# US 2024/0189232 A1 · Proteolipid vesicles formulated with fusion associated small transmembrane proteins

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- Number: US 2024/0189232 A1
- Document ID: US20240189232A1
- Status: Published application
- Kind: A1
- Application number: 18/029,823
- Filed: 2021-10-01
- Published: 2024-06-13
- Earliest claimed: 2020-10-01
- Assignees: Entos Pharmaceuticals Inc.
- Applicants: Entos Pharmaceuticals Inc.
- Inventors: John David Lewis; Arun Raturi
- Web page: https://patentgrep.com/patent/US20240189232A1

## CPC

- A61K 9/1271: Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911})
- C12N 15/88: using microencapsulation, e.g. using {amphiphile} liposome vesicle
- 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 31/7088: Compounds having three or more nucleosides or nucleotides
- A61K 31/7105: Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
- A61K 31/711: Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 38/1709: {from mammals}
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61K 47/6917: {the form being a lipoprotein vesicle, e.g. HDL or LDL proteins}
- A61K 48/0025: {wherein the non-active part clearly interacts with the delivered nucleic acid}
- A61K 48/0033: {the non-active part being non-polymeric}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid}
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 35/00: Antineoplastic agents
- A61P 35/04: specific for metastasis
- 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 2310/14: interfering nucleic acids [NA]
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2720/12222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Disclosed is a proteolipid vesicle (PLV) for delivering a therapeutic cargo, such as nucleic acids, polypeptides and molecules, to a cell, the proteolipid vesicle having a lipid nanoparticle comprising one or more ionizable lipids and one or more of a fusion associated small transmembrane (FAST) family of proteins and chimerics thereof, where the molar ratio of ionizable lipid to therapeutic cargo is between 2.5:1 and 20:1. Incorporation of a chimeric FAST protein into a PLV platform enhances intracellular delivery and expression of mRNA and pDNA both in vitro and in vivo. These PLVs also display a favorable immune profile and are significantly less toxic than conventional LNPs.

## Claims

### Claim 1

A proteolipid vesicle for delivering a therapeutic cargo to a cell comprising:
a lipid nanoparticle comprising one or more ionizable lipids; and
one or more of a fusion associated small transmembrane (FAST) family of proteins and chimerics thereof,
wherein the molar ratio of ionizable lipid to therapeutic cargo is between 2.5:1 and 20:1.

### Claim 2 (depends on 1)

The proteolipid vesicle of claim 1, wherein the one or more ionizable lipids is Dlin-KC2-DMA (KC2), DODMA, DODAP, DOBAQ, DOTMA, 18:1 EPC, DOTAP, DDAB, 18:0 EPC, 18:0 DAP or 18:0 TAP.

### Claim 3 (depends on 2)

The proteolipid vesicle of claim 2, wherein the one or more ionizable lipids is DODAP and/or DODMA.

### Claim 4 (depends on 3)

The proteolipid vesicle of claim 3,
a) wherein the FAST protein is p10, p13, p14, p15, p16, p22, or chimerics thereof; or
b) wherein the FAST protein is a p14/p15 chimera, p10/p14 chimera or a p10/p15 chimera.

### Claim 5

(canceled)

### Claim 6 (depends on 5)

The proteolipid vesicle of claim 5, wherein the p14p15 chimera comprises the ectodomain and transmembrane of p14 and the endodomain of p15; the ectodomain of p14, the transmembrane domain and endodomain of p15; or the ectodomain and endodomain of p14 and the transmembrane of p15.

### Claim 7

(canceled)

### Claim 8 (depends on 6)

The proteolipid vesicle of claim 6, further comprising a therapeutic cargo, wherein the therapeutic cargo is a nucleic acid, polypeptides or molecule.

### Claim 9

(canceled)

### Claim 10 (depends on 8)

The proteolipid vesicle of claim 8,
a) wherein when the therapeutic cargo is the nucleic acid, the nucleic acid is pDNA, mRNA, siRNA, miRNA, self-amplifying mRNA, genetic adjuvants, promoters or molecular gene editing tools;
b) wherein when the therapeutic cargo is the polypeptide, the polypeptide is a peptide, epitope, or antigenic molecule; or
c) wherein when the therapeutic cargo is the molecule, the molecule is a small drug molecule, biological molecule, structural macromolecule, or therapeutic molecule.

### Claim at position 11

11-12. (canceled)

### Claim 13 (depends on 10)

The proteolipid vesicle of claim 10, wherein the molar ratio is 5:1, 7.5:1, 10:1 or 15:1.

### Claim 14 (depends on 13)

The proteolipid vesicle of claim 13,
a) wherein the lipid nanoparticle comprises DOTAP, DODAP, DOPE and DMG-PEG;
b) wherein the lipid nanoparticle comprises DOTAP, DODAP, DOPE and DMG-PEG in a mole percentage of 24:42:30:4;
c) wherein the lipid nanoparticle comprises DOTAP, DODMA, DOPE and DMG-PEG;
d) wherein the lipid nanoparticle comprises DOTAP, DODMA, DOPE and DMG-PEG in a mole percentage of 24:42:30:4;
e) wherein the lipid nanoparticle comprises DOTAP, DODAP, DODMA, DOPE and DMG-PEG;
f) wherein the lipid nanoparticle comprises DOTAP, DODAP, DODMA, DOPE and DMG-PEG in a mole percentage of 24:21:21:30:4;
q) wherein the lipid nanoparticle comprises DOTAP, DODAP, DOPE and DMG-PEG,
h) wherein the lipid nanoparticle comprises DOTAP, DODAP, DOPE and DMG-PEG in a mole percentage of 6:60:30:4 or 3:63:30:4;
i) wherein the lipid nanoparticle comprises DODAP, DOPE and DMG-PEG;
j) wherein the lipid nanoparticle comprises DODAP, DOPE and DMG-PEG in a mole percentage of 66:30:4;
h) wherein the lipid nanoparticle comprises DODAP, cholesterol, DOPE and DMG-PEG; or
i) wherein the lipid nanoparticle comprises DODAP, cholesterol, DOPE and DMG-PEG in a mole percentage of 49.5:24.75:23.75:2, 49.5:38.5:10:2 or 61.7:26.3:19:3.

### Claim at position 14

15-25. (canceled)

### Claim 26 (depends on 13)

The proteolipid vesicle of claim 13 further comprising bombesin attached to the C-terminal of the FAST protein or chimeric thereof.

### Claim 27 (depends on 1)

A composition for delivering a therapeutic cargo to a cell comprising:
the proteolipid vesicle of claim 1; and
a therapeutic cargo encapsulated by the proteolipid vehicle.

### Claim 28 (depends on 27)

The composition of claim 27, wherein the therapeutic cargo is a nucleic acid, polypeptide or molecule.

### Claim 29 (depends on 28)

The composition of claim 28, wherein the nucleic acid is pDNA, mRNA or siRNA.

### Claim 30 (depends on 29)

The composition of claim 29, wherein the lipid nanoparticle comprises;
a) DOTAP:DODAP:DOPE:DMG-PEG in a mole percentage of 24:42:30:4 and the molar ratio of ionizable lipid to pDNA is between 5:1 and 10:1;
b) DOTAP:DODAP:DOPE:DMG-PEG in a mole percentage of 24:42:30:4 and the molar ratio of ionizable lipid to mRNA is between 5:1 and 10:1
c) DOTAP:DODMA:DOPE:DMG-PEG in a mole percentage of 24:42:30:4 and the molar ratio of ionizable lipid to pDNA is between 4:1 to 7.5:1;
d) DOTAP:DODMA:DOPE:DMG-PEG in a mole percentage of 24:42:30:4 and the molar ratio of ionizable lipid to mRNA is between 2.5:1 to 7.5:1;
e) DOTAP:DODAP:DODMA:DOPE:DMG-PEG in a mole percentage of 24:21:21:30:4 and the molar ratio of ionizable lipid to pDNA is between 3:1 to 7.5:1;
f) DOTAP:DODAP:DODMA:DOPE:DMG-PEG in a mole percentage of 24:21:21:30:4 and the molar ratio of ionizable lipid to mRNA is between 2.5:1 to 7.5:1;
q) DOTAP:DODAP:DOPE:DMG-PEG in a mole percentage of 3:63:30:4 and the molar ratio of ionizable lipid to pDNA is between 7.5:1 to 15:1;
h) DOTAP:DODAP:DOPE:DMG-PEG in a mole percentage of 3:63:30:4 and the molar ratio of ionizable lipid to pDNA is between 5:1 to 12:1;
i) DOTAP:DODAP:DOPE:DMG-PEG in a mole percentage of 6:60:30:4 and the molar ratio of ionizable lipid to pDNA is between 5:1 to 15:1;
i) DOTAP:DODAP:DOPE:DMG-PEG in a mole percentage of 6:60:30:4 and the molar ratio of ionizable lipid to mRNA is between 5:1 to 15:1;
k) DODAP:DOPE:DMG-PEG in a mole percentage of 66:30:4 and the molar ratio of ionizable lipid to pDNA is between 5:1 to 20:1;
l) DODAP:DOPE:DMG-PEG in a mole percentage of 66:30:4 and the molar ratio of ionizable lipid to mRNA is between 5:1 to 20:1;
m) DODAP:cholesterol:DOPE:DMG-PEG in a mole percentage of 49.5:24.75:23.75:2 and the molar ratio of ionizable lipid to pDNA is between 5:1 to 15:1;
n) DODAP:cholesterol:DOPE:DMG-PEG in a mole percentage of 49.5:24.75:23.75:2 and the molar ratio of ionizable lipid to mRNA is between 2.5:1 to 15:1;
o) DODAP:cholesterol:DOPE:DMG-PEG in a mole percentage of 49.5:38.5:10:2 and the molar ratio of ionizable lipid to pDNA is between 5:1 to 15:1;
p) DODAP:cholesterol:DOPE:DMG-PEG in a mole percentage of 49.5:38.5:10:2 and the molar ratio of ionizable lipid to mRNA is between 2.5:1 to 15:1;
q) DODAP:cholesterol:DOPE:DMG-PEG in a mole percentage of 61.7:26.3:19:3 and the molar ratio of ionizable lipid to pDNA is between 5:1 to 15:1; or
r) DODAP:cholesterol:DOPE:DMG-PEG in a mole percentage of 61.7:26.3:19:3 and the molar ratio of ionizable lipid to mRNA is between 2.5:1 to 15:1.

### Claim at position 20

31-47. (canceled)

### Claim 48 (depends on 30)

The composition of claim 30, wherein the therapeutic cargo is c14orf132 siRNA.

### Claim at position 22

49-56. (canceled)

### Claim 57 (depends on 28)

A method of delivering a therapeutic cargo to a host cell, comprising:
administering the composition of claim 28 to a cell.

### Claim 58 (depends on 57)

The method of claim 57, wherein the therapeutic cargo is a nucleic acid, polypeptides or molecule.

### Claim 59 (depends on 58)

The method of claim 58,
a) wherein when the therapeutic cargo is the nucleic acid, the nucleic acid is pDNA, mRNA, siRNA, miRNA, self-amplifying mRNA, genetic adjuvants, promoters or molecular gene editing tools;
b) wherein when the therapeutic cargo is the polypeptide, the polypeptide is a peptide, epitope, or antigenic molecule; or
c) wherein when the therapeutic cargo is the molecule, the molecule is a small drug molecule, biological molecule, structural macromolecule, or therapeutic molecule.

### Claim at position 26

60-61. (canceled)

### Claim 62 (depends on 59)

The method of claim 59, wherein the host cell is a cancer cell, immortalized cell, primary cell or muscle cell.

### Claim 63

(canceled)