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US-12624367-B2 - AAV6 variants

US12624367B2US 12624367 B2US12624367 B2US 12624367B2US-12624367-B2

Abstract

This disclosure relates to variant AAV6 particles engineered to escape host neutralizing antibodies but retain or improve transduction efficiency, and their use as gene delivery vehicles.

Inventors

  • Mavis Agbandje-McKenna
  • Antonette D. Bennett
  • Robert McKenna

Assignees

  • UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INCORPORATED

Dates

Publication Date
20260512
Application Date
20190329

Claims (19)

  1. 1 . A method comprising: administering to a subject a recombinant AAV6 (rAAV6) particle comprising an AAV6 capsid protein having a single substitution consisting of a substituted amino acid at position 531, wherein the substituted amino acid at position 531 is glutamic acid (E), aspartic acid (D), histidine (H), tyrosine (Y), methionine (M), or leucine (L), wherein the rAAV6 particle comprises a gene of interest, wherein the subject is seropositive for an adeno-associated virus (AAV) capsid protein of serotype 6 (AAV6) antigen comprising lysine 531, and wherein the subject is not seropositive for the capsid protein comprising the substituted amino acid.
  2. 2 . The method of claim 1 , wherein the subject is human.
  3. 3 . The method of claim 1 , wherein the method further comprises determining the subject has anti-AAV6 antibodies that bind to the AAV6 antigen comprising lysine 531.
  4. 4 . The method of claim 3 , wherein determining the subject has anti-AAV6 antibodies comprises: incubating a sample of serum obtained from the subject with one or more AAV6 capsid antigens, and measuring the amount of anti-AAV6 antibodies bound to the one or more AAV6 capsid antigens, wherein the one or more AAV6 capsid antigens are bound to a solid support.
  5. 5 . The method of claim 4 , wherein the AAV6 capsid antigen is a protein or peptide, wherein the protein or peptide has a sequence that is comprised in AAV6 VP1, VP2 or VP3 capsid protein.
  6. 6 . The method of claim 1 , wherein the subject was previously exposed to a rAAV particle and/or had been previously administered a rAAV particle.
  7. 7 . The method of claim 1 , wherein the gene of interest encodes a therapeutic protein, an antibody, a peptibody, a growth factor, a clotting factor, a hormone, a membrane protein, a cytokine, a chemokine, an activating or inhibitory peptide acting on cell surface receptors or ion channels, a cell-permeant peptide targeting intracellular processes, a thrombolytic, an enzyme, a bone morphogenetic protein, a differentiation factor, a nuclease or other protein used for gene editing, an Fc-fusion protein, an anticoagulant, a nuclease, guide RNA or other nucleic acid or protein for gene editing.
  8. 8 . The method of claim 1 , wherein the rAAV6 particle is administered to the subject subcutaneously, intraocularly, intravitreally, subretinally, parenterally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracisternally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection.
  9. 9 . A method comprising: administering to a subject a subsequent rAAV6 particle, wherein the subsequent rAAV6 particle comprises an AAV6 capsid protein having a single substitution consisting of a substituted amino acid at position 531, wherein the substituted amino acid at position 531 is glutamic acid (E), aspartic acid (D), histidine (H), tyrosine (Y), methionine (M), or leucine (L), wherein the subject had previously received a recombinant AAV (rAAV) particle comprising an AAV6 capsid protein that comprises lysine at position 531, wherein the subsequent rAAV6 particle comprises a gene of interest, wherein the subject is seropositive for an adeno-associated virus (AAV) capsid protein of serotype 6 (AAV6) antigen comprising lysine 531 and the subject is not seropositive for the capsid protein comprising the substituted amino acid.
  10. 10 . The method of claim 9 , wherein the subject is human.
  11. 11 . The method of claim 9 , wherein the method further comprises determining the subject has anti-AAV6 antibodies that bind to the AAV6 antigen comprising lysine 531.
  12. 12 . The method of claim 11 , wherein determining the subject has anti-AAV6 antibodies comprises: incubating a sample of serum obtained from the subject with one or more AAV6 capsid antigens, and measuring the amount of anti-AAV6 antibodies bound to the one or more AAV6 capsid antigens, wherein the one or more AAV6 capsid antigens are bound to a solid support.
  13. 13 . The method of claim 12 , wherein the AAV6 capsid antigen is a protein or peptide, wherein the protein or peptide has a sequence that is comprised in an AAV6 VP1, VP2 or VP3 capsid protein.
  14. 14 . The method of claim 9 , wherein the gene of interest encodes a therapeutic protein, an antibody, a peptibody, a growth factor, a clotting factor, a hormone, a membrane protein, a cytokine, a chemokine, an activating or inhibitory peptide acting on cell surface receptors or ion channels, a cell-permeant peptide targeting intracellular processes, a thrombolytic, an enzyme, a bone morphogenetic protein, a differentiation factor, a nuclease or other protein used for gene editing, an Fc-fusion protein, an anticoagulant, a nuclease, guide RNA or other nucleic acid or protein for gene editing.
  15. 15 . A recombinant AAV6 (rAAV6) capsid protein comprising one of the following amino acids at position 531 according to SEQ ID NO: 1: histidine (H), methionine (M), or leucine (L).
  16. 16 . The method of claim 1 , wherein the substituted amino acid at position 531 is glutamic acid, aspartic acid, histidine, or tyrosine.
  17. 17 . The method of claim 1 , wherein the substituted amino acid at position 531 is negatively charged.
  18. 18 . The method of claim 9 , wherein the substituted amino acid at position 531 is glutamic acid, aspartic acid, histidine, or tyrosine.
  19. 19 . The method of claim 9 , wherein the substituted amino acid at position 531 is negatively charged.

Description

RELATED APPLICATIONS This application is national stage filing under 35 U.S.C. § 371 of international application number PCT/US2019/025045, filed Mar. 29, 2019, which claims the benefit of U.S. provisional application No. 62/650,213, filed Mar. 29, 2018, each of which is incorporated herein by reference in its entirety. GOVERNMENT SUPPORT This invention was made with government support under R01 GM082946 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 28, 2020, is named U119670054US01-SEQ-PRW.txt, and is 71 bytes in size. BACKGROUND Adeno-associated virus (AAV) of serotype 6 are being developed as vectors for the treatment of various diseases, disorders, and conditions, especially those targeting the lung and skeletal muscle. However, pre-existing antibodies for AAV6 present a significant limitation to achieving optimal efficacy for the AAV6 gene delivery system. SUMMARY The present disclosure is based, at least in part, on the identification of an AAV6 capsid residue that is important for interactions with antibodies that recognize AAV6 capsids and which would result in reduced transduction and gene expression in target cells when AAV6 particles are administered to a subject. The inventors of this disclosure identified amino acid K531 (VP1 numbering) common to all three capsid proteins Vp1, VP2, and VP3, as a determinant of monoclonal antibody ADK6 recognition of AAV6 by determining the structure of AAV6-ADK6 complex using cyro-electron microscopy (cryo-EM). The footprint of the interaction between AAV6 and ADK6 was also confirmed by cell-based assays. Using this footprint, the inventors of the present disclosure developed rAAV particles with substituted amino acids residues in this footprint (e.g., at residue K531) that are able to evade neutralizing antibodies when administered to subjects. One advantage of an neutralizing antibody-evading AAV particle is that fewer particles are needed to be administered to a subject for delivering a gene compared to an AAV particle that do not evade neutralizing antibodies. Accordingly, the present disclosure provides a method with which to deliver one or more genes of interest to target cells of a subject in which it is anticipated that AAV particles used to comprise and deliver the one or more genes of interest will be recognized by neutralizing antibodies. In some aspects, provided herein is a method comprising administering to a subject that is seropositive for AAV6 a recombinant rAAV6 particle that is able to evade anti-AAV6 antibodies. In some embodiments, a variant recombinant rAAV6 particle that is able to evade anti-AAV6 antibodies comprises a capsid protein comprising a substituted amino acid at position 531, and the rAAV particle comprises a gene of interest. In some embodiments, the subject has preexisting anti-AAV6 antibodies. In some embodiments, a method of administering to a subject a rAAV6 particle comprising a substitution at position 531K of one or more of its capsid proteins further comprises determining whether the subject is seropositive for AAV6. In some embodiments, determining whether the subject is seropositive for AAV6 comprises determining whether a subject has anti-AAV6 antibodies. In some embodiments, determining whether a subject has anti-AAV6 antibodies comprises incubating a sample of serum obtained from the subject with one or more AAV6 capsid antigens, and measuring the amount of anti-AAV6 antibodies bound to the one or more AAV6 capsid antigens. In some embodiments, the one or more AAV6 capsid antigens are bound to a solid support. In some embodiments, the antibodies in a subjects serum sample are bound to a solid support. In some embodiments, AAV6 capsid antigens is a protein or peptide. In some embodiments, proteins or peptides that are AAV6 capsid antigens have a sequence that is comprised in AAV6 VP1, VP2, or VP3 capsid proteins. In some embodiments, a variant rAAV6 particle as disclosed herein comprises a capsid protein comprising amino acid sequence of a serotype other than serotype 6. In some embodiments, the substituted amino acid at position 531 is glutamic acid, aspartic acid, histidine, tyrosine, arginine, methionine, or leucine. In some embodiments, the substituted amino acid at position 531 is glutamic acid, aspartic acid, histidine, tyrosine, or arginine. In some embodiments, the substituted amino acids at position 531 of a variant rAAV6 particle is negatively charged. In some embodiments, the substituted amino acids at position 531 of a variant rAAV6 particle that is negatively charged is glutamic acid or aspartic acid. In some embodiments, the subject was previously exposed to a rAAV particle. In some embodime