# US 10,945,954 B2 · ARRDC1-mediated microvesicles (ARMMS) and uses thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,945,954 B2
- Document ID: US10945954B2
- Status: Granted
- Kind: B2
- Application number: 15/669,720
- Filed: 2017-08-04
- Published: 2018-03-01
- Granted: 2021-03-16
- Earliest claimed: 2012-02-06
- Assignees: President and Fellows of Harvard College; The Board of Trustees of the Leland Stanford Junior University
- Applicants: President and Fellows of Harvard College; The Board of Trustees of the Leland Stanford Junior University
- Inventors: Quan Lu; Joseph F. Nabhan; Stanley N. Cohen
- Web page: https://patentgrep.com/patent/US10945954B2

## CPC

- A61K 9/107: Emulsions {; Emulsion preconcentrates; Micelles (composition of emulsions A61K47/00)}
- A61K 47/42: Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein (oligopeptides having up to five amino acids {A61K47/183}; polyamino acids A61K47/34)
- C07K 14/47: from mammals
- G01N 33/6893: {related to diseases not provided for elsewhere}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/70: containing domain for protein-protein interaction
- G01N 2333/47: Assays involving proteins of known structure or function as defined in the subgroups
- G01N 2333/726: G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention provide isolated arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicles (ARMMs). Methods for generating and for isolating ARMMs are also provided herein. ARMMs can be used to deliver agents, for example, nucleic acids (e.g., siRNAs, microRNAs, lincRNAs), proteins (e.g., transcription factors, chromatin modulators, kinases, phosphorylases, or recombinases), or small molecules to target cells in vitro and in vivo, and methods for such ARMM-mediated delivery are provided herein. Diagnostic and therapeutic methods using ARMMs are also described herein.

## Claims

### Claim 1

An arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM), comprising
(a) a lipid bilayer; and
(i) an ARRDC1 protein, or fragment thereof, associated with an enzyme; or
(ii) a TSG101 protein, or fragment thereof, associated with an enzyme.

### Claim 2 (depends on 1)

The microvesicle of claim 1, wherein the enzyme is selected from the group consisting of kinases, phosphatases, ligases, recombinases, chromatin modulators, reprogramming factors, proteases, RNA editing enzymes, and nucleases.

### Claim 3 (depends on 1)

The microvesicle of claim 1, wherein the enzyme is a nuclease.

### Claim 4 (depends on 3)

The microvesicle of claim 3, wherein the nuclease is a zinc finger nuclease.

### Claim 5 (depends on 1)

The microvesicle of claim 1, wherein the enzyme is covalently bound to the ARRDC1 protein or fragment thereof.

### Claim 6 (depends on 1)

The microvesicle of claim 1, wherein the enzyme is non-covalently bound to
(i) the ARRDC1 protein, or fragment thereof; or
(ii) the TSG101 protein, or fragment thereof.

### Claim 7 (depends on 1)

The microvesicle of claim 1, wherein the ARRDC1 protein fragment comprises an ARRDC1 PSAP domain.

### Claim 8 (depends on 1)

The microvesicle of claim 1, wherein the enzyme is covalently bound to the TSG101 protein or fragment thereof.

### Claim 9 (depends on 1)

The microvesicle of claim 1, wherein the microvesicle further comprises an integrin, a receptor tyrosine kinase, a G-protein coupled receptor, or a membrane-bound immunoglobulin.

### Claim 10 (depends on 1)

The microvesicle of claim 1, wherein the microvesicle further comprises a protein selected from the group consisting of AHCYL2, AIP, AKR7L, AP1B1, AP1M2, C8orf30A, CASK, CNNM4, CPM, CSNK1A1, CSNK1A1L, CSNK2A1, CXADR, EFCAB4B, EIF4B, ENAH, EPB41L5, ERBB2IP, EXOC1, EXOC2, EXOC7, EXOC8, FAM102A, FAM83F, FAM84B, FERMT2, FNBP1L, GALNT7, GRB7, GRB7, ILK-2, INPP5A, IPI:IPI00328587.4, IPI:IPI00719051.3, ITCH, KIAA0174, KIAA1244, KIAA1522, KIAA1598, KIF13B, LASP1, LNPEP, LRRC1, LSS, MAPK3, MARK2, MLPH, MON2, MTOR, MTSS1, MYO10, MYO18A, NF2, NOTCH2, OCLN, PARVA, PIK3R2, PKM2, PPCS, PPID, PRKAG1, PSMC3, PTPRD, PVRL2, QARS, RAD23B, RHPN2, SH3GL1, SNTB2, SPAG1, SPINT1, SRM, STARD10, STUB1, STX16, SYTL1, TLN2, TNS3, TRIM3, TUBB2C, TWF1, USPS, VAV2, VAV3, XPOT, YARS, ZDHHC5, and ZYX.

### Claim 11 (depends on 9)

The microvesicle of claim 9, wherein the microvesicle comprises an integrin selected from the group consisting of α1β1, α2β1, α4β1, α5β1, α6β1, αLβ2, αMβ2, αIIbβ3, αVβ3, αVβ5, αVβ6, and α6β4 integrins; a receptor tyrosine kinase chosen from the group consisting of an EGF receptor (ErbB family), insulin receptor, PDGF receptor, FGF receptor, VEGF receptor, HGF receptor, Trk receptor, Eph receptor, AXL receptor, LTK receptor, TIE receptor, ROR receptor, DDR receptor, RET receptor, KLG receptor, RYK receptor, and MuSK receptor; a G-protein coupled receptor chosen from the group consisting of a Rhodopsin-like receptor, Secretin receptor, metabotropic glutamate/pheromone receptor, cyclic AMP receptor, frizzled/smoothened receptor, CXCR4, CCR5, or beta-adrenergic receptor; and/or an exocyst protein chosen from EXOC7, EXOC8, EXOC1, and EXOC2.

### Claim 12 (depends on 1)

The microvesicle of claim 1, wherein the enzyme is bound to
(i) the ARRDC1 protein, or fragment thereof via a linker; or
(ii) the TSG101 protein, or fragment thereof via a linker.

### Claim 13 (depends on 12)

The microvesicle of claim 12, wherein the linker is a cleavable linker.

### Claim 14 (depends on 13)

The microvesicle of claim 13, wherein the cleavable linker comprises a protease recognition site or a UV-cleavable moiety.

### Claim 15

The microvesicle of 87, wherein the microvesicle diameter is from about 30 nm to about 500 nm.

### Claim 16 (depends on 1)

A method of delivering an enzyme to a target cell, the method comprising contacting the target cell with the microvesicle of claim 1.

### Claim 17 (depends on 16)

The method of claim 16, wherein the target cell is a human cell.

### Claim 18 (depends on 16)

The method of claim 16, wherein the target cell is a stem cell.

### Claim 19 (depends on 16)

The method of claim 16, wherein the target cell is a cell in a subject, and wherein the method comprises administering the microvesicle to the subject.

### Claim 20 (depends on 19)

The method of claim 19, wherein the subject is a human subject.