# US 2019/0167809 A1 · METHODS OF TREATING A SUBJECT WITH mRNA ENCODING COAGULATION FACTOR VIII (F8)

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- Number: US 2019/0167809 A1
- Document ID: US20190167809A1
- Status: Published application
- Kind: A1
- Application number: 16/278,666
- Filed: 2019-02-18
- Published: 2019-06-06
- Earliest claimed: 2007-09-04
- Assignees: CureVac AG
- Applicants: CureVac AG
- Inventors: Mariola FOTIN-MLECZEK; Patrick BAUMHOF
- Web page: https://patentgrep.com/patent/US20190167809A1

## CPC

- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- 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 38/45: Transferases (2)
- A61K 38/4846: {Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)}
- A61K 47/61: the organic macromolecular compound being a polysaccharide or a derivative thereof
- A61K 47/6455: {Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids}
- A61K 48/0033: {the non-active part being non-polymeric}
- A61K 48/0041: {the non-active part being polymeric}
- 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}
- A61K 48/0075: {characterised by an aspect of the delivery route, e.g. oral, subcutaneous}
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 37/08: Antiallergic agents (antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14)
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12Y 113/12007: Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
- C12Y 204/01007: Sucrose phosphorylase (2.4.1.7)
- C12Y 304/21022: Coagulation factor IXa (3.4.21.22)
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 2039/53: {DNA (RNA) vaccination}
- 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
- 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)
- C12Y: ENZYMES

## Abstract

The present invention relates to a complexed RNA, comprising at least one RNA complexed with one or more oligopeptides, wherein the oligopeptide has a length of 8 to 15 amino acids and has the empirical formula (Arg)l;(;Lys)m;(His)n;(Orn)o;(Xaa)x. The invention further relates to a method for transfecting a cell or an organism, thereby applying the inventive complexed RNA. Additionally, pharmaceutical compositions and kits comprising the inventive complexed RNA, as well as the use of the inventive complexed RNA for transfecting a cell, tissue or an organism and/or for modulating, preferably inducing or enhancing, an immune response are disclosed herein.

## Claims

### Claim 1

A method of treating a subject having haemophilia A comprising administering an effective amount of a pharmaceutical composition comprising a purified mRNA encoding coagulation factor VIII (F8) to the subject, wherein the mRNA comprises a 5′ cap structure; wherein the mRNA comprises a poly-A tail; wherein the mRNA is comprised in a liposomal system.

### Claim 2 (depends on 1)

The method of claim 1, wherein the mRNA has been purified by chromatography.

### Claim 3 (depends on 2)

The method of claim 2, wherein the mRNA has been purified by affinity chromatography.

### Claim 4 (depends on 3)

The method of claim 3, wherein the mRNA has been purified by HPLC using a porous reverse phase as the stationary phase.

### Claim 5 (depends on 1)

The method of claim 1, wherein the subject is a human subject.

### Claim 6 (depends on 1)

The method of claim 1, wherein the pharmaceutical composition is administered by injection.

### Claim 7 (depends on 6)

The method of claim 6, wherein the pharmaceutical composition is administered by intravenous injection.

### Claim 8 (depends on 1)

The method of claim 1, wherein the poly-A tail comprises 10 to 200 adenosine nucleotides.

### Claim 9 (depends on 1)

The method of claim 1, wherein the mRNA additionally comprises a poly-C tail of 10 to 200 cytosine nucleotides.

### Claim 10 (depends on 1)

The method of claim 1, wherein the mRNA further comprises a 5′ and/or a 3′ untranslated region (UTR).

### Claim 11 (depends on 1)

The method of claim 1, wherein the mRNA has been modified by introduction of a non-native nucleotide compared with a corresponding native mRNA nucleotide and/or by covalent coupling of the mRNA with a further chemical moiety.

### Claim 12 (depends on 1)

The method of claim 1, wherein the mRNA comprises a G/C content in the F8 coding region which is greater than the G/C content of a coding region of the native mRNA sequence encoding F8.

### Claim 13 (depends on 1)

The method of claim 1, wherein the mRNA comprises an F8 coding sequence that is modified, compared with a native mRNA encoding F8, such that at least one codon of the native mRNA which codes for a tRNA which is relatively rare in the subject is exchanged for a codon which codes for a tRNA which is relatively frequent in the subject.

### Claim 14 (depends on 11)

The method of claim 11, wherein the mRNA comprises a chemical modification relative to a naturally occurring mRNA.

### Claim 15 (depends on 11)

The method of claim 11, wherein the mRNA comprises at least one nucleotide that is substituted with a nucleotide analog selected from the group consisting of: 2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5-triphosphate), 2′-deoxy-2′-deamine oligoribonucleotide (2′-amino-2′-deoxycytidine-5′-triphosphate, 2′-amino-2′-deoxyuridine-5′-triphosphate), 2′-O-alkyl oligoribonucleotide, 2′-deoxy-2′-C-alkyl oligoribonucleotide (2′-O-methylcytidine-5′-triphosphate, 2′-methyluridine-5′-triphosphate), 2′-C-alkyl oligoribonucleotide, and isomers thereof (2′-aracytidine-5′-triphosphate, 2′-arauridine-5′-triphosphate), or azidotriphosphate (2′-azido-2′-deoxycytidine-5′-triphosphate, 2′-azido-2′-deoxyuridine-5′-triphosphate)4-thio-uridine-5′-(mono)phosphate, 2-Aminopurine-riboside-5′-(mono)phosphate, 5-Aminoallylcytidine-5′-(mono)phosphate, 5-Aminoallyluridine-5′-(mono)phosphate, 5-Bromocytidine-5′-(mono)phosphate, 5-Bromo-2′-deoxycytidine-5′-(mono)phosphate, 5-Bromouridine-5′-(mono)phosphate, 5-Bromo-2′-deoxyuridine-5′-(mono)phosphate, 5-Iodocytidine-5′-(mono)phosphate, 5-Iodo-2′-deoxycytidine-5′-(mono)phosphate, 5-Iodouridine-5′-(mono)phosphate, 5-Iodo-2′-deoxyuridine-5′-(mono)phosphate, 5-Propynyl-2′-deoxycytidine-5′-(mono)phosphate, 5-Propynyl-2′-deoxyuridine-5′-(mono)phosphate, 5-formylcytidine-5′-(mono)phosphate, 5,2′-O-dimethylcytidine-5′-(mono)phosphate, 5-hydroxymethylcytidine-5′-(mono)phosphate, 5-formyl-2-O-methylcytidine-5′-(mono)phosphate, 5,2′-O-dimethyluridine-5′-(mono)phosphate, 5-methyl-2-thiouridine-5′-(mono)phosphate, 5-hydroxyuridine-5′-(mono)phosphate, 5-methoxyuridine-5′-(mono)phosphate, uridine 5-oxyacetic acid-5′-(mono)phosphate, uridine 5-oxyacetic acid methyl ester-5′-(mono)phosphate, 5-(carboxyhydroxymethyl)uridine-5′-(mono)phosphate, 5-(carboxyhydroxymethyl)uridine methyl ester-5′-(mono)phosphate, 5-methoxycarbonylmethyluridine-5′-(mono)phosphate, 5-methoxycarbonylmethyl-2′-O-methyluridine-5′-(mono)phosphate, 5-methoxycarbonylmethyl-2-thiouridine-5′-(mono)phosphate, 5-aminomethyl-2-thiouridine-5′-(mono)phosphate, 5-methylaminomethyluridine-5′-(mono)phosphate, 5-methylaminomethyl-2-thiouridine-5′-(mono)phosphate, 5-methylaminomethyl-2-selenouridine-5′-(mono)phosphate, 5-carbamoylmethyluridine-5′-(mono)phosphate, 5-carbamoylmethyl-2′-O-methyluridine-5′-(mono)phosphate, 5-carboxymethylaminomethyluridine-5′-(mono)phosphate, 5-carboxymethylaminomethyl-2′-O-methyluridine-5′-(mono)phosphate, 5-carboxymethylaminomethyl-2-thiouridine-5′-(mono)phosphate, 5-carboxymethyluridine-5′-(mono)phosphate, 5-methyldihydrouridine-5′-(mono)phosphate, 5-taurinomethyluridine-5′-(mono)phosphate, 5-taurinomethyl-2-thiouridine-5′-(mono)phosphate, 5-(isopentenylaminomethyl)uridine-5′-(mono)phosphate, 5-(isopentenylaminomethyl)-2-thiouridine-5′-(mono)phosphate, 5-(isopentenylaminomethyl)-2′-O-methyluridine-5′-(mono)phosphate, 6-Azacytidine-5′-(mono)phosphate, 7-Deazaadenosine-5′-(mono)phosphate, 7-Deazaguanosine-5′-(mono)phosphate, 8-Azaadenosine-5′-(mono)phosphate, 8-Azidoadenosine-5′-(mono)phosphate, Pseudouridine-5′-(mono)phosphate, 2′-Amino-2′-deoxycytidine-(mono)phosphate, 2′-Fluorothymidine-5′-(mono)phosphate, inosine-5′-(mono)phosphate, and 2′-O-Methyl-inosine-5′-(mono)phosphate.

### Claim 16 (depends on 11)

The method of claim 11, wherein the mRNA comprises at least one nucleotide that is substituted with a nucleotide analog selected from the group consisting of: 2-amino-6-chloropurineriboside-5′-triphosphate, 2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate, 2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5-aminoallylcytidine-5′-triphosphate, 5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, 5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate, 7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate, benzimidazole-riboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, puromycin-5′-triphosphate, and xanthosine-5′-triphosphate.

### Claim 17 (depends on 16)

The method of claim 16, wherein the nucleotide analog is chosen from the group consisting of: 5-methylcytidine 5′-triphosphate and pseudouridine 5′-triphosphate.

### Claim 18 (depends on 1)

The method of claim 1, wherein the liposomal system comprises a cationic lipid.

### Claim 19 (depends on 18)

The method of claim 18, wherein the mRNA is provided in complex with the cationic lipid.

### Claim 20

A method of inducing or enhancing an innate immune response in a subject in need thereof, comprising administering to the subject a complexed ribonucleic acid (RNA) comprising at least one RNA molecule complexed with one or more oligopeptides, wherein the at least one RNA molecule is not covalently bound to the one or more oligopeptides, wherein the nitrogen/phosphate ratio (N/P-ratio) of the RNA to the one or more oligopeptides is in the range of 0.75-25, and wherein the oligopeptide is 8 to 15 amino acids in length and comprises the following formula:
(Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x (formula I)
wherein
l+m+n+o+x=8-15, and
l, m, n or o independently of each other is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide; and
Xaa is any amino acid selected from native or non-native amino acids except Arg, Lys, His or Orn; and
x is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8, provided, that the overall content of Xaa does not exceed 50% of all amino acids of the oligopeptide.

### Claim 21

A method of treating a subject having an ornithine transcarbamylase deficiency comprising administering an effective amount of a pharmaceutical composition comprising mRNA encoding human ornithine transcarbamylase (OTC) to the subject, wherein the mRNA comprises a 5′ cap structure; wherein the mRNA comprises a poly-A tail; wherein the mRNA is comprised in a liposomal system.