# US 12,180,477 B2 · Dynamic pharmacokinetic-modifying anchors

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- Number: US 12,180,477 B2
- Document ID: US12180477B2
- Status: Granted
- Kind: B2
- Application number: 17/725,102
- Filed: 2022-04-20
- Published: 2022-11-24
- Granted: 2024-12-31
- Earliest claimed: 2019-01-18
- Assignees: University of Massachusetts
- Applicants: University of Massachusetts
- Inventors: Anastasia Khvorova; Bruno Miguel da Cruz Godinho; Matthew Hassler
- Web page: https://patentgrep.com/patent/US12180477B2

## CPC

- 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 31/713: Double-stranded nucleic acids or oligonucleotides
- C12N 2310/11: Antisense
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/16: Aptamers
- C12N 2310/312: Phosphonates
- C12N 2310/314: Phosphoramidates
- C12N 2310/315: Phosphorothioates
- C12N 2310/3181: Peptide nucleic acid, PNA
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/322: 2'-R Modification
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2310/343: having patterns, e.g. ==--==--==--
- C12N 2310/344: Position-specific modifications, e.g. on every purine, at the 3'-end
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2310/351: Conjugate
- C12N 2310/3513: Protein; Peptide
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N 2310/3519: Fusion with another nucleic acid
- C12N 2310/51: in polymeric form, e.g. multimers, concatemers
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- 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)

## Abstract

Therapeutic oligonucleotides comprising pharmacokinetic (PK)-modifying anchors are provided. Methods for treating diseases or disorders comprising administering to a subject a therapeutic oligonucleotide comprising one or more PK-modifying anchors are provided.

## Claims

### Claim 1

A method for treating a disease or a disorder in a patient in need thereof, comprising administering to the patient a compound comprising a first oligonucleotide, a second oligonucleotide, and a pharmacokinetic (PK)-modifying anchor, wherein:
the first oligonucleotide is between 16 to 25 nucleotides in length, and comprises a 5′ end, a 3′ end, and complementarity to a target gene or a target mRNA;
the second oligonucleotide is between 12 to 17 nucleotides in length, and comprises a 5′ end and a 3′ end; and
the pharmacokinetic (PK)-modifying anchor comprises an anchor oligonucleotide between 5 to 8 nucleotides in length and comprising a 3′ end and a 5′ end, and at least one polymer conjugated to the 5′ end, wherein the at least one polymer has a molecular weight between about 2,000 Da and about 40,000 Da, and wherein the at least one polymer is not a polypeptide,
wherein a portion of the 5′ end of the first oligonucleotide is complementary to the second oligonucleotide and a portion of the 3′ end of the first oligonucleotide is complementary to the anchor oligonucleotide.

### Claim 2 (depends on 1)

The method of claim 1, wherein the pharmacokinetic (PK)-modifying anchor further comprises a linker between the 5′ end of the anchor oligonucleotide and the at least one polymer, the linker comprising an ethylene glycol chain, an alkyl chain, or a combination thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein the first oligonucleotide comprises perfect complementarity to the target gene or the target mRNA.

### Claim 4 (depends on 1)

The method of claim 1, wherein the at least one polymer is selected from the group consisting of a hydrophobic polycarbonate, a polyester, an amphiphilic block copolymer, a hydrophobic block copolymer, and a polysaccharide.

### Claim 5 (depends on 4)

The method of claim 4, wherein:
the hydrophobic polycarbonate is poly(DTR carbonate).

### Claim 6 (depends on 4)

The method of claim 4, wherein the polyester is selected from the group consisting of a polyhydroxyalkanoate, a polycaprolactone, a poly(hyroxybuterate-hydroxyvalerate), a polyglycolic acid, and a polylactic acid.

### Claim 7 (depends on 4)

The method of claim 4, wherein the amphiphilic block copolymer is selected from the group consisting of a polyvinylpyrrolidone, a poly(2-ethyl-2-oxazoline), an acrylonitrile styrene acrylate, an N-(2-hydroxypropyl) methacrylamide, and a polyethylene glycol (PEG).

### Claim 8 (depends on 4)

The method of claim 4, wherein the hydrophobic block copolymer is selected from the group consisting of a poly(N,N-dimethylacrylamide), a poly(N,N-diethylaniline), a poly(diphenylamine), and a poly(tetrahydrofurfuryl methacrylate).

### Claim 9 (depends on 4)

The method of claim 4, wherein the polysaccharide is selected from the group consisting of a soluble polyglucose, a non-soluble polyglucose, a cellulose, a glycogen, and an amylopectin.

### Claim 10 (depends on 1)

The method of claim 1, wherein the PK-modifying anchor comprises more than one polymer.

### Claim 11 (depends on 10)

The method of claim 10, wherein the PK-modifying anchor comprises 2, 3, or 4 polymers.

### Claim 12 (depends on 1)

The method of claim 1, wherein the first oligonucleotide is fully chemically modified.

### Claim 13 (depends on 1)

The method of claim 1, wherein:
the number of nucleotides in the first oligonucleotide is the same as the number of nucleotides in the second oligonucleotide and the anchor oligonucleotide combined.

### Claim 14 (depends on 1)

The method of claim 1, wherein the anchor oligonucleotide consists of 8 nucleotides complementary to the 3′ end of the first oligonucleotide.

### Claim 15 (depends on 1)

The method of claim 1, wherein the first oligonucleotide is not conjugated at the 5′ end or at the 3′ end.

### Claim 16 (depends on 1)

The method of claim 1, wherein the first oligonucleotide comprises a greater number of nucleotides than the second oligonucleotide and the anchor oligonucleotide combined.

### Claim 17 (depends on 1)

The method of claim 1, wherein the first oligonucleotide comprises fewer nucleotides than in the second oligonucleotide and the anchor oligonucleotide combined.

### Claim 18

A method of delivering a compound to the liver of a subject, comprising administering to the subject a compound comprising:
a first oligonucleotide, wherein the first oligonucleotide comprises 16 to 25 contiguous nucleotides, a 5′ end, a 3′ end, and complementarity to a target gene or target mRNA;
a second oligonucleotide comprising 12 to 17 contiguous nucleotides, a 5′ end, and a 3′ end, wherein a portion of the 5′ end of the first oligonucleotide is complementary to a portion of the second oligonucleotide, and wherein the second oligonucleotide comprises an N-Acetylgalactosamine (GalNAc) moiety or a derivative thereof conjugated to the 3′ end; and
a pharmacokinetic (PK)-modifying anchor comprising an anchor oligonucleotide comprising a 5′ end, a 3′ end, and at least one polymer conjugated to the 5′ end, wherein the anchor oligonucleotide comprises about 5 to about 15 nucleotides that are complementary to the 3′ end of the first oligonucleotide, and wherein the at least one polymer is at least about 2,000 Da.

### Claim 19

A method for treating a disease or a disorder in a patient in need thereof, comprising administering to the patient a compound comprising a first oligonucleotide, a second oligonucleotide, and a pharmacokinetic (PK)-modifying anchor, wherein:
the first oligonucleotide is between 16 to 25 nucleotides in length, and comprises a 5′ end, a 3′ end, a complementarity to a target gene or target mRNA;
the second oligonucleotide is between 12 to 17 nucleotides in length, and comprises a 5′ end and a 3′ end; and
the pharmacokinetic (PK)-modifying anchor comprises an anchor oligonucleotide between 5 to 8 nucleotides in length and comprising a 3′ end and a 5′ end, and at least one polymer conjugated to the 5′ end, wherein the at least one polymer is at least about 2,000 Da,
wherein a portion of the 5′ end of the first oligonucleotide is complementary to the second oligonucleotide and a portion of the 3′ end of the first oligonucleotide is complementary to the anchor oligonucleotide, and
wherein the second oligonucleotide further comprises conjugate moiety Xc at the 3′ end, at the 5′ end, at an internal position, or a mixture thereof.

### Claim 20 (depends on 19)

The method of claim 19,
wherein the second oligonucleotide is attached to Xc with a linker L.

### Claim 21 (depends on 20)

The method of claim 20, wherein L comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

### Claim 22 (depends on 20)

The method of claim 20, wherein Xc is attached at the 3′ end of the second oligonucleotide.

### Claim 23 (depends on 19)

The method of claim 19, wherein Xc is selected from the group consisting of a fatty acid, a steroid, a secosteroid, a lipid, a ganglioside, a nucleoside analog, and an endocannabinoid.

### Claim 24 (depends on 19)

The method of claim 19, wherein Xc comprises an N-Acetylgalactosamine (GalNAc) moiety or a derivative thereof.

### Claim 25 (depends on 19)

The method of claim 19, wherein Xc has an affinity for one or both of a low-density lipoprotein and an intermediate density lipoprotein.

### Claim 26 (depends on 19)

The method of claim 19, wherein Xc is a saturated or unsaturated moiety having fewer than three double bonds.

### Claim 27 (depends on 19)

The method of claim 19, wherein Xc has an affinity for high density lipoprotein.

### Claim 28 (depends on 19)

The method of claim 19, wherein Xc is a polyunsaturated moiety having three or more double bonds.

### Claim 29

A method for treating a disease or a disorder in a patient in need thereof, comprising administering to the patient a compound comprising a first oligonucleotide, a second oligonucleotide, and a pharmacokinetic (PK)-modifying anchor, wherein:
the first oligonucleotide comprises a 5′ end, a 3′ end, and complementarity to a target gene or target mRNA;
the second oligonucleotide comprises a 5′ end and a 3′ end; and
the pharmacokinetic (PK)-modifying anchor comprises an anchor oligonucleotide comprising a 3′ end and a 5′ end, and at least one polymer conjugated to the 5′ end, wherein the at least one polymer is at least about 2,000 Da,
wherein a portion of the 5′ end of the first oligonucleotide is complementary to the second oligonucleotide and a portion of the 3′ end of the first oligonucleotide is complementary to the anchor oligonucleotide, wherein:
a) the first oligonucleotide is between 21 nucleotides to 25 nucleotides in length;
b) the second oligonucleotide is between 13 nucleotides and 17 nucleotides in length; and
c) the anchor oligonucleotide is between 5 nucleotides and 8 nucleotides in length;
a) the first oligonucleotide is 21 nucleotides in length;
b) the second oligonucleotide is 13 nucleotides in length; and
c) the anchor oligonucleotide is 8 nucleotides in length;
a) the first oligonucleotide is 23 nucleotides in length;
b) the second oligonucleotide is 15 nucleotides in length; and
c) the anchor oligonucleotide is 8 nucleotides in length; or
a) the first oligonucleotide is 25 nucleotides in length;
b) the second oligonucleotide is 17 nucleotides in length; and
c) the anchor oligonucleotide is 8 nucleotides in length.

### Claim 30

A method for treating a disease or a disorder in a patient in need thereof, comprising administering to the patient:
a first oligonucleotide, wherein the first oligonucleotide is 21 nucleotides in length, with a 5′ end and a 3′ end, and is complementary to a target gene or a target mRNA;
a second oligonucleotide, wherein the second oligonucleotide is 13 nucleotides in length, with a 5′ end and a 3′ end, and is complementary to nucleotides 1-13 of the first oligonucleotide; and
an anchor oligonucleotide, wherein the anchor oligonucleotide is 8 nucleotides in length, with a 5′ end, a 3′ end, and is complementary nucleotides 14-21 of the first oligonucleotide,
wherein the second oligonucleotide is attached to a molecule comprising a cholesterol, a docosanoic acid, a docosahexaenoic acid, or an N-acetylgalactosamine at the 3′ end,
wherein the anchor oligonucleotide is attached to a polyethylene glycol polymer comprising a molecular weight of between 10,000 and 40,000 Daltons at the 5′ end,
wherein the first oligonucleotide comprises complementarity to both the second oligonucleotide and the anchor oligonucleotide to form a duplex, and wherein the 5′ end of the second oligonucleotide is not connected to the 3′ end of the anchor oligonucleotide.

### Claim 31

A method for treating a disease or a disorder in a patient in need thereof, comprising administering to the patient:
an oligonucleotide duplex comprising a first oligonucleotide, wherein the first oligonucleotide is 25 nucleotides in length, with a 5′ end and a 3′ end, and comprises complementarity to a target gene or a target mRNA;
a second oligonucleotide, wherein the second oligonucleotide is 17 nucleotides in length, with a 5′ end and a 3′ end, is hydrophobically modified, and is complementary to nucleotides 1-17 of the first oligonucleotide; and
an anchor oligonucleotide, wherein the anchor oligonucleotide is 8 nucleotides in length, with a 5′ end and a 3′ end, and is complementarity to nucleotides 18-25 of the first oligonucleotide, wherein the anchor oligonucleotide comprises a pharmacokinetic-modifying moiety.

### Claim 32

A method for treating a disease or a disorder in a patient in need thereof, comprising administering to the patient an siRNA duplex comprising:
a first RNA strand between 21 nucleotides and 25 nucleotides in length and comprising complementarity to a target gene or a target mRNA;
a second RNA strand between 13 nucleotides and 17 nucleotides in length and having homology with a target sequence; and
an anchor oligonucleotide strand between 5 nucleotides and 8 nucleotides in length, wherein the anchor oligonucleotide strand comprises a polymeric pharmacokinetic-modifying moiety selected from the group consisting of a hydrophilic polycarbonate, a polyethylene glycol (PEG), a block co-polymer, a poloxamer, a polysaccharide, and a poly(lactic-co-glycolic acid) (PLGA),
wherein the first RNA strand can pair with the second RNA strand to form an asymmetric siRNA, and the anchor oligonucleotide strand binds to a single stranded region of the siRNA.