# US 2023/0271997 A1 · Stabilized Nucleic Acids Encoding Messenger Ribonucleic Acid (mRNA)

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- Number: US 2023/0271997 A1
- Document ID: US20230271997A1
- Status: Published application
- Kind: A1
- Application number: 18/310,298
- Filed: 2023-05-01
- Published: 2023-08-31
- Earliest claimed: 2017-08-15
- Assignees: Intellia Therapeutics, Inc.
- Applicants: Intellia Therapeutics, Inc.
- Inventors: Christian Dombrowski
- Web page: https://patentgrep.com/patent/US20230271997A1

## CPC

- C07H 21/02: with ribosyl as saccharide radical
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C07K 14/475: Growth factors; Growth regulators
- C07K 14/521: {Chemokines}
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

This disclosure relates to the field of poly-adenylated (poly-A) tails. In some embodiments, a DNA encodes a poly-A tail located 3′ to nucleotides encoding a protein of interest, wherein the poly-A tail comprises one or more non-adenine nucleotide.

## Claims

### Claim 1

A DNA comprising nucleotides encoding a poly-adenylated (poly-A) tail located 3′ to nucleotides encoding a protein of interest, wherein the poly-A tail comprises a first homopolymer sequence of at least 8 consecutive adenine (A) nucleotides and an interrupting sequence comprising one or more non-adenine (A) nucleotides.

### Claim 2 (depends on 1)

The DNA of claim 1, wherein the poly-A tail further comprises a second homopolymer sequence of at least consecutive adenine (A) nucleotides.

### Claim 3 (depends on 1, 2)

The DNA of claim 1 or 2, wherein the poly-A tail comprises three or more homopolymer sequences of at least 8 consecutive adenine (A) nucleotides.

### Claim 4 (depends on 1)

The DNA of claim 1, wherein the first and/or subsequent homopolymer sequence comprises at least 10, 15, 20, 25, 30, 35, or 40 consecutive adenine nucleotides.

### Claim 5 (depends on 1)

The DNA of claim 1, wherein the one or more non-adenine nucleotide prevents the loss of one or more adenine nucleotide during DNA replication as compared to the loss that occurs in a DNA comprising a 3′ tail of a similar or same length that contains only adenine nucleotides.

### Claim 6 (depends on 1)

The DNA of claim 1, wherein the one or more non-adenine nucleotide is positioned to interrupt the consecutive adenine nucleotides so that a poly(A) binding protein can bind to a stretch of consecutive adenine nucleotides.

### Claim 7 (depends on 1)

The DNA of claim 1, wherein the poly-A tail comprises at least 50 total adenine nucleotides.

### Claim 8 (depends on 1)

The DNA of claim 1, wherein the poly-A tail comprises 40-1000, 40-900, 40-800, 40-700, 40-600, 40-500, 40-400, 40-300, 40-200, or 40-100 total adenine nucleotides.

### Claim 9 (depends on 1)

The DNA of claim 1, wherein the poly-A tail comprises 95-100 total adenine nucleotides.

### Claim 10 (depends on 1)

The DNA of claim 1, wherein the poly-A tail comprises or contains 90, 91, 92, 93, 94, 95, 96, or 97 total adenine nucleotides.

### Claim 11 (depends on 1)

The DNA of claim 1, wherein the poly-A tail comprises or contains 96 or 97 total adenine nucleotides.

### Claim 12 (depends on 1)

The DNA of claim 1, wherein the one or more interrupting sequence comprises or contains one non-adenine nucleotide or one consecutive stretch of 2-10 non-adenine nucleotides.

### Claim 13 (depends on 1)

The DNA of claim 1, wherein the one or more interrupting sequence comprises or contains one non-adenine nucleotide or one consecutive stretch of 2-10 nucleotides that includes two or more non-adenine nucleotides.

### Claim 14 (depends on 12, 13)

The DNA of claim 12 or 13, wherein the non-adenine nucleotide(s) is located after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides.

### Claim 15 (depends on 1)

The DNA of claim 1, wherein the one or more non-adenine nucleotide is located after at least 8-50 consecutive adenine nucleotides.

### Claim 16 (depends on 1)

The DNA of claim 1, wherein the one or more non-adenine nucleotide is located after at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The DNA of any one of claims 1-16, wherein the interrupting sequence is a trinucleotide, dinucleotide or mononucleotide interrupting sequence.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The DNA of any one of claims 1-16, wherein the poly-A tail comprises or contains one non-adenine nucleotide or one consecutive stretch of 2-10 non-adenine nucleotides every 8-50 consecutive adenine nucleotides.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

The DNA of any one of claims 1-16, wherein the poly-A tail comprises or contains one non-adenine nucleotide or one consecutive stretch of 2-10 non-adenine nucleotides every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

### Claim 20

The DNA of any preceding claim, wherein the poly-A tail comprises or contains 1, 2, 3, 4, or 5 consecutive non-adenine nucleotides every 8-50 consecutive adenine nucleotides.

### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)

The DNA of any one of claims 1-20, wherein the poly-A tail comprises or contains 1, 2, 3, 4, or 5 consecutive non-adenine nucleotides every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)

The DNA of any one of claims 1-21, wherein the poly-A tail comprises or contains more than one non-adenine nucleotide or more than one consecutive stretch of 2-10 non-adenine nucleotides.

### Claim 23

The DNA of any preceding claim, wherein the more than one non-adenine nucleotide or more than one consecutive stretch of 2-10 non-adenine nucleotides are irregularly spaced within the poly-A tail.

### Claim 24

The DNA of any preceding claim, wherein the poly-A tail comprises or contains one non-adenine nucleotide or 2, 3, 4, or 5 consecutive non-adenine nucleotides every 12 consecutive adenine nucleotides.

### Claim 25

The DNA of any preceding claim, wherein the poly-A tail comprises or contains one non-adenine nucleotide or 2, 3, 4, or 5 consecutive non-adenine nucleotides every 16 consecutive adenine nucleotides.

### Claim 26

The DNA of any preceding claim, wherein the poly-A tail comprises or contains one non-adenine nucleotide or 2, 3, 4, or 5 consecutive non-adenine nucleotides every 25 consecutive adenine nucleotides.

### Claim 27

The DNA of any preceding claim, wherein the poly-A tail comprises or contains one non-adenine nucleotide or 2, 3, 4, or 5 consecutive non-adenine nucleotides every 30 consecutive adenine nucleotides.

### Claim 28

The DNA of any preceding claim, wherein the poly-A tail comprises or contains one non-adenine nucleotide or 2, 3, 4, or 5 consecutive non-adenine nucleotides every 39 consecutive adenine nucleotides.

### Claim 29

The DNA of any preceding claim, wherein the non-adenine nucleotide is guanine, cytosine, or thymine.

### Claim 30 (depends on 29)

The DNA of claim 29, wherein the non-adenine nucleotide is a guanine nucleotide.

### Claim 31 (depends on 29)

The DNA of claim 29, wherein the non-adenine nucleotide is a cytosine nucleotide.

### Claim 32 (depends on 29)

The DNA of claim 29, wherein the non-adenine nucleotide is a thymine nucleotide.

### Claim 33 (depends on 29)

The DNA of claim 29, comprising more than one non-adenine nucleotide selected from:
a. guanine and thymine nucleotides;
b. guanine and cytosine nucleotides;
c. thymine and cytosine nucleotides; or
d. guanine, thymine and cytosine nucleotides.

### Claim 34

The DNA of any preceding claim, wherein the non-adenine nucleotide consists of one non-adenine nucleotide selected from guanine, cytosine, and thymine.

### Claim 35

The DNA of any preceding claim, wherein the non-adenine nucleotides comprise two non-adenine nucleotides selected from one or more of guanine, cytosine, and thymine.

### Claim 36

The DNA of any preceding claim, wherein the non-adenine nucleotides comprise three non-adenine nucleotides selected from one or more of guanine, cytosine, and thymine.

### Claim 37

The DNA of any preceding claim, wherein the adenine nucleotides are adenosine monophosphate.

### Claim 38

The DNA of any preceding claim, wherein the protein is a therapeutic protein.

### Claim 39 (depends on 38)

The DNA of claim 38, wherein the protein a cytokine or chemokine.

### Claim 40 (depends on 38)

The DNA of claim 38, wherein the protein a growth factor.

### Claim 41 (depends on 38)

The DNA of claim 38, wherein the protein is Cas9 or modified Cas9.

### Claim 42

A mRNA encoded by the DNA of any one of the preceding claims.

### Claim 43

The DNA of any preceding claim, wherein the DNA is within a vector.

### Claim 44 (depends on 43)

The DNA of claim 43, wherein the vector is within a host cell.

### Claim 45 (depends on 43)

The DNA of claim 43, wherein the one or more non-adenine nucleotide prevents loss of nucleotides encoding the poly-A tail within the vector during growth of the host cell as compared to the loss that occurs in a DNA comprising nucleotides encoding a poly-A tail of a similar or same length that contains only adenine nucleotides.

### Claim 46 (depends on 43)

A method of producing mRNA from the DNA vector of claim 43, comprising:
a. linearizing the vector downstream of the poly-A tail;
b. denaturing the linearized vector; and
c. contacting the denaturized DNA with an RNA polymerase in the presence of guanine, cytosine, uracil, and adenine nucleotides.