Polypeptide assemblies and methods for the production thereof

Patent metadata

Field	Value
Publication number	US-11028383-B2
Application number	US-201916676253-A
Country	US
Kind code	B2
Filing date	Nov 6, 2019
Priority date	Feb 27, 2015
Publication date	Jun 8, 2021
Grant date	Jun 8, 2021

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”); and
(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 ESCRT machinery to the site of budding by binding directly or indirectly to one or more ESCRT or ESCRT-associated proteins (“L domain”);
wherein one or more protein in the multimeric assembly comprises one or more polypeptide domain that is capable of interacting with a lipid bilayer (“M domain”), wherein the one or more M domains are selected from the group consisting of SEQ ID NOs: 52-151, 283-297, and 299-300 and wherein (i) at least one protein in each oligomeric substructure comprises one or more M domain, or (ii) wherein each protein comprises one or more 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 the one or more O interfaces of different proteins in the oligomeric substructure 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.

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

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

5. The multimeric assembly of claim 1 , wherein the one or more L domains are capable of non-covalently interacting with one or more proteins in the ESCRT pathway.

6. The multimeric assembly of claim 1 , wherein the one or more L domains comprise a linear amino acid sequence motif selected from the group consisting of SEQ ID NOs: 152-197 or 305-306, or overlapping combinations thereof.

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

8. The multimeric assembly of claim 7 , further comprising a cargo interacting with the packaging moiety.

9. 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.

10. The multimeric assembly of claim 9 , further comprising one or more transmembrane protein or membrane-anchored protein embedded in the lipid bilayer.

11. The multimeric assembly of claim 10 , 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.

12. The multimeric assembly of claim 10 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.

13. A recombinant polypeptide comprising
(a) a polypeptide domain that is capable of interacting with a lipid bilayer (“M domain”), wherein the one or more M domains are selected from the group consisting of SEQ ID NOs: 52-151, 283-297, and 299-300;
(b) a polypeptide-polypeptide interface (“O interface”);
(c) a 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
(d) a packaging moiety;
wherein the M domain, the L domain, and the O interface are not each present in a single naturally occurring protein.

14. The recombinant polypeptide of claim 13 , wherein the M domain is capable of non-covalently interacting with a lipid bilayer.

15. The recombinant polypeptide of claim 13 , wherein the L domain is capable of non-covalently interacting with one or more proteins in the ESCRT machinery or proteins known to recruit the ESCRT machinery to the site of budding by binding to one or more ESCRT proteins directly or indirectly.

16. The recombinant polypeptide of claim 13 , wherein the L domains comprise a linear amino acid sequence motif selected from the group consisting of SEQ ID NOs: 152-197 or 305-306, or overlapping combinations thereof.

17. A recombinant polypeptide comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 304, wherein the polypeptide includes at least 1, 2, 3, 4, 5, or more amino acid substitutions compared to SEQ NO: 21.

18. A recombinant nucleic acid encoding the recombinant polypeptide of claim 13 .

19. A recombinant expression vector comprising the recombinant nucleic acid of claim 18 operatively linked to a promoter.

20. A recombinant host cell comprising the recombinant expression vector of claim 19 .

Assignees

• Univ Washington
• Univ Utah
• 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

• 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

• A61K9/127

  Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant · CPC title

• C07K2319/06

  containing a lysosomal/endosomal localisation signal · CPC title

• C07K2319/735

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

• C07K14/435

  from animals; from humans · CPC title