# US 10,118,944 B2 · Cell penetrating peptides for intracellular delivery of molecules

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- Number: US 10,118,944 B2
- Document ID: US10118944B2
- Status: Granted
- Kind: B2
- Application number: 14/433,592
- Filed: 2013-10-04
- Published: 2016-05-26
- Granted: 2018-11-06
- Earliest claimed: 2012-10-04
- Assignees: Aadigen, LLC
- Applicants: Aadigen, LLC
- Inventors: Gilles Divita; Sebastien Deshayes; Karidia Konate; May Catherine Morris
- Web page: https://patentgrep.com/patent/US10118944B2

## CPC

- C07K 7/06: having 5 to 11 amino acids
- A61K 9/5123: {Organic compounds, e.g. fats, sugars}
- A61K 47/542: {Carboxylic acids, e.g. a fatty acid or an amino acid}
- A61K 47/554: {the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid}
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 47/62: the modifying agent being a protein, peptide or polyamino acid
- 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)
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- A61K 9/5169: {Proteins, e.g. albumin, gelatin}
- 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)
- 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)

## Abstract

A cell-penetrating peptide characterized in that it comprises an amino acid sequence consisting of a beta-alanine or a serine in N-terminal position, linked to a sequence consisting of 9 consecutive amino acids from the sequence RXWXRLWXRLR (SEQ ID NO:7), wherein X in position 2 is R or S and X in positions 4 and 8 are, independently from each other, W or F.

## Claims

### Claim 1

A 10 amino acid cell-penetrating peptide characterized in that it comprises an amino acid sequence consisting of a beta-alanine or a serine in the N-terminal position, linked to a sequence consisting of 9 consecutive amino acids from the sequence
(SEQ ID NO: 7)
RXWXRLWXRLR,
wherein X in position 2 is R or S and X in positions 4 and 8 are, independently from each other, W or F.

### Claim 2 (depends on 1)

The cell-penetrating peptide of claim 1, wherein the amino acid sequence is selected from the group consisting of:
(SEQ ID No: 1)
X1WWRLWWRLR,
(SEQ ID No: 2)
X1WFRLWFRLR,
(SEQ ID No: 3)
X1WFRLWWRLR,
(SEQ ID No: 4)
X1WWRLWFRLR,
(SEQ ID No: 5)
X1RWWRLWWRL,
and
(SEQ ID No: 6)
X1RSWFRLWFR,
wherein X1 is beta-A or S.

### Claim 3 (depends on 1)

The cell-penetrating peptide of claim 1, further comprising, covalently linked to the N-terminal end of the amino acid sequence, one or several chemical entities selected from the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide and a targeting molecule.

### Claim 4 (depends on 1)

The cell-penetrating peptide of claim 1, further comprising, covalently linked to the C-terminal end of said amino acid sequence, one or several groups selected from the group consisting of a cysteamide, a cysteine, a thiol, an amide, an optionally substituted nitrilotriacetic acid, a carboxyl, a linear or branched optionally substituted C1-C6 alkyl, a primary or secondary amine, an osidic derivative, a lipid, a phospholipid a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide and a targeting molecule.

### Claim 5 (depends on 1)

A complex comprising a cell-penetrating peptide according to claim 1 and a cargo selected from the group consisting of proteins, nanobodies, peptides, peptide-analogues, uncharged oligonucleotides, PNAs and small hydrophobic molecules.

### Claim 6 (depends on 5)

The complex of claim 5, wherein said cargo is a molecule of at most 1.5 kDa.

### Claim 7 (depends on 5)

The complex of claim 5, wherein said cargo is an anticancer or an anti viral drug.

### Claim 8 (depends on 5)

The complex of claim 5, wherein said cargo is selected from the group consisting of amino acids, di- or tri- peptides, daunomycin, Paclitaxel, doxorubicin, AZT, porphyrin, fluorescently labelled nucleosides or nucleotides, hydrophobic maghemite and fluorescent dyes.

### Claim 9 (depends on 5)

The complex of claim 5, wherein said cargo is a cosmetic agent.

### Claim 10 (depends on 5)

The complex of claim 5, wherein the size of the complex is between 50 and 200 nm.

### Claim 11 (depends on 5)

A nanoparticle comprising a complex according to claim 5, coated by a layer of peripheral cell-penetrating peptides, wherein said peripheral cell-penetrating peptides have a peptide sequence different from SEQ ID Nos: 1 to 6.

### Claim 12 (depends on 1)

A nanoparticle comprising a core which comprises a cargo complexed to a first entity selected from the group consisting of cell-penetrating peptides, liposomes, polycationic structures and carbon nanoparticles, wherein said core is coated by peripheral cell-penetrating peptides, and wherein the peripheral cell-penetrating peptides have the peptide sequence of a cell-penetrating peptide according to claim 1.

### Claim 13 (depends on 12)

The nanoparticle of claim 12, wherein said first entity is a cell-penetrating peptide selected from the group consisting of VEPEP-3, VEPEP-6, VEPEP-9, CADY, MPG, PEP-1, PPTG1, and poly Arginine.

### Claim 14 (depends on 12)

The nanoparticle of claim 12, wherein the size of the nanoparticle is between 20 and 300 nm.

### Claim 15 (depends on 12)

The nanoparticle of claim 12, wherein the peripheral cell-penetrating peptides comprise a poly-ethylene glycol group covalently linked to their N-terminus, and/or a cysteamide group covalently linked to their C-terminus.

### Claim 16 (depends on 12)

The nanoparticle of claim 12, wherein at least some of the peripheral cell-penetrating peptides are bound to a targeting molecule.

### Claim 17 (depends on 12)

The nanoparticle of claim 12, for use as a medicament.

### Claim 18 (depends on 12)

The nanoparticle of claim 12, for use as a marker or an imaging agent.

### Claim 19 (depends on 12)

A therapeutic, cosmetic or diagnostic composition comprising a nanoparticle according to claim 12.

### Claim 20 (depends on 19)

The composition of claim 19, which is formulated for intravenous, intratumoral, topical, intrarectal, intranasal, transdermal, or intradermal administration, or for administration via a mouth spray, or for administration as a subcutaneous implant for slow release of a drug.

### Claim 21 (depends on 12)

A method for delivering a molecule into a cell in vitro, comprising a step of putting said cell into contact with a nanoparticle according to claim 12, wherein the cargo of the nanoparticle is said molecule.