# US 11,998,635 B2 · Targeted extracellular vesicles comprising membrane proteins with engineered glycosylation sites

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- Number: US 11,998,635 B2
- Document ID: US11998635B2
- Status: Granted
- Kind: B2
- Application number: 16/852,894
- Filed: 2020-04-20
- Published: 2020-12-17
- Granted: 2024-06-04
- Earliest claimed: 2015-09-28
- Assignees: Northwestern University
- Applicants: Northwestern University
- Inventors: Joshua N. Leonard; Michelle E. Hung
- Web page: https://patentgrep.com/patent/US11998635B2

## CPC

- A61K 9/1075: {Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers (A61K9/0026 takes precedence)}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 47/46: Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C12N 15/88: using microencapsulation, e.g. using {amphiphile} liposome vesicle
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/06: containing a lysosomal/endosomal localisation signal
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- C07K 2319/85: containing an RNA binding domain
- C07K 2319/91: containing a motif for glycosylation
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N 2810/6081: rhabdoviridae, e.g. VSV
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed are extracellular vesicles comprising an engineered targeting protein for targeting the extracellular vesicles to target cells. The targeting protein is a fusion protein that includes a ligand, an engineered glycosylation site, and an exosome-targeting domain. Exemplary extracellular vesicles may include but are not limited to exosomes.

## Claims

### Claim 1

A method of loading an extracellular vesicle with a cargo nucleic acid, the method comprising the steps of:
expressing within a vesicle-producing cell that contains the cargo nucleic acid (i)
a fusion protein comprising:
(i) a nucleic acid-binding domain;
(ii) a vesicle-targeting domain, and
(iii) an engineered glycosylation site, wherein the engineered glycosylation site comprises an amino acid sequence that is not naturally present in the fusion protein or any of the components of the fusion protein;
wherein:
the cargo nucleic acid includes a binding motif that binds to the nucleic acid-binding domain of the fusion protein, and
the vesicle-targeting domain of the fusion protein localizes to the extracellular vesicles released by the vesicle-producing cell,
such that the cargo nucleic acid is loaded into the vesicle when the fusion protein and cargo nucleic acid are both present in by the vesicle-producing cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the vesicle-targeting domain of the fusion protein is a domain of a lysosome-associated protein.

### Claim 3 (depends on 2)

The method of claim 2, wherein the lysosome-associated protein is a lysosome-associated membrane protein (LAMP).

### Claim 4 (depends on 3)

The method of claim 3, wherein the LAMP comprises a luminal N-terminus and a cytoplasmic C-terminus.

### Claim 5 (depends on 3)

The method of claim 3, wherein the nucleic acid-binding domain of the fusion protein comprises a binding domain of a bacteriophage.

### Claim 6 (depends on 5)

The method of claim 5, wherein the bacteriophage is a MS2 bacteriophage, a R17 bacteriophage, a lambdoid bacteriophage, a P22 bacteriophage, or a phi21 bacteriophage.

### Claim 7 (depends on 1)

The method of claim 1, wherein the vesicle-targeting domain of the fusion protein comprises a sequence selected from a group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

### Claim 8 (depends on 1)

The method of claim 1, wherein the vesicle-targeting domain of the fusion protein comprises a sequence having at least 80% amino acid sequence identity to a sequence selected from a group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.

### Claim 9 (depends on 1)

The method of claim 1, wherein the nucleic acid-binding domain of the fusion protein comprises a binding domain of a bacteriophage.

### Claim 10 (depends on 9)

The method of claim 9, wherein the bacteriophage is a MS2 bacteriophage, a R17 bacteriophage, a lambdoid bacteriophage, a P22 bacteriophage, or a phi21 bacteriophage.

### Claim 11 (depends on 1)

The method of claim 1, wherein the cargo nucleic acid is an RNA.

### Claim 12 (depends on 11)

The method of claim 11, wherein the RNA is a therapeutic RNA.

### Claim 13 (depends on 11)

The method of claim 11, wherein the binding motif comprises one or more high affinity binding loops comprising a sequence and structure selected from the group consisting of:
where N—N is any two base-paired RNA nucleotides.

### Claim 14 (depends on 1)

The method of claim 1, wherein the vesicle-targeting domain of the fusion protein comprises a domain of a lysosome-associated membrane protein (LAMP), the nucleic acid-binding domain of the fusion protein comprises a binding domain of a bacteriophage, and the cargo nucleic acid is an RNA.

### Claim 15 (depends on 1)

The method of claim 1, wherein the vesicle-targeting domain of the fusion protein comprises a sequence selected from a group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36; the nucleic acid-binding domain of the fusion protein comprises a binding domain of a bacteriophage selected from a group consisting of a MS2 bacteriophage, a R17 bacteriophage, a lambdoid bacteriophage, a P22 bacteriophage, and a phi21 bacteriophage; and the cargo nucleic acid is an RNA.

### Claim 16 (depends on 15)

The method of claim 15, wherein the RNA is a therapeutic RNA.

### Claim 17 (depends on 15)

The method of claim 15, wherein the binding motif comprises one or more high affinity binding loops comprising a sequence and structure selected from the group consisting of:
where N—N is any two base-paired RNA nucleotides.

### Claim 18

In a method of loading an extracellular vesicle with a cargo nucleic acid, the method comprising the steps of:
expressing within a vesicle-producing cell that contains the cargo nucleic acid
a fusion protein comprising:
(i) a nucleic acid-binding domain; and
(ii) a vesicle-targeting domain, and
wherein:
the cargo nucleic acid includes a binding motif that binds to the nucleic acid-binding domain of the fusion protein, and
the vesicle-targeting domain of the fusion protein localizes the extracellular vesicles released by the vesicle-producing cell,
such that the cargo nucleic acid is loaded into the vesicle when the fusion protein and cargo nucleic acid are both present in the vesicle-producing cell;
the improvement that comprises engineering the fusion protein to comprise an engineered glycosylation site, wherein the engineered glycosylation site comprises an amino acid sequence that is not naturally present in the fusion protein or any of the components of the fusion protein.