from about 50 amino acids to about 100 amino acids in length. In some embodiments, the at least one peptide ligand is from about 25 amino acids to about 50 amino acids in length. [093] In some embodiments, the at least one peptide ligand comprises an isoelectric point from about 3.5 to about 9.5. In some embodiments, the at least one peptide ligand comprises an isoelectric point from about 4.0 to about 9.0. In some embodiments, the at least one peptide ligand comprises an isoelectric point from about 5.0 to about 8.0. In some embodiments, the at least one peptide ligand comprises an isoelectric point from about 4.0 to about 7.0.
• the at least one peptide ligand comprises an isoelectric point from about 6.0 to about 9.0. [094] In some embodiments, the at least one peptide ligand comprises a polarity value from about -1.2 to about 1.2. In some embodiments, the at least one peptide ligand exhibits a disassociation constant (K D ) less than or equal to about 1.0 -4 M at pH 7.4. In some embodiments, the at least one peptide ligand exhibits a disassociation constant (KD) less than or equal to about 10 -5 M at a pH that is higher than or equal to 7.0.
• K D disassociation constant
• KD disassociation constant
• the at least one peptide ligand exhibits a disassociation constant (K D ) higher than or equal to about 10 -4 M at a pH that is lower than or equal to 6.5. In some embodiments, the at least one peptide ligand from (a) and/or (b) exhibits a dynamic binding capacity (DBC10%) of at least 10 13 vp/mL of resin.
• K D disassociation constant
• DRC10% dynamic binding capacity
• the at least one peptide ligand of the present disclosure from (a) comprises: (i) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 1; (ii) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 2; (iii) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 3; (iv) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 4; (v) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 5; (vi) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 6; (vii) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7; (viii) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8; (ix) an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9; (x) an amino acid sequence having at least 80% sequence NCSU-2023-105-02 NCSU-4
• the at least one peptide ligand of the present disclosure from (a) comprises: (i) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; (ii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 2; (iii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 3; (iv) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4; (v) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5; (vi) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 6; (vii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7; (viii) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 8; (ix) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 9; (x) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10; (xi) an amino acid sequence having at least
• the at least one peptide ligand of the present disclosure from (a) comprises: (i) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1; (ii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 2; (iii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 3; (iv) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 4; (v) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 5; (vi) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 6; (vii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7; (viii) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 8; (ix) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 9; (x) an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1; (ii) an amino acid sequence having
• the at least one peptide ligand of the present disclosure from (a) comprises: (i) an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 1; (ii) an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 2; (iii) an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 3; (iv) an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 4; (v) an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 5; (vi) an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 6; (vii) an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 7; (viii) an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 8; (ix) an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 9; (x) an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 10; (i) an amino acid sequence having at
• the composition comprises: (i) at least two peptide ligands from SEQ ID NOs: 1-17 and 33-35; (ii) at least three peptide ligands from SEQ ID NOs: 1-17 and 33- 35; (iii) at least four peptide ligands from SEQ ID NOs: 1-17 and 33-35; (iv) at least five peptide ligands from SEQ ID NOs: 1-17 and 33-35; (v) at least six peptide ligands from SEQ ID NOs: 1- 17 and 33-35; (vi) at least seven peptide ligands from SEQ ID NOs: 1-17 and 33-35; (vii) at least eight peptide ligands from SEQ ID NOs: 1-17 and 33-35; (viii) at least nine peptide ligands from SEQ ID NOs: 1-17 and 33-35; (ix) at least ten peptide ligands from SEQ ID NOs:
• the peptide ligands of the present disclosure can purify an AAV from a biological fluid.
• the biological fluid is a cell culture fluid.
• the biological fluid comprises a supernatant and/or a cellular lysate.
• the biological fluid is derived from a virus production cell line.
• the virus production cell line is selected from the group consisting of CHO cells, HEK293 cells, MDCK-S, MDCK-A, Vero cells, LLC-MK2D, PER.C6, EB66, AGE1.CR cells, Spodoptera frugiperda (Sf9) cells, and HeLa cells, or any derivatives or variants thereof.
• Embodiments of the present disclosure also include an adsorbent (e.g., adsorption chromatography) comprising any of the compositions described herein. Further described herein are adsorbents comprising a composition as described above, where each peptide ligand of the composition is conjugated to a support. Supports may comprise, but are not limited to, particles, beads, plastic surfaces, resins, fibers, and/or membranes.
• the solid support comprises a non-porous or porous particle, a membrane, a plastic surface, a fiber or a woven or non-woven fibermat, a hydrogel, a microplate, and/or a microfluidic device.
• the solid support comprises polymethacrylate and derivatives, polyolefin and derivatives, polyesters and derivatives, polyethers and derivatives, polystyrene, crosslinked polysaccharides, iron oxide, silica, titania, and/or zirconia.
• supports may include microparticles and/or nanoparticles. Each support may be made out of any suitable material including, but not limited to, synthetic or natural polymers, metals, and metal oxides.
• Some supports may be magnetic, such as a magnetic bead, microparticle and/or nanoparticle.
• Suitable synthetic polymers include, but are not limited to, polymethacrylate, polyethersulfone, and polyethyleneglycole.
• Suitable natural polymers include, but are not limited to, cellulose, crosslinked agarose, and chitosan.
• Suitable metal oxides include, but are not limited to, iron oxide, silica, titania, and zirconia. Further described herein are adsorbents comprising a composition as described above conjugated to a support.
• the adsorbent comprises a single type of support made from a single type of support material, where all of the peptides in the composition are conjugated to supports formed of the single type of support material.
• the composition may comprise one or more different types of peptides, each conjugated to the single type of support made from the single type of support material.
• the adsorbent comprises a plurality of types of support. Each type of support may be made of the same type of support material or different types of support materials.
• the composition may comprise one or more different types of peptides, as described further herein, each conjugated to a different type of support.
• the peptides of the composition can be conjugated to a soluble compound, for example stimuli-responsive polymer chains to remove AAVs by affinity precipitation.
• a soluble compound for example stimuli-responsive polymer chains to remove AAVs by affinity precipitation.
• the method includes contacting a composition comprising any of the peptide ligands described herein, or an adsorbent comprising any of the peptides described herein, with a biological fluid comprising the AAV, wherein the at least one peptide ligand binds the AAV.
• the method includes eluting the AAV from the peptide ligand, thereby purifying the AAV.
• the methods of the present disclosure can further comprise washing the composition or adsorbent to remove one or more product- and/or process-related impurities or contaminants from the AAVs bound to the peptide ligands.
• the method can be performed under any binding conditions suitable for use with the composition or adsorbent, including both static binding conditions and dynamic binding conditions.
• the peptide ligands of the present disclosure exhibit unique operational features that provide distinct technological advantages over the compositions and methods currently available to purify AAVs. For example, currently available ligands that are used to purify AAVs require harsh elution conditions (e.g., pH ⁇ 3.0), which damages the AAV products being eluted. In contrast, the peptide ligands of the present disclosure release bound AAVs under much gentler conditions, which do not damage the AAV product.
• compositions comprising at least one peptide ligand described herein that can be used to elute AAVs at a pH ranging from about 6.0 to about 7.5.
• a composition comprising at least one peptide ligand described herein, or an adsorbent comprising at least one peptide ligand described herein is substantially resistant to NaOH, or a similar alkaline agent, at a concentration from about 0.1M to about 0.5M.
• a composition comprising at least one peptide ligand described herein, or an adsorbent comprising at least one peptide ligand described herein is substantially reusable for up to about 10 cycles.
• the method further comprises a washing step before eluting the AAV from the at least one peptide ligand.
• the AAV purification methods of the present disclosure result in at least a 50% yield for the AAV.
• the method produces at least an 80-fold reduction in host cell proteins.
• the binding affinity of the compositions and/or adsorbent for the AAVs, as compared to one or more product- and/or process-related impurities or contaminants, can be adjusted by changes in the following: properties and concentration of the AAVs, properties and concentration of the one or more product- and/or process-related impurities or contaminants; the properties and concentration of the host cell proteins; the composition, concentration, and pH of the mixture; the loading conditions and residence time of the contacting and washing steps; and/or the composition, concentration, and pH of the aqueous buffers utilized to conduct the purification of the AAV from the mixture using the compositions and/or adsorbent.
• the contacting step can comprise a low pH buffer of between pH 7-8.
• the elution is performed at pH from about 5.0 to about 8.0. In some embodiments, the elution is performed at pH from about 5.0 to about 7.5. In some embodiments, the elution is performed at pH from about 5.0 to about 7.0. In some embodiments, the elution is performed at pH from about 5.0 to about 6.5. In some embodiments, the elution is performed at pH from about 5.0 to about 6.0. In some embodiments, the elution is performed at pH from about 5.0 to about 5.5. In some embodiments, the elution is performed at pH from about 5.5 to about 8.0.
• the elution is performed at pH from about 6.0 to about 8.0. In some embodiments, the elution is performed at pH from about 6.5 to about 8.0. In some embodiments, the elution is performed at pH from about 7.0 to about 8.0. In some embodiments, the elution is performed at pH from about 7.5 to about 8.0. In some embodiments, the elution is performed at pH from about 6.0 to about 7.0. In some embodiments, the elution is performed at pH from about 5.5 to about 7.5.
• the methods of the present disclosure result in at least a 50% yield for the AAV (e.g., as compared to methods in which the peptide ligands of the present disclosure are not used). In some embodiments, the methods of the present disclosure result in at least a 60% yield for the AAV. In some embodiments, the methods of the present disclosure result in at least a 70% yield for the AAV. In some embodiments, the methods of the present disclosure result in at least an 80% yield for the AAV. In some embodiments, the methods of the present disclosure result in at least a 90% yield for the AAV.
• Embodiments of the present disclosure also include an adeno-associated virus (AAV) purified using any of the methods described herein.
• AAV adeno-associated virus
• the AAV exhibits at least 25% transduction efficiency (i.e., a measure of biological activity).
• the AAV exhibits at least 30% transduction efficiency.
• the AAV exhibits at least 35% transduction efficiency.
• the AAV exhibits at least 40% transduction activity.
• the AAV exhibits at least 45% transduction efficiency.
• the AAV exhibits at least 50% transduction efficiency. In some embodiments, the AAV exhibits at least 55% transduction efficiency. In some embodiments, the AAV exhibits at least 60% transduction efficiency. In some embodiments, the AAV exhibits at least 65% transduction efficiency. In some embodiments, the AAV exhibits at least 70% transduction efficiency. In some embodiments, the AAV exhibits at least 75% transduction efficiency. In some embodiments, the AAV exhibits at least 80% transduction efficiency. In some NCSU-2023-105-02 NCSU-41892.601 3199.0019WO embodiments, the AAV exhibits at least 85% transduction efficiency. In some embodiments, the AAV exhibits at least 90% transduction efficiency.
• the AAV exhibits at least 95% transduction efficiency.
• Materials and Methods [0111] In silico design of peptide mimetics of the AAV receptor (AAVR) and anti-AAV antibody A20. The crystal structures of AAVR in complex with AAV1 (PDB ID: 6JCQ and 7TI5), AAV2 (3J1S, 6IHB and 6NZ0), AAV5 (7KP3 and 7KPN), and AAV9 (7WJX and 7WQP) as well as the complex of AAV2 with monoclonal antibody A20 (3J1S) were analyzed to identify the residues on the protein ligands (AAVR and A20) and the AAV virion protein (VP1) involved in the affinity interaction and calculate their pairwise contributions to the binding energy.
• Each peptide sequence was placed in a simulation box with periodic NCSU-2023-105-02 NCSU-41892.601 3199.0019WO boundary containing 1,500 TIP3P water molecules and equilibrated with 10,000 steps of steepest gradient descent; heated to 300 K in an NVT ensemble for 250 ps using 1 fs time steps; and equilibrated to 1 atm via a 500-ps NPT simulation with 2 fs time steps.
• the energetic landscape associated with the various peptide conformations was sampled to identify the structures with absolute energy minima.
• the structure of the VP1 from AAV1 (PDB ID: 6JCR), AAV2 (6IH9), AAV3 (3KIC), AAV4 (2G8G), AAV5 (7KP3), AAV6 (5EGC), AAV7 (7JOT), AAV8 (2QA0), and AAV9 (7WJX) were initially prepared using Protein Prep Wizard (PPW, Schrödinger, New York, NY) by correcting missing residues or atoms, adding explicit hydrogens, removing salt ions, and optimizing the hydrogen-bonding network.
• PW Protein Prep Wizard
• the resulting VPs were utilized to construct triangular clusters of VP1-VP2-VP3 proteins, whose ionization states at pH 6.0 and 7.4 were obtained - and the corresponding structural minimization of the clusters were performed using PROPKA.
• the construct triangular clusters were finally draped on the spherical cap of the corresponding AAV capsid.
• the peptide ligands were then docked in silico against the AAVR binding sites using the docking software HADDOCK (High Ambiguity Driven Protein-Protein Docking) v.2.4.
• the bound AAV2, AAV3, AAV6, AAV9, and AAVrh.10 were eluted 0.4 M MgCl2 in 10 mM Bis-Tris HCl buffer at pH 6.5; whereas the bound AAV1, AAV5, AAV7, and AAV8 were released in a first elution step using 20 mM NaCl in 10 mM Bis-Tris buffer at pH 7.0 followed by a second elution step using 0.4 M MgCl2 in 10 mM Bis-Tris HCl buffer at pH 6.5.
• the chromatographic steps of loading, washing, and AAV elution were conducted as described herein.
• Cleaning-in-Place (CIP) was then conducted by flowing 15 CVs of 0.1 M NaOH (aq) at 1 mL/min followed by static incubation for 15 min.
• the resin was finally washed with 5 CVs of binding buffer.
• the collected flow-through and elution fractions were analyzed by ELISA Kit to determine the values of AAV yield, HEK293 ELISA Kit to quantify the values of HCP removal, and size exclusion chromatography to measure global product purity, and fluorescence flow cytometry to quantify the transduction efficiency of the eluted AAVs.
• the AAVs in the HEK293 cell lysate and the eluted samples were serially diluted in DMEM (no FBS NZQ NZ ⁇ VOV[ ⁇ VP_$ NZQ NQQRQ cV ⁇ U ⁇ [XeO ⁇ RZR N ⁇ 1 uT(YA' 7 b[XaYR [S )'* YA [S QVXa ⁇ RQ 77K _NY ⁇ XR was incubated with the HT1080 cells. After 24 hrs, spent medium was replaced with fresh DMEM supplemented with 10% v/v FBS and the cells were cultured for 72 hrs.
• Equation 2 The fraction of cells expressing GFP (GFP + ) was quantified using a CytoFlex flow cytometer (Beckman Coulter, Brea, CA) and the number of transduction units per mL (TU/mL) was calculated using Equation 2: [0145] Equation 2 [0146] Wherein NHT1080 is the number of cells incubated with the diluted AAV sample, V is the volume of the diluted AAV sample, and DF is the dilution factor. [0147] In silico design of alkaline-stable variants of peptide A10.
• the structures with absolute energy minima were docked against the triangular clusters using the docking software HADDOCK (High Ambiguity Driven Protein-Protein Docking) v.2.4.
• the AAVR-binding residues and the A20-binding residues on the VP proteins and residues X1X2[...]Xn on the peptides were marked NCSU-2023-105-02 NCSU-41892.601 3199.0019WO as “active”, while all other residues were marked as “passive”.
• the top AAV:peptide clusters were ranked using the dMM-PBSA score.
• Adeno-associated viruses have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs are being introduced with improved tissue targeting, potency, and safety, the current technology for AAV manufacturing is modeled after conventional antibody bioprocessing: in particular, the purification pipeline hinges on protein ligands with high binding strength for the affinity-based capture step. While providing high AAV-binding capacity and selectivity, these ligands require strong acid (pH ⁇ 3) elution conditions, thus compromising product activity and stability, and their low biochemical stability limits their lifetime in spite of their high cost.
• embodiments of the present disclosure provide a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, while (ii) enabling product elution under near-physiological conditions (pH 6.0) and (iii) granting extended reusability by withstanding multiple regenerations.
• A20-mimetic CYIHFSGYTNYNPSLKSC (SEQ ID NO: 1) and AAVR-mimetic CVIDGSQSTDDDKIC (SEQ ID NO: 10) demonstrated excellent capture of serotypes belonging to distinct clones/clades – AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 – corroborating the in silico models documenting their ability to target regions of the virion proteins that are conserved across all serotypes.
• CVIDGSQSTDDDKIC-Toyopearl resin (SEQ ID NO: 10) features values of binding capacity ( ⁇ 10 14 vp per mL) and product yields ( ⁇ 60-80%) on par with commercial adsorbents, and purified AAV2 from HEK293 and Sf9 cell lysates affording high recovery (up to 78%) and reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%) of the purified viruses.
• the accompanying Examples are offered as illustrative as a partial scope and particular embodiments of the disclosure and are not meant to be limiting of the scope of the disclosure.
• a number of biological ligands targeting AAV are known to date, including transmembrane receptor proteins and engineered proteins. Tissue targeting and cell access by AAVs is mediated by attachment factors, namely glycan moieties (e.g., sucrose octasulfate, sialic acid, and galactose), also known as ‘primary receptors’, which feature promiscuous low-affinity capsid binding and whose role is to accumulate AAV at the cell surface; and cell surface receptors that specifically interact with AAV and whose binding is required to initiate viral cell entry.
• glycan moieties e.g., sucrose octasulfate, sialic acid, and galactose
• primary receptors also known as ‘primary receptors’, which feature promiscuous low-affin
• AAV2 oligosaccharide heparin
• AAV-DJ fondaparinux
• AAV receptor also known as KIAA0319L
• single chain camelid antibody fragments and small protein scaffolds have been developed as affinity ligands for purifying AAVs from recombinant cell lysates, including serotype-agnostic AAVX, AAV8- and AAV9-targeted CSAL8 and CSAL9 developed by ThermoFisher; serotype-agnostic AVB by Cytiva; and AVIPure ® AAV2, AAV8, and AAV9 for the corresponding serotypes.
• Representative complexes formed by the selected peptides on the target serotypes are shown in FIG. 1, while the values of binding energy and the corresponding dissociation constant (KD,in silico) are listed in Table 5; finally, detailed results of A1, A4, and A10 docking on the target AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 are reported in FIG.6.
• the shortest A20-mimetics (A6 – A9) showed the ability to form multiple low-affinity binding poses on the various serotypes that do not overlap with the binding sites of either A20 or AAVR.
• AAV capsids present multiple sites that are highly conserved across serotypes and “ligandable” (i.e., and whose physicochemical features – namely, pocket surface and volume as well as balance of electrostatic, hydrophobic and hydrogen bond-forming residues – are suitable to accommodate peptide ligands); since the target regions of AAVR and A20 are included in the list of ligandable sites, it did not surprise that A20-mimetic peptides interact with multiple sites on all serotypes.
• a milder binding strength does not necessarily translate into weaker binding; the peptide density on the surface of the resin is sufficient to form multiple interactions with a single capsid, wherein multiple affinity interactions with modest binding energy are synergized into a strong avidity-like binding that efficient AAV capture (note: the values of peptide density on the resin ( ⁇ 0.12 – 0.15 mmol per gram), the resin’s specific surface ( ⁇ 30 m 2 /g), and the projection area of the triangular unit formed by 3 VPs on the icosahedral capsid ( ⁇ 81 nm 2 ), in fact, suggest that up to 30 peptides are displayed on pore surface that is impacted by a single capsid, enabling the formation of 3 - 5 VP:peptide interactions per bound capsid).
• A20-mimetics A1 – A4 and AAVR-mimetics A10 m 7*+ N ⁇ R ⁇ UR _R]aRZPR_ cV ⁇ U ⁇ UR _ ⁇ [ZTR_ ⁇ bN ⁇ VN ⁇ V[Z VZ OVZQVZT RZR ⁇ Te #ss b > 3 kcal/mol, ⁇ NZ_XN ⁇ VZT VZ N s@D > 150-fold increase), and thus the highest likelihood of releasing the bound capsids under the desired conditions.
• the mechanism of VP:peptide dissociation portrayed by the MD simulations is a combined results of the variation in pH and ionic strength. Contrary to what generally observed with peptide ligands, the Coulombic interactions formed by A20 and its mimetics A1 – A4 provided a rather minor contribution (11-15%) to the binding energy at pH 7.4: the only interactions found were formed by cationic Lys in A1 and A2 with Asp514 and Asp 711 on AAV1, Asp269 and Asp 711 on AAV2, Asp704 on AAV5, Asp268 on AAV6, Asp270 on AAV8, and Asp231 on AAV9; conversely, A3 and A4 do not contain ionizable residues (except NCSU-2023-105-02 NCSU-41892.601 3199.0019WO His, which is neutral at pH 7.4); finally, the triplet (DDD) of A10 – A12 only targeted Lys508 on AAV1 and AAV2, Lys501 on AAV5, Lys
• Example 2 Evaluation of AAV binding by the designed peptide ligands in non-competitive mode.
• the results of molecular docking and dynamics of designed peptides on multiple serotypes NCSU-2023-105-02 NCSU-41892.601 3199.0019WO yielded a shortlist of sequences – namely, A20-mimetic peptides CYIHFSGYTNYNPSLKSC (A1; SEQ ID NO: 1), CYVHFSGYSNYSPSC (A3; SEQ ID NO: 3), CYGHFSGYGNYGPC (A4; SEQ ID NO: 4), and CYIHFSGYTNYNPC (A6; SEQ ID NO: 6), and AAVR-mimetic peptides CVIDGSQSTDDDKIC (A10; SEQ ID NO: 10) and CDSQSTDDDKIC (A12; SEQ ID NO: 12) – to be evaluated in dynamic mode against target serotypes AAV1, AAV2, AAV
• serotypes 1 and 2 target skeletal, muscle and cardiac cells, and are currently utilized in clinical trials against heart failure, Pompe disease, Hemophilia B, and AAT deficiency; serotypes 1, 2, 5, 8, 9 target the cells in the central nervous system, especially neurons, and are currently being tested in clinical trials against Alzheimer, Canavan, and Parkinson diseases; finally, serotypes 6 targets epithelial, skeletal cells, and hepatocytes, while serotype 8 targets cardiac cells, skeletal cells, hepatocytes.
• affinity resins marketed as serotype-agnostic show excellent binding of AAV1, AAV2, AAV5, and AAV6, but may struggle to capture AAV8 and AAV9, and dedicated adsorbents for their purification have been developed.
• the model AAVs adopted in the present disclosure while only representing half of the wild serotypes, provide a broad coverage of the AAV atlas and thus adequate evaluation of the AAV-targeting activity of the designed sequences.
• the peptides were conjugated on Toyopearl NH2-750F resin, whose large pore diameter (> 100 nm) and small particle size ensures efficient AAV transport into and binding onto the adsorbent pores.
• the feedstocks were formulated as pure AAVs at ⁇ 5.0 ⁇ 10 11 - 5.0 ⁇ 10 12 vp/mL in 10 mM Bis-Tris buffer at pH 7.0 and loaded at the ratio of ⁇ 10 13 vp per mL of resin (the expected to be the average binding capacity of the resins).
• the bound AAVs were eluted from the peptide-Toyopearl resins under the same conditions adopted for peptide design – namely, 1 M MgCl2 in 10 mM Bis-Tris buffer at pH 6.0 – whereas a strong acidic buffer (i.e., 200 mM MgCl2 in 200 mM citrate buffer at pH 2.2 and PBS at pH 2.0, respectively, as recommended by the manufacturers) was used for elution from the POROSTM CaptureSelectTM AAVX and AVB Sepharose HP resins used as reference adsorbents.
• a strong acidic buffer i.e., 200 mM MgCl2 in 200 mM citrate buffer at pH 2.2 and PBS at pH 2.0, respectively, as recommended by the manufacturers
• peptides A1 and A4 afforded excellent binding and gentle release of AAV1 and AAV6: the values of product yield were respectively 66.2 and 54.1% with A1-Toyopearl resin, which performed comparably to AAVX POROSTM resin (74.8 and 67.4%) and outperformed AVB Sepharose (55.5 and 15.0%); and 43.9% and 49.6% with A4-Toyopearl resin.
• A1-Toyopearl resin which performed comparably to AAVX POROSTM resin (74.8 and 67.4%) and outperformed AVB Sepharose (55.5 and 15.0%); and 43.9% and 49.6% with A4-Toyopearl resin.
• Both serotypes belong to Clade A, which is closely related to Clade B, to which AAV2 belongs: AAV1 and AAV6 feature, in fact, a 91-92% structural homology with AAV2 (note: sequence homology, however, is 81-83%).
• NCSU-2023-105-02 NCSU-41892.601 3199.0019WO A10 returned a remarkable yield of 66.5%, comparable with that of AAVX POROSTM resin (69.7%) and significantly higher than that of AVB Sepharose resin (3.5%).
• the capture of AAV5 proved the most challenging: the most unique among all AAVs, AAV5 exhibits a poor structural (58-71%) and sequence (58-79%) homology with the other serotypes evaluated in the present disclosure. It is therefore remarkable that peptide A1 captured (0.2% loss) and released it efficiently (41.6% yield).
• Three general conclusions can be drawn from the experimental evaluation of the in silico-selected peptides.
• peptides A3, A6, and A12 behaved differently with different serotypes, affording excellent capture and yield of AAV2 and AAV6, but high product loss and consequently low yield of AAV1, AAV5, AAV8, and AAV9.
• the low capture of these serotypes is matched by the in silico results, which predicted a low binding strength of these peptides at pH 7.4 (K D > 3.510 -5 M); conversely, a higher binding strength (K D ⁇ 7.510 -7 M – 2.810 -6 M) was predicted for AAV2 and AAV6, suggesting that, while moderate affinity is still desirable, binding strength must be above a minimum threshold to ensure sufficient product capture.
• the SEC and SXC results demonstrate the high purity of the AAV2 eluted from the peptide-based adsorbents.
• the comparative analysis of the SEC chromatograms returns global values of impurity decrease of 150-fold for A1-Toyopearl resin, 730-fold for A4-Toyopearl resin, and 550-fold for A10-Toyopearl resin.
• Similar results were provided by the SXC chromatograms, confirming that peptide-based adsorbents deliver eluates whose purity is comparable to that afforded by the affinity adsorbents utilized in the gene therapy industry.
• the Sf9 harvest features a significantly higher HCP titer (0.3 vs. 1.1 mg/mL, respectively), which motivates why the values of HCP LRV are lower than those reported in FIG. 3A. Nonetheless, the concentration of residual HCPs in the eluates from the peptide-based NQ_[ ⁇ ORZ ⁇ _ cR ⁇ R P[Z_V_ ⁇ RZ ⁇ Xe ORX[c ,. uT(YA% VZ XVZR cV ⁇ U RXaN ⁇ R_ [S NSSVZV ⁇ e ⁇ R_VZ_ PUN ⁇ NP ⁇ R ⁇ V_ ⁇ VP of chromatographic processes for biotherapeutics.
• POROSTM CaptureSelectTM AAVX resin (note: the gene encapsidated in the target AAV2 encodes for green fluorescence protein (GFP), which NCSU-2023-105-02 NCSU-41892.601 3199.0019WO enables quantifying the transduction activity via fluorescence flow cytometry).
• the transduction activity – namely, the ability of a virus to effectively deliver its gene payload to the target cells – is a critical quality parameter of viral vectors and is significantly impacted by the process parameters.
• AAVs like most viral vectors, are prone to lose their activity in response to variations in buffer conductivity and pH used to control adsorption and elution during chromatographic purification.
• the DBC10% of (A1; SEQ ID NO: 1) CYIHFSGYTNYNPSLKS-, (A4; SEQ ID NO: 4) CYGHFSGYGNYGPC-, and (A10; SEQ ID NO: 10) CVIDGSQSTDDDKIC-Toyopearl resins were measured via frontal loading of a clarified lysate containing AAV2 at the titer of 2.51 ⁇ 10 12 vp/mL at the residence time (RT) of 3 min (note: the clarified lysate was adopted in lieu of a pure AAV2 solution to provide a realistic evaluation of the binding capacity of the resins, whose operation is intended for competitive conditions; the adopted RT is recommended for POROSTM AAVX and AVB Sepharose resins and was therefore adopted to ensure comparability).
• the binding capacity and selectivity of an affinity resin can decrease over time due to several factors, such as chemical degradation, physical damage, and fouling, leading to a loss of product yield and purity as well as additional costs related to the replacement and validation of the adsorbent.
• Protein A-based resins for antibody purification whose lifetime has now reached 150 – 200 cycles with intermediate caustic cleaning in place
• the commercial affinity resins for AAV purification cannot withstand harsh alkaline treatment and rapidly lose their binding capacity, mandating frequent column replacement.
• the reusability of the peptide- based adsorbents to withstand 20 cycles of AAV2 purification followed by regeneration and cleaning in place was evaluated.
• embodiments of the present disclosure provide an ensemble of small peptide affinity ligands designed to transform AAV purification as they provide (i) selective as well as flexible product capture, being serotype-agnostic and applicable to both HEK293 and Sf9 fluids; (ii) gentle elution, allowing product release under near-physiological pH; and (iii) robust reusability, maintaining a high binding capacity over multiple purification cycles.
• the proposed adsorbents are undoubtedly more scalable and affordable, and they leverage the ability to mass manufacture GMP-quality peptides at relatively low cost ( ⁇ US$8 per gram per amino acid residue, when manufactured at > 10 kg scale per year).
• Peptides were cyclized to enhance their binding affinity and selectivity by reducing the entropic penalty of the binding energy. Additionally, single amino acid mutations were introduced in the A20-mimetic peptides to expand their targeting beyond AAV2, although that did not manage to reach the pan-selective biorecognition of their AAVR-mimetic counterparts.
• Adeno-associated viruses have emerged as a central family of vectors in the realm of gene delivery, providing therapeutic options to diseases once deemed incurable. At the same time, they necessitate efficient and affordable purification methods that can be platformed to serve all AAV serotypes.
• Embodiments of the present disclosure address these challenges by exploring the potential of peptide ligands discovered for serotype- agnostic AAV purification via affinity chromatography.
• Previous studies reveal a pH-dependent affinity interaction: AAV2, AAV3, AAV6, AAV9, and AAVrh.10 are effectively captured at neutral pH, while binding AAV1, AAV5, AAV7, and AAV8 is stronger in acidic environment (pH 5).
• AAV binding to cell-displayed AAVR varies among serotypes in a pH-dependent fashion. These differences, rooted in the amino acid sequence of the virion proteins (VPs), are manifested in the structural and functional features unique to each serotype, and govern AAV’s response to the environment, tissue tropism, and cell interaction. For example, the N-terminus of VP1 undergoes a reversible pH-induced unfolding and refolding process, while the N-termini of VP3 and VP2 are reversibly externalized as the pH is lowered from 7.4 to 5.
• All feedstocks were adjusted to an AAV titer of ⁇ 5 ⁇ 10 12 vp/mL, which resembles the concentration of cell lysates found in the gene therapy industry, and loaded on the resins at ⁇ 5 ⁇ 10 13 vp per mL of resin.
• the AAVs bound at neutral pH were eluted from the peptide-functionalized chromatographic resins at pH 6.5 (400 mM MgCl2 in 10 mM Bis-Tris buffer), whereas the AAVs bound at pH 5 were initially eluted at pH 7 (20 mM NaCl and 2 mM MgCl2 in 10 mM Bis-Tris buffer) followed by a second elution step at pH 6.5.
• elution was conducted in two steps, the first at low conductivity and pH 7 and the second in 0.4 M MgCl2 and pH 6.5.
• the first elution step afforded appreciable values of yield, yet insufficient in the context of bioprocessing, necessitating the second elution step to achieve global yields up to 90% (Table 10).
• the differential release of AAVs in buffers with different conductivities and pH results from the heterogeneity of the capsids (e.g., VP ratio and structural arrangement, identity of genetic payload, etc.). This suggests the applicability of peptide ligands not only for purifying, but also for fractionating capsids and isolating the population with higher transduction activity.
• AAV1 and AAV6 despite belonging to clade A and their VPs sharing 99% sequence and structural homology, require different binding pH. A similar effect was observed with AAV8 and AAVrh.10, which belong to clade E and share 93% sequence-based and 97% structural homology. This indicates, as observed above, that the peptide ligands target highly defined binding sites on the capsid surface, which require optimal display for effective AAV capture.
• the chromatograms of AAV purification collated in FIGS.18A-18D provide a visual representation of the separation dynamics, offering insights into the efficiency and effectiveness of the purification process.
• the flow-through segment of the various chromatograms indicate different adsorption dynamics, likely rooted in the material properties of the chromatographic substrates (i.e.% ⁇ [XeYR ⁇ UeX&YR ⁇ UNP ⁇ eXN ⁇ R ORNQ_ [S /.
• the AAVR-mimetic ligands consistently afforded yields of 50-to-60% and 400-to-500-fold reduction of HCPs, on par with AAVX and AVB affinity resins (FIGS. 12A-12D).
• the A20- mimetic peptides afforded comparable values of yields of serotypes 3, 6, and rh.10, but lower values of purity (200-to-300-fold reduction of HCPs).
• Ligand A10 emerged as a top performing ligand by providing a consistently high purification performance. Particularly remarkable was the AAV9 purification by A10, which outperformed all other ligands in terms of product yield and purity (72.8% and 230-fold reduction of HCPs).
• FIGS.14A-14D This is confirmed by the values of capsid yields and HEK293 HCP removal summarized in FIGS.14A-14D, which stood respectively at 50-60% and 200-to-500-fold for serotypes 1, 5, and 8 – placing the purification performance of peptide ligands on par with that of AAVX (note: AVB failed to bind effectively serotypes 5, 6, 7, 9, for which the yield was limited to 15%).
• AAV9 peptide A10 afforded remarkable results with AAV7, outperforming all other ligand with a yield of 73% and a 250-fold reduction of HCPs.
• FIGS.20A-20H and FIGS.21A-21D confirm these finding by providing an at-a-glance demonstration of capsid concentration and clearance of all soluble contaminants across the entire spectrum of composition and size.
• Example 7 [0190] Optimizing AAV recovery and purity using AAVR-mimetic ligand A10. The first set of purification tests was conducted by loading the resin to a ratio of 10 13 vp per mL of resin (due to limited availability of feedstock). Prior studies, however, indicated that higher resin loads and the combination of loading in up-flow and elution in down-flow is conducive to higher values of yield.
• the purification of all serotypes was therefore repeated using the lead ligand A10 by increasing the load to approach the dynamic binding capacity (DBC10% ⁇ 1 – 3 ⁇ 10 14 vp per mL of resin).
• A10 was demonstrated to capture preferentially assembled AAV particles and release an early elution fraction enriched in transgene-loaded capsids, thus affording a concomitant enrichment and purification of transducing virions. Accordingly, the elution of bound viruses was conducted in two steps.

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