Protein enriched microvesicles and methods of making and using the same

Patent metadata

Field	Value
Publication number	US-10793828-B2
Application number	US-201715415754-A
Country	US
Kind code	B2
Filing date	Jan 25, 2017
Priority date	Jun 11, 2013
Publication date	Oct 6, 2020
Grant date	Oct 6, 2020

Abstract

Protein enriched micro-vesicles and methods of making and using the same are provided. Aspects of the methods include maintaining a cell having a membrane-associated protein comprising a first dimerization domain and a target protein having a second dimerization domain under conditions sufficient to produce a micro-vesicle from the cell, wherein the micro-vesicle includes the target protein. Also provided are cells, reagents and kits that find use in making the micro-vesicles, as well as methods of using the micro-vesicles, e.g., in research and therapeutic applications.

First claim

What is claimed is:

1. A micro-vesicle comprising:
a membrane-associated chimeric protein comprising a first dimerization domain; and
a target chimeric protein that is not membrane-associated comprising a second dimerization domain,
wherein the first and second dimerization domains are specifically bound to each other in a dimerized complex or the first and second dimerization domains are bound to each other by a single dimerization mediator.

2. The micro-vesicle according to claim 1 , wherein the first and second dimerization domains are specifically bound to each other in the dimerized complex.

3. The micro-vesicle according to claim 1 , wherein first and second dimerization domains are bound to each other by a single dimerization mediator.

4. The micro-vesicle according to claim 3 , wherein the dimerization mediator is a modifiable dimerization mediator.

5. The micro-vesicle according to claim 1 , wherein the micro-vesicle further comprises a micro-vesicle inducer.

6. The micro-vesicle according to claim 5 , wherein the micro-vesicle inducer comprises a viral membrane fusion protein.

7. The micro-vesicle according to claim 6 , wherein the viral membrane fusion protein is VSV-G.

8. The micro-vesicle according to claim 1 , wherein the membrane-associated chimeric protein is selected from the group consisting of a myristoylated protein, a farnesylated protein, a membrane anchor protein, a transmembrane protein and membrane lipid protein.

9. The micro-vesicle according to claim 1 , wherein the first and second dimerization domains are heterodimeric.

10. The micro-vesicle according to claim 1 , wherein the first and second dimerization domains are homodimeric.

11. The micro-vesicle according to claim 1 , wherein the first and second dimerization domains are selected from DmrA and DmrC domains, DmrB domains, DmrD domains, dimerization domains of the dihydrofolate reductase system, dimerization domains of TAg and p53, and dimerization domains of Src Homology 2 and a phosphotyrosine-binding protein.

12. The micro-vesicle according to claim 1 , wherein the micro-vesicle further comprises a second target protein comprising a third dimerization domain.

13. The micro-vesicle according to claim 1 , wherein the target chimeric protein is a research protein selected from the group consisting of: a genomic modification protein, a transcription modulator of an inducible expression system, a member of a signal production system, a hormone, a prohormone, a protease, an enzyme activity modulator, a peptide aptamer, an antibody, a modulator of a protein-protein interaction, and a cellular reprogramming protein.

14. The micro-vesicle according to claim 13 , wherein the research protein is a genomic modification protein.

15. The micro-vesicle according to claim 14 , wherein the genomic modification protein is selected from the group consisting of: a CRE recombinase, a meganuclease, a Zinc-finger nuclease, a clustered regularly interspaced short palindromic repeats/CRISPER associated protein 9 (CRISPR/Cas-9) nuclease and a transcription activator-like (TAL) effector nuclease.

16. The micro-vesicle according to claim 1 , wherein the membrane associated with the membrane-associated protein is separate from a cell.

Assignees

• Takara Bio Usa Inc

Inventors

• Haugwitz Michael
• Quinn Thomas Patrick
• Farmer Andrew Alan
• Fernández Montserrat Morell

Classifications

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title

• C12N2760/20222

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• C12N5/0602 Primary

  Vertebrate cells · CPC title

• C12N9/1241

  Nucleotidyltransferases (2.7.7) · CPC title