Polypeptide assemblies and methods for the production thereof

Patent metadata

Field	Value
Publication number	US-12545903-B2
Application number	US-202117336889-A
Country	US
Kind code	B2
Filing date	Jun 2, 2021
Priority date	Feb 27, 2015
Publication date	Feb 10, 2026
Grant date	Feb 10, 2026

Abstract

The application discloses multimeric assemblies including multiple oligomeric substructures, where each oligomeric substructure includes multiple proteins that self-interact around at least one axis of rotational symmetry, where each protein includes one or more polypeptide-polypeptide interface (“O interface”); and one or more polypeptide domain that is capable of effecting membrane scission and release of an enveloped multimeric assembly from a cell by recruiting the ESCRT machinery to the site of budding by binding to one or more proteins in the eukaryotic ESCRT complex (“L domain”); and where the multimeric assembly includes one or more subunits comprising one or more polypeptide domain that is capable of interacting with a lipid bilayer (“M domain”), as well as membrane-enveloped versions of the multimeric assemblies.

First claim

We claim:

1 . A multimeric assembly, comprising a plurality of oligomeric substructures, wherein each oligomeric substructure comprises a plurality of proteins that self-interact around at least one axis of rotational symmetry, wherein each protein comprises:
(a) one or more polypeptide-polypeptide interface (“O interface”); (b) one or more polypeptide domain that is capable of effecting membrane scission and release of an enveloped multimeric assembly from a cell by recruiting the Endosomal Sorting Complex Required for Transport (ESCRT) machinery to the site of budding by binding directly or indirectly to one or more ESCRT or ESCRT-associated proteins (“L domain”), wherein the one or more L domains comprise a linear amino acid sequence motif selected from the group consisting of SEQ ID NOS: 152-466, 168-497 or 305-306; wherein one or more proteins in the multimeric assembly comprises one or more polypeptide domain that is capable of interacting with a lipid bilayer (“M domain”); wherein the M domain, L domain, and O interface are not each present in a single naturally occurring protein, wherein the plurality of oligomeric substructures interact with each other at the one or more O interfaces.

2 . The multimeric assembly of claim 1 , wherein each oligomeric substructure comprises one or more M domain, or wherein each protein comprises one or more M domain.

3 . The multimeric assembly of claim 1 , wherein the one or more O interfaces orient the plurality of oligomeric substructures such that their symmetry axes are aligned with symmetry axes of the same kind in a designated mathematical symmetry group.

4 . The multimeric assembly of claim 1 , wherein the one or more O interfaces of each oligomeric substructure are identical.

5 . The multimeric assembly of claim 1 , wherein the one or more M domains are capable of non-covalently interacting with a lipid bilayer.

6 . The multimeric assembly of claim 1 , wherein the one or more M domains comprise a polypeptide having an acylation motif (including but not limited to N-terminal myristoylation motifs, palmitoylation motifs, farnesylation motifs, and geranylgeranylation motifs), a polar headgroup-binding domain, envelope proteins of enveloped viruses, membrane protein transporters, membrane protein channels, B-cell receptors, T-cell receptors, transmembrane antigens of human pathogens, growth factors receptors, G-protein coupled receptors (GPCRs), complement regulatory proteins including but not limited to CD55, CD59, and transmembrane protein domains.

7 . The multimeric assembly of claim 1 , wherein the one or more M domains are selected from the group consisting of SEQ ID NOS: 52-151 and 280-300.

8 . The multimeric assembly of claim 1 , wherein the one or more O interfaces are non-naturally occurring.

9 . The multimeric assembly of claim 1 , Wherein the one or more O interfaces comprise or consist of the amino acid sequence of SEQ ID NO: 1-5, 7-9, 20, or 304.

10 . The multimeric assembly of claim 1 , wherein the one or more O interfaces comprise or consist of the amino acid sequence of SEQ ID NO: 20 or 304.

11 . The multimeric assembly of claim 1 , further comprising a packaging moiety in one or more of the proteins.

12 . The multimeric assembly of claim 1 , further comprising a cargo interacting with the packaging moiety.

13 . The multimeric assembly of claim 12 , wherein the cargo is selected from the group consisting of proteins, nucleic acids, and small organic compounds.

14 . The multimeric assembly of claim 12 , wherein the cargo comprises a polypeptide or polynucleotide selected from the group consisting of SEQ ID NOS: 202-219.

15 . The multimeric assembly of claim 1 , further comprising a lipid bilayer enveloping the multimeric assembly, wherein one or more of the M domains is bound to the lipid bilayer.

16 . The multimeric assembly of claim 15 , further comprising one or more transmembrane protein or membrane-anchored protein embedded in the lipid bilayer.

17 . The multimeric assembly of claim 16 , wherein the transmembrane or membrane-anchored protein is selected from the group consisting of the envelope proteins of enveloped viruses, membrane protein transporters, membrane protein channels, B-cell receptors, T-cell receptors, transmembrane antigens of human pathogens, growth factors receptors, G-protein coupled receptors (GPCRs), and complement regulatory proteins.

18 . The multimeric assembly of claim 15 , wherein the one or more transmembrane protein or membrane-anchored protein embedded in the lipid bilayer comprises one or more polypeptide selected from the group consisting of SEQ ID NOS: 307-315.

19 . The multimeric assembly of claim 1 , wherein the multimeric assembly comprises a cargo, wherein the cargo is not bound to the multimeric assembly.

20 . The multimeric assembly of claim 19 , wherein the cargo is a protein, nucleic acid, lipid, or small molecule.

21 . The multimeric assembly of claim 19 , Wherein the cargo comprises 2′,3′-cyclic GMP-AMP (cGAMP).

Assignees

• Univ Washington
• Univ Utah Res Found

Inventors

• King Neil
• Sundquist Wesley
• Votteler Joerg
• Hsia Yang
• Baker David
• Bale Jacob
• Lajoie Marc
• Butterfield Gabriel
• Gray Elizabeth
• Stetson Daniel

Classifications

• C12Y401/02014

  2-Dehydro-3-deoxy-phosphogluconate aldolase (4.1.2.14) · CPC title

• C07K2319/735

  containing a domain for self-assembly, e.g. a viral coat protein (includes phage display) · CPC title

• C07K2319/06

  containing a lysosomal/endosomal localisation signal · CPC title

• C07K2319/03

  containing a transmembrane segment · CPC title

• A61K9/14

  Particulate form, e.g. powders, {Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles (microspheres A61K9/16; microcapsules A61K9/50; nanocapsules, nanoparticles of the matrix type A61K9/51)} · CPC title