# US 10,945,955 B2 · Artificial exosome composition and related methods

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- Number: US 10,945,955 B2
- Document ID: US10945955B2
- Status: Granted
- Kind: B2
- Application number: 16/393,582
- Filed: 2019-04-24
- Published: 2019-11-14
- Granted: 2021-03-16
- Earliest claimed: 2018-04-25
- Assignees: University of Massachusetts
- Applicants: University of Massachusetts
- Inventors: Reka Agnes Haraszti; Anastasia Khvorova; Neil Aronin
- Web page: https://patentgrep.com/patent/US10945955B2

## CPC

- A61K 9/1272: comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543})
- A61K 9/1271: Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911})
- A61K 31/7115: Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
- A61K 31/7125: Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- A61K 38/1709: {from mammals}
- A61K 38/1719: {Muscle proteins, e.g. myosin or actin}
- A61K 38/1741: {alpha-Glycoproteins}
- A61K 47/28: Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
- A61K 47/542: {Carboxylic acids, e.g. a fatty acid or an amino acid}
- A61K 47/62: the modifying agent being a protein, peptide or polyamino acid
- A61K 47/6911: {the form being a liposome}
- A61K 47/6917: {the form being a lipoprotein vesicle, e.g. HDL or LDL proteins}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/86: Viral vectors
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/312: Phosphonates
- C12N 2310/313: Phosphorodithioates
- C12N 2310/3183: Diol linkers, e.g. glycols or propanediols
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2310/343: having patterns, e.g. ==--==--==--
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2710/10343: viral genome or elements thereof as genetic vector
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Novel artificial exosomes and methods for producing novel artificial exosomes are provided. Methods of delivering cargo molecules to a cell using artificial exosomes are also provided.

## Claims

### Claim 1

An artificial exosome comprising:
(a) one or more proteins comprising rab7, desmoplakin, alpha 2-HS glycoprotein (AHSG), or a mixture thereof, wherein each protein further comprises a lipid anchor; and
(b) a cardiolipin or a variant thereof,
wherein the artificial exosome is loaded with exogenously-derived, heterologous cargo selected from the group consisting of a peptide, a polypeptide, a nucleic acid, a virus, a small molecule, a fluorophore, and a combination thereof.

### Claim 2 (depends on 1)

The artificial exosome of claim 1, wherein the artificial exosome further comprises dioleoylphosphatidylcholine (DOPC) and cholesterol.

### Claim 3 (depends on 1)

The artificial exosome of claim 1, wherein:
the lipid anchor comprises one or more of a myristoylation, a palmitoylation, a prenylation, and a glypiation; and/or
the lipid anchor is a transmembrane domain, a PIP2-binding domain, or a PIP3-binding domain.

### Claim 4 (depends on 1)

The artificial exosome of claim 1, wherein the cardiolipin or variant thereof is selected from the group consisting of cardiolipin, monolysocardiolipin, and dilysocardiolipin.

### Claim 5 (depends on 1)

The artificial exosome of claim 1, wherein the cardiolipin or the variant thereof is dilysocardiolipin.

### Claim 6 (depends on 1)

The artificial exosome of claim 1, wherein the cardiolipin or variant thereof comprises about 10% w/w to about 50% w/w of the total lipid content of the artificial exosome.

### Claim 7 (depends on 1)

The artificial exosome of claim 1, wherein the cardiolipin or variant thereof comprises about 30% w/w of the total lipid content of the artificial exosome, optionally wherein the artificial exosome comprises a DOPC:cholesterol:cardiolipin ratio of about 40:30:30% w/w.

### Claim 8 (depends on 1)

The artificial exosome of claim 1, wherein the exogenously-derived cargo molecule comprises an oligonucleotide.

### Claim 9 (depends on 8)

The artificial exosome of claim 8, wherein the oligonucleotide comprises one or more hydrophobic modifications.

### Claim 10 (depends on 8)

The artificial exosome of claim 8, wherein the oligonucleotide comprises one or more modified nucleotides comprising a modified ribose group, a modified phosphate group, a modified nucleobase, or a mixture thereof.

### Claim 11

A method of isolating artificial exosomes comprising an exogenously-derived cargo from a sample comprising a population of exosomes, wherein the method comprises incubating the sample with a binding agent that binds to a protein comprising one or more of rab7, desmoplakin, and alpha 2-HS glycoprotein (AHSG), and a cardiolipin or a variant thereof; wherein each protein further comprises a lipid anchor; wherein the binding agent binds to the one or more proteins of the artificial exosome and thereby isolates the sample, and wherein the artificial exosome is loaded with exogenously-derived, heterologous cargo selected from the group consisting of a peptide, a polypeptide, a nucleic acid, a virus, a small molecule, a fluorophore, and a combination thereof.

### Claim 12 (depends on 11)

The method of claim 11, wherein the binding agent is an antibody or an antigen-binding fragment thereof.

### Claim 13

A method of producing an artificial exosome comprising an exogenously-derived cargo comprising proteins comprising rab7, desmoplakin, and alpha 2-HS glycoprotein (AHSG), and a cardiolipin or variants thereof comprising the steps of:
a) mixing DOPC, cholesterol, and a cardiolipin or a variant thereof to form a cardiolipin-containing liposome; and
b) incubating the cardiolipin-containing liposome with rab7, desmoplakin, and AHSG to form an artificial exosome.

### Claim 14 (depends on 13)

The method of claim 13, further comprising c) centrifuging the artificial exosome to isolate the artificial exosome.

### Claim 15 (depends on 14)

The method of claim 14, wherein the mixing step a) further comprises mixing a cargo molecule to produce a loaded cardiolipin-containing liposome.

### Claim 16 (depends on 14)

The method of claim 14, further comprising step d) wherein the isolated artificial exosome is incubated with a cargo molecule to produce a loaded artificial exosome.

### Claim 17 (depends on 13)

The method of claim 13, further comprising drying the mixture of step a) to form a dry lipid film and rehydrating the dry lipid film in an aqueous buffer to form the cardiolipin-containing liposome.

### Claim 18 (depends on 13)

The method of claim 13, wherein mixing step a) occurs in an organic solvent.

### Claim 19 (depends on 13)

The method of claim 13, wherein the incubation step b) occurs for about 1 hour and at about 37° C.

### Claim 20

A method of delivering a cargo molecule to a neuronal cell, comprising contacting the neuronal cell with an artificial exosome containing the cargo molecule, wherein the artificial exosome comprises one or more proteins comprising rab7, desmoplakin, AHSG, and a cardiolipin or variant thereof; wherein each protein further comprises a lipid anchor; and wherein the cargo molecule is delivered to the neuronal cell.