# US 11,078,247 B2 · RNA encoding a therapeutic protein

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- Number: US 11,078,247 B2
- Document ID: US11078247B2
- Status: Granted
- Kind: B2
- Application number: 16/098,844
- Filed: 2017-05-04
- Published: 2019-08-08
- Granted: 2021-08-03
- Earliest claimed: 2016-05-04
- Assignees: CureVac AG
- Applicants: CureVac AG
- Inventors: Mariola Fotin-Mleczek; Ingmar Hoerr
- Web page: https://patentgrep.com/patent/US11078247B2

## CPC

- C07K 14/505: Erythropoietin [EPO]
- 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 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- 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)}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 2310/531: Stem-loop; Hairpin
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to an RNA encoding a therapeutic protein. In particular, the present invention relates to RNA suitable for use as a medicament. The present invention concerns such novel RNA as well as compositions and kits comprising the RNA. Furthermore, the present invention relates to the RNA, compositions or kits as disclosed herein for use as a medicament. The present invention also provides the use of the RNA, compositions or kits as disclosed herein far increasing the expression of said encoded protein, in particular in gene therapy.

## Claims

### Claim 1

A method of treating or preventing hereditary spastic paraplegia, wherein the method comprises administering to a subject in need thereof an effective amount of a purified RNA comprising at least one coding sequence encoding a CYP7B1 protein that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2846, wherein the coding sequence comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences according to any one of SEQ ID NOs: 28960, 42017, 55074, 15903, and 68131 or a sequence at least 80% identical to any one of SEQ ID NOs: 28960, 42017, 55074, 15903, and 68131.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the at least one coding sequence encodes a CYP7B1 protein according to the amino acid sequence of SEQ ID NO: 2846.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the RNA is an mRNA, a viral RNA or a replicon RNA.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the RNA is a modified RNA.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the
the G/C content of the at least one coding sequence of the RNA is increased compared to the G/C content of the corresponding coding sequence of the corresponding wild type RNA, and/or wherein the C content of the at least one coding sequence of the RNA is increased compared to the C content of the corresponding coding sequence of the corresponding wild type RNA, and/or wherein the codons in the at least one coding sequence of the RNA are adapted to human codon usage, wherein the codon adaptation index (CAI) is increased or maximised in the at least one coding sequence of the RNA,
wherein the amino acid sequence encoded by the RNA is not modified compared to the amino acid sequence encoded by the corresponding wild type RNA.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the RNA comprises a 5′-CAP structure, a 5′-untranslated region (5′-UTR), and/or at least one 3′-untranslated region element (3′-UTR element).

### Claim 7 (depends on 1)

The method according to claim 1, wherein the RNA comprises at least one histone stem-loop.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the at least one histone stem loop comprises a nucleic acid sequence according to SEQ ID NO: 2451.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the at least one RNA comprises a poly(A) sequence, and/or a poly(C) sequence.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the poly(A) sequence comprises 10 to 200, 10 to 100, 40 to 80 or 50 to 70 adenosine nucleotides.

### Claim 11 (depends on 9)

The method according to claim 9, wherein the poly(C) sequence comprises 10 to 200, 10 to 100, 20 to 70, 20 to 60 or 10 to 40 cytosine nucleotides.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the RNA comprises, in 5′ to 3′ direction, the following elements:
a) a 5′-CAP structure,
b) at least one coding sequence comprising a nucleic acid sequence selected from the group consisting of nucleic acid sequences according to any one of SEQ ID NO: 28960, 42017, 55074, 15903, and 68131 or a having at least 80% identity to any one of SEQ ID NO: 28960, 42017, 55074, 15903, and 68131,
c) a poly(A) tail,
d) optionally a poly(C) tail,
e) optionally a histone stem-loop.

### Claim 13 (depends on 6)

The method according to claim 6, wherein the RNA comprises a 3′-UTR element and wherein the 3′-UTR element comprises a nucleic acid sequence derived from a 3′-UTR of a gene.

### Claim 14 (depends on 13)

The method according to claim 13, wherein the 3′-UTR element comprises a nucleic acid sequence derived from a 3′-UTR of a gene selected from the group consisting of an albumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, or from a homolog, a fragment or a variant thereof.

### Claim 15 (depends on 14)

The method according to claim 14, wherein the 3′-UTR element comprises a nucleic acid sequence derived from a 3′-UTR of an α-globin gene.

### Claim 16 (depends on 1)

The method of claim 1, wherein the purified RNA is formulated in a composition with one or more liposome, lipoplex or lipid nanoparticle (LNP).

### Claim 17 (depends on 16)

The method of claim 16, wherein the purified RNA is formulated in a composition with one or more LNP comprising an ionizable or cationic lipid.

### Claim 18 (depends on 17)

The method of claim 17, wherein the composition is administered by injection.