# US 11,299,511 B2 · Oligonucleotide compositions and methods of making the same

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- Number: US 11,299,511 B2
- Document ID: US11299511B2
- Status: Granted
- Kind: B2
- Application number: 15/705,021
- Filed: 2017-09-14
- Published: 2018-01-18
- Granted: 2022-04-12
- Earliest claimed: 2014-05-01
- Assignees: Geron Corporation
- Applicants: Geron Corporation
- Inventors: Premchandran H. Ramiya
- Web page: https://patentgrep.com/patent/US11299511B2

## CPC

- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- 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)}
- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 2310/113: targeting other non-coding nucleic acids, e.g. antagomirs
- C12N 2310/3145: with the nitrogen in 3' or 5'-position
- C12N 2310/315: Phosphorothioates
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12Y 207/07049: RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The present disclosure provides a solid phase method of making oligonucleotides via sequential coupling cycles including at least one coupling of a dinucleotide dimer subunit to a free 3′-terminal group of a growing chain. The oligonucleotides include at least two nucleoside subunits joined by a N3′→P5′ phosphoramidate linkage. The method may include the steps of (a) deprotecting the protected 3′ amino group of a terminal nucleoside attached to a solid phase support, said deprotecting forming a free 3′ amino group; (b) contacting the free 3′ amino group with a 3′-protected amino-dinucleotide-5′-phosphoramidite dimer in the presence of a nucleophilic catalyst to form an internucleoside N3′→P5′ phosphoramidite linkage; and (c) oxidizing (e.g., sulfurizing) the linkage. The compositions produced by the subject methods may include a reduced amount of one or more (N−x) oligonucleotide products. Also provided are pharmaceutical compositions including the subject oligonucleotide compositions.

## Claims

### Claim 1

An unpurified synthetic composition comprising:
a polynucleotide compound having a sequence of N nucleoside subunits wherein at least two of the nucleoside subunits are joined by a N3′→P5′ phosphoramidate inter-subunit linkage, or a salt thereof; and
a 3′-protected amino-dinucleotide phosphoramidate-5′-phosphoramidite dimer,
wherein the unpurified synthetic composition has less than 1 part in 10 by weight of (N−1) product relative to the polynucleotide compound or a salt thereof, wherein N is 10 or more.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the N3′→P5′ phosphoramidate inter-subunit linkage is a N3′→P5′ thiophosphoramidate inter-subunit linkage having the structure:
3′-NH—P(S)(OR)—O-5′
wherein R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl and a phosphate protecting group, or a salt thereof.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the compound comprises a polynucleotide having a sequence of 10 or more nucleoside subunits complementary to the RNA component of human telomerase.

### Claim 4 (depends on 3)

The composition of claim 3, wherein the polynucleotide comprises between 3 and 50 contiguous nucleoside subunits complementary to the RNA component of human telomerase.

### Claim 5 (depends on 3)

The composition of claim 3, wherein the nucleoside subunits are all joined by N3′→P5′ phosphoramidate inter-subunit linkages.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the polynucleotide comprises a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4), TAGGGTTAGACAA (SEQ ID NO:3) and CAGTTAGGGTTAG (SEQ ID NO:5).

### Claim 7 (depends on 6)

The composition of claim 6, wherein the compound has the structure:
or a salt thereof;
wherein “nps” represents a thiophosphoramidate linkage —NH—P(═O)(SH)—O—, connecting the 3′-carbon of one nucleoside to the 5′-carbon of the adjacent nucleoside.

### Claim 8 (depends on 6)

The composition of claim 6, wherein the compound has the structure:
wherein each MX+ is independently hydrogen or a counterion of a salt, each x of MX+ is independently 1, 2 or 3 and n is an integer from 5 to 13.

### Claim 9 (depends on 6)

The composition of claim 6, wherein the compound has the structure:

### Claim 10 (depends on 1)

The composition of claim 1, having less than 1 part in 20 by weight of (N−1) product relative to the compound.

### Claim 11 (depends on 1)

The composition of claim 1, having less than 1 part in 10 by weight of any one (N−x) product relative to the compound, wherein x of (N−x) is 1 to (N−1).

### Claim 12 (depends on 11)

The composition of any one of claim 11, having less than 40 parts in 100 by total weight of all (N−x) polynucleotide-containing products relative to the compound, wherein x of (N−x) is 1 to (N−1).

### Claim 13 (depends on 1)

The composition of claim 1, having the following profile of (N−x) polynucleotide-containing products, wherein x is 1 to (N−1):
less than 1 part in 10 by weight of (N−1) product relative to the compound;
at least 10 parts in 100 by weight of (N−2) and (N−3) products relative to the compound.

### Claim 14 (depends on 1)

The composition of claim 1, wherein the nucleoside subunits of the polynucleotide compound independently have the structure:
wherein:
B is a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof;
X is O or S;
R is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an aryl, a substituted aryl, a phosphate protecting group; and
R3 is selected from hydrogen, O—R2, and halogen, wherein R2 is H, an alkyl, a substituted alkyl and a hydroxyl protecting group.

### Claim 15 (depends on 1)

The composition of claim 1, having less than 1 part in 15 by weight of (N−1) product relative to the compound.

### Claim 16 (depends on 1)

The composition of claim 1, having less than 1 part in 25 by weight of (N−1) product relative to the compound.

### Claim 17 (depends on 1)

The composition of claim 1, wherein N is between 10 and 40.

### Claim 18 (depends on 17)

The composition of claim 17, wherein N is between 10 and 30.

### Claim 19 (depends on 18)

The composition of claim 18, wherein N is between 10 and 20.

### Claim 20 (depends on 1)

The composition of claim 1, having less than 9% by weight of the (N−1) product.

### Claim 21 (depends on 20)

The composition of claim 20, having less than 5% by weight of the (N−1) product.

### Claim 22 (depends on 1)

The composition of claim 1, having less than 11% of any (N−x) product, wherein x of (N−x) is 1 to (N−1).

### Claim 23 (depends on 1)

The composition of claim 1, having the following profile of (N−x) polynucleotide-containing products, wherein x of (N−x) is 1 to (N−1):
less than 5% by weight of a (N−1) product; and
at least 10% by weight of (N−2) and (N−3) products.