# US 9,522,176 B2 · MRNA therapy for phenylketonuria

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- Number: US 9,522,176 B2
- Document ID: US9522176B2
- Status: Granted
- Kind: B2
- Application number: 14/521,323
- Filed: 2014-10-22
- Published: 2015-04-23
- Granted: 2016-12-20
- Earliest claimed: 2013-10-22
- Assignees: Shire Human Genetic Therapies, Inc.
- Applicants: Shire Human Genetic Therapies, Inc.
- Inventors: Frank DeRosa; Michael Heartlein; Anusha Dias
- Web page: https://patentgrep.com/patent/US9522176B2

## CPC

- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- 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 38/44: Oxidoreductases (1)
- 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}
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 13/02: of urine or of the urinary tract, e.g. urine acidifiers
- A61P 25/00: Drugs for disorders of the nervous system
- C12Y 114/16001: Phenylalanine 4-monooxygenase (1.14.16.1)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES

## Abstract

The present invention provides, among other things, methods of treating phenylketonuria (PKU), including administering to a subject in need of treatment a composition comprising an mRNA encoding phenylalanine hydroxylase (PAH) at an effective dose and an administration interval such that at least one symptom or feature of PKU is reduced in intensity, severity, or frequency or has delayed in onset. In some embodiments, the mRNA is encapsulated in a liposome comprising one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids.

## Claims

### Claim 1

A method of treating phenylketonuria (PKU), the method comprising administering systemically to a subject in need of treatment a composition comprising an mRNA encoding a phenylalanine hydroxylase (PAH),
wherein the mRNA comprises a coding nucleotide sequence consisting of SEQ ID NO:3,
wherein the mRNA is encapsulated within a liposome with a size of less than 100 nm, and
wherein administration of the composition at an effective dose and at an administration interval results in increased PAH protein level in the liver and in reduced phenylalanine level in the serum as compared to the baseline of PAH and phenylalanine levels before the treatment.

### Claim 2 (depends on 1)

The method of claim 1, wherein the liposome comprises a cationic lipid, a non-cationic lipid, a cholesterol-based lipid and a polyethylene glycol (PEG)-modified lipid.

### Claim 3 (depends on 2)

The method of claim 2, wherein the cationic lipid is selected from the group consisting of 1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), N,N-dimethyl-2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propan-1-amine (DLinDMA), 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLinKC2DMA, [XTC2]), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12), 10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-5-yl)propanoate (ICE), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (4Z,15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dimyristyloxyproyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), dioleoyloxy-N-[2-sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA) and N,N-dimethyl-(2,3-dimyristyloxy)propylamine (DMDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), (2S)-2-(4-((10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)butoxy)-N,N-dimethyl-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propan-1-amine (CLinDMA), 2-[5′-(cholest-5-en-3[betal]-oxy)-3′-oxapentoxy)-3-dimethyl-1-1(cis,cis-9′,12′-octadecadienoxy) propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), (9Z,9′Z,12Z,12′Z)-3-(dimethylamino)propane-1,2-diyl bis(octadeca-9,12-dienoate) (DLinDAP), 1,2-dilinoleylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2-((2,3-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propyl)disulfanyl)-N,N-dimethylethanamine (HGT4003), and combinations thereof.

### Claim 4 (depends on 3)

The method of claim 3, wherein the cationic lipid comprises cKK-E12, which has the structure:

### Claim 5 (depends on 2)

The method of claim 2, wherein the cholesterol-based lipid is cholesterol or PEGylated cholesterol.

### Claim 6 (depends on 2)

The method of claim 2, wherein the cationic lipid constitutes about 30-50% of the liposome by molar ratio.

### Claim 7 (depends on 2)

The method of claim 2, wherein the ratio of cationic lipid:non-cationic lipid:cholesterol lipid:PEGylated lipid is approximately 40:30:25:5 by molar ratio.

### Claim 8 (depends on 1)

The method of claim 1, wherein the liposome comprises a combination selected from the group consisting of:
cKK-E12, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol and 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (DMG-PEG2K);
C12-200, DOPE, cholesterol and DMG-PEG2K;
HGT4003, DOPE, cholesterol and DMG-PEG2K; or
ICE, DOPE, cholesterol and DMG-PEG2K.

### Claim 9 (depends on 1)

The method of claim 1, wherein the mRNA is administered at the effective dose ranging from about 0.01-5.0 mg/kg body weight.

### Claim 10 (depends on 1)

The method of claim 1, wherein the composition is administered once a week.

### Claim 11 (depends on 1)

The method of claim 1, wherein the mRNA comprises a 5′ UTR sequence of SEQ ID NO:4.

### Claim 12 (depends on 1)

The method of claim 1, wherein the mRNA comprises a 3′ UTR sequence of SEQ ID NO:5 or SEQ ID NO:6.