# US 12,178,855 B2 · Compositions and methods for facilitating delivery of synthetic nucleic acids to cells

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- Number: US 12,178,855 B2
- Document ID: US12178855B2
- Status: Granted
- Kind: B2
- Application number: 16/961,187
- Filed: 2019-01-10
- Published: 2021-02-25
- Granted: 2024-12-31
- Earliest claimed: 2018-01-10
- Assignees: Translate Bio MA, Inc.
- Applicants: Translate Bio MA, Inc.
- Inventors: Balkrishen Bhat; Jia Tay; Saswata Karmakar; Nelson Chau
- Web page: https://patentgrep.com/patent/US12178855B2

## CPC

- A61K 38/45: Transferases (2)
- 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 31/712: Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
- A61K 31/7125: Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
- A61K 47/549: {Sugars, nucleosides, nucleotides or nucleic acids}
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 37/00: Drugs for immunological or allergic disorders
- C12N 9/1018: {Carboxy- and carbamoyl transferases (2.1.3)}
- 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 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N 2310/3231: having an additional ring, e.g. LNA, ENA
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N 2310/3519: Fusion with another nucleic acid
- C12Y 201/03003: Ornithine carbamoyltransferase (2.1.3.3)
- 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
- 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

Provided herein are compositions and methods for facilitating or enhancing delivery of nucleic acids, such as synthetic mRNAs, into cells or tissues. Such compositions and methods may include use of a targeting moiety-conjugated, such as an N-acetylgalactosamine (GalNAc)-conjugated, oligonucleotide to facilitate or enhance delivery.

## Claims

### Claim 1

A composition comprising:
(a) a single-stranded oligonucleotide of 5 to 50 nucleotides in length covalently linked to a targeting moiety; and
(b) a synthetic RNA comprising at least one binding region that is complementary to a contiguous stretch of at least 5 nucleotides of the oligonucleotide, wherein the single-stranded oligonucleotide is non-covalently associated with the synthetic RNA through the at least one binding region in the synthetic RNA; and
wherein the targeting moiety comprises one or more one or more sugar moieties and has of one of the following formulae:
wherein:
each instance of LA, LB, and LC is independently either absent or is a linker selected from the group consisting of substituted alkylene, substituted alkenylene, substituted alkynylene, substituted heteroalkylene, substituted heteroalkenylene, substituted heteroalkynylene, substituted acylene, substituted carbocyclylene, substituted heterocyclylene, substituted arylene, substituted heteroarylene, and combinations thereof; and
each instance of G1, G2, and G3 is independently a ligand.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the synthetic RNA comprises at least two copies of the binding region.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the copies of the binding region are separated from one another by a spacer region comprising at least one nucleotide.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the oligonucleotide comprises at least one modified nucleotide.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the targeting moiety comprises one or more ligands selected from a sugar moiety, a folate moiety, and a cell-penetrating peptide.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the targeting moiety comprises one or more N-acetylgalactosamine ligands.

### Claim 8 (depends on 1)

The composition of claim 1, wherein each instance of LA, LB, and LC independently comprises a triazole diradical.

### Claim 9 (depends on 1)

The composition of claim 1, wherein each instance of G1, G2, and G3 is independently a ligand selected from a sugar moiety, a folate moiety, or a cell-penetrating peptide.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the targeting moiety comprises a group of the formula:
wherein:
each instance of L′ and L″ is independently a bond that is substituted alkylene or substituted heteroalkylene;
each instance of G1, G2, and G3 is independently a sugar moiety, a folate moiety, or a cell-penetrating peptide; and
n is an integer from 1 to 10, inclusive.

### Claim 11 (depends on 1)

The composition of claim 1, wherein the targeting moiety comprises a group of the following formula:
wherein:
each instance of RN is independently hydrogen, substituted alkyl, substituted acyl, or a nitrogen protecting group; and
each instance of RO is independently hydrogen, substituted alkyl, substituted acyl, or an oxygen protecting group.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the synthetic RNA comprises at least one modified nucleotide and/or modified internucleotide linkage.

### Claim 13 (depends on 1)

A method of delivering a synthetic RNA to a cell, the method comprising delivering the composition of claim 1 to the cell.

### Claim 14

A pharmaceutical preparation comprising a complex, wherein the complex comprises:
a single-stranded nucleic acid non-covalently associated with an oligonucleotide;
wherein the single-stranded nucleic acid is at least 50 to 10,000 nucleotides longer than the oligonucleotide; and
wherein the oligonucleotide is covalently linked to one or more targeting moieties, wherein the one or more targeting moieties is of one of the following formulae:
wherein:
each instance of LA, LB, and LC is independently either absent or is a linker selected from the group consisting of substituted alkylene, substituted alkenylene, substituted alkynylene, substituted heteroalkylene, substituted heteroalkenylene, substituted heteroalkynylene, substituted acylene, substituted carbocyclylene, substituted heterocyclylene, substituted arylene, substituted heteroarylene, and combinations thereof; and
each instance of G1, G2, and G3 is independently a ligand.

### Claim 15 (depends on 14)

The pharmaceutical preparation of claim 14, wherein the complex is of the following formula:
wherein:
NA is a single-stranded nucleic acid;
OL is an oligonucleotide;
----- represents one or more non-covalent bonds;
LT is a covalent bond or a linker moiety; and
T is a targeting moiety;
wherein the single-stranded nucleic acid is 50 to 10,000 nucleotides longer than the oligonucleotide.

### Claim 16 (depends on 14)

The pharmaceutical preparation of claim 14, wherein the single-stranded nucleic acid is hybridized to the oligonucleotide.

### Claim 17 (depends on 14)

The pharmaceutical preparation of claim 14, wherein the targeting moiety comprises one or more ligands selected from a sugar moiety, a folate moiety, and a cell-penetrating peptide.

### Claim 18 (depends on 1)

The composition of claim 1, wherein the synthetic RNA is a synthetic mRNA that comprises at least one modified nucleotide and/or modified internucleotide linkage.

### Claim 19 (depends on 18)

The composition of claim 18, wherein the synthetic mRNA comprises a 3′ untranslated region (3′UTR).

### Claim 20 (depends on 19)

The composition of claim 19, wherein the 3′UTR comprises one or more binding sites for binding to the single-stranded oligonucleotide.

### Claim 21 (depends on 19)

The composition of claim 19, wherein the binding sites are not naturally present in human mRNA 3′UTRs.