# US 12,109,223 B2 · Polymer nanoparticle and DNA nanostructure compositions and methods for non-viral delivery

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,109,223 B2
- Document ID: US12109223B2
- Status: Granted
- Kind: B2
- Application number: 18/444,146
- Filed: 2024-02-16
- Published: 2024-06-13
- Granted: 2024-10-08
- Earliest claimed: 2020-12-03
- Assignees: Battelle Memorial Institute
- Applicants: Battelle Memorial Institute
- Inventors: Anthony D. Duong; Cherry Gupta; Kenneth R. Sims, Jr.; Danielle Huk; Jacob Lilly; Stephanie M. Kute; Mike Koeris
- Web page: https://patentgrep.com/patent/US12109223B2

## CPC

- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- A61K 47/6455: {Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids}
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 47/6929: {the form being a nanoparticle, e.g. an immuno-nanoparticle}
- A61K 47/6933: {the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol}
- A61K 47/6935: {the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol}
- C08F 293/005: {using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent}
- 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)}
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- B82Y 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites
- B82Y 35/00: Methods or apparatus for measurement or analysis of nanostructures
- B82Y 40/00: Manufacture or treatment of nanostructures
- C08F 2438/03: Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]
- C12N 2310/11: Antisense
- C12N 2310/16: Aptamers
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/531: Stem-loop; Hairpin
- 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)
- C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES

## Abstract

The invention relates to polymer nanoparticle and DNA nanostructure delivery compositions for non-viral delivery, and methods therefor. More particularly, the invention relates to polymer nanoparticle delivery compositions, such as reversible addition-fragmentation chain transfer (RAFT) polymer compositions, and DNA nanostructure delivery compositions, such as DNA origami compositions, for the delivery of more than one payload, or for the delivery of a nucleic acid construct payload of 3 kB or more, and methods therefor.

## Claims

### Claim 1

A RAFT block copolymer comprising
a. a first terminus comprising a first capping unit derived from a first chain transfer agent in a RAFT copolymerization process;
b. a first block prepared from one or more monomer units covalently attached to the first reactive functional unit, and having a molecular weight (Mn) in the range of about 20 kDa to about 80 kDa and a degree of polymerization in the range of about 20 to about 400;
c. a second block prepared from one or more monomer units covalently attached to the first block, and having a molecular weight (Mn) in the range of about 5 kDa to about 80 kDa and a degree of polymerization in the range of about 10 to about 500; and
d. a second terminus comprising a second capping unit derived from a second chain transfer agent;
wherein the RAFT block copolymer has one or more of an overall molecular weight (Mn) in the range of about 25 kDa to about 160 kDa, and overall degree of polymerization in the range of about 30 to about 900, a size in the range of about of about 10 to about 60 nm, and a maximum corona-to-core ratio (CCR) of about 1 to about 4.

### Claim 2 (depends on 1)

The RAFT block copolymer of claim 1, wherein the first block is prepared from one or more monomer units selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

### Claim 3 (depends on 1)

The RAFT block copolymer of claim 1, wherein the first block is prepared from one of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, or methyl methacrylate.

### Claim 4 (depends on 1)

The RAFT block copolymer of claim 1, wherein the second block is prepared from one or more monomer units selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

### Claim 5 (depends on 1)

The RAFT block copolymer of claim 1, wherein the second block is a random copolymer prepared from two different monomer units independently selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

### Claim 6 (depends on 1)

The RAFT block copolymer of claim 1, wherein the second block is a random copolymer prepared from three different monomer units independently selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

### Claim 7 (depends on 1)

The RAFT block copolymer of claim 1, wherein each chain transfer agent is independently selected from the group consisting of bis(carboxymethyl)trithiocarbonate, bis(2-amino-2-oxoethyl) trithiocarbonate, bis[4-(2-hydroxyethoxycarbonyl)benzyl] trithiocarbonate, 4-cyano-4-(ethylsulfanylthiocarbonyl) sulfanylvpentanoic acid, 4-cyano-4-((phenylcarbonothioyl)thio)pentanoic acid, and 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanoic acid.

### Claim 8 (depends on 1)

The RAFT block copolymer of claim 1, wherein the first capping unit is of the formula
wherein * represents a point of covalent attachment to the first block.

### Claim 9 (depends on 1)

The RAFT block copolymer of claim 1, wherein the second capping unit is of the formula
wherein * represents a point of covalent attachment to the second block, and R is —SC2-C12 alkyl or C6H5.

### Claim 10 (depends on 1)

A RAFT block copolymer conjugate comprising a RAFT block copolymer according to claim 1, and further comprising a biomolecule, drug, or label covalently attached to the RAFT block copolymer through a functional group on one of the first or second chain transfer agents that is incorporated into the RAFT block copolymer.

### Claim 11 (depends on 10)

The RAFT block copolymer complex of claim 10, wherein the payload comprises nucleic acids.

### Claim 12 (depends on 10)

The RAFT block copolymer complex of claim 10, wherein the nucleic acids comprise DNA or RNA.

### Claim 13 (depends on 10)

The RAFT block copolymer complex of claim 10, wherein the nucleic acids comprise a ribonucleoprotein.

### Claim 14 (depends on 10)

The RAFT block copolymer complex of claim 10, wherein the payload is a nucleic acid and the nucleic acid payload is of a size selected from the group consisting of 3 kB or more, 3.5 kB or more, 4 kB or more, 4.5 kB or more, 5 kB or more, 5.5 kB or more, 6 kB or more, 6.5 kB or more, 7 kB or more, 7.5 kB or more, 8 kB or more, and 8.5 kB or more.

### Claim 15 (depends on 1)

A RAFT block copolymer complex comprising a RAFT block copolymer according to claim 1, and further comprising a payload complexed to the RAFT block copolymer through electrostatic interaction.

### Claim 16 (depends on 15)

The RAFT block copolymer complex of claim 15, wherein the payload comprises nucleic acids.

### Claim 17 (depends on 15)

The RAFT block copolymer complex of claim 15, wherein the nucleic acids comprise DNA or RNA.

### Claim 18 (depends on 15)

The RAFT block copolymer complex of claim 15, wherein the payload is a nucleic acid and the nucleic acid payload is of a size selected from the group consisting of 3 kB or more, 3.5 kB or more, 4 kB or more, 4.5 kB or more, 5 kB or more, 5.5 kB or more, 6 kB or more, 6.5 kB or more, 7 kB or more, 7.5 kB or more, 8 kB or more, and 8.5 kB or more.

### Claim 19

A method of preparing a RAFT block copolymer comprising:
i. contacting a first chain transfer agent, a first initiator and one or more monomer units to provide a first block;
ii. contacting a second chain transfer agent, a second initiator and one or more monomer units to provide a second block
iii. contacting the first block and the second block under conditions capable of coupling the blocks to provide the RAFT block copolymer.

### Claim 20 (depends on 19)

The method of claim 19, wherein the first chain transfer agent is a diamino or dihydroxy chain transfer agent, and the second chain transfer agent is a dicarboxylic acid chain transfer agent.

### Claim 21 (depends on 19)

The method of claim 19, wherein the first chain transfer agent is bis(2-amino-2-oxoethyl) trithiocarbonate or bis[4-(2-hydroxyethoxycarbonyl)benzyl] trithiocarbonate, and the second chain transfer agent is bis(carboxymethyl)trithiocarbonate.

### Claim 22 (depends on 19)

The method of claim 19, wherein the first chain transfer agent is a dicarboxylic acid chain transfer agent, and the second chain transfer agent is a diamino or dihydroxy chain transfer agent.

### Claim 23 (depends on 19)

The method of claim 19, wherein the first chain transfer agent is bis(carboxymethyl)trithiocarbonate, and the second chain transfer agent is bis(2-amino-2-oxoethyl) trithiocarbonate or bis[4-(2-hydroxyethoxycarbonyl)benzyl] trithiocarbonate.

### Claim 24 (depends on 19)

The method of claim 19, wherein the one or more monomer units in step (i) are independently selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

### Claim 25 (depends on 19)

The method of claim 19, wherein the one or more monomer units in step (i) is one of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, or methyl methacrylate.

### Claim 26 (depends on 19)

The method of claim 19, wherein the one or more monomer units in step (ii) are independently selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

### Claim 27 (depends on 19)

The method of claim 19, wherein the one or more monomer units in step (ii) are two different monomer units independently selected from the group consisting of 2-dimethylaminoethyl acrylate, 2-(diethylamino) ethyl methacrylate, 2-(diisopropylamino) ethyl methacrylate, butyl methacrylate, ethyl acrylic acid, propyl acrylic acid, (hydroxyethyl)methacrylate, and methyl methacrylate.

### Claim 28 (depends on 19)

The method of claim 19, wherein the one or more monomer units in step (ii) are 2-dimethylaminoethyl acrylate, butyl methacrylate, and propyl acrylic acid; or 2-dimethylaminoethyl acrylate and butyl methacrylate; or 2-dimethylaminocthyl acrylate, butyl methacrylate, and ethyl acrylic acid.

### Claim 29 (depends on 19)

The method of claim 19, wherein the RAFT block copolymer has one or more of an overall molecular weight (Mn) in the range of about 25 kDa to about 160 kDa, and overall degree of polymerization in the range of about 30 to about 900, a size in the range of about of about 10 to about 60 nm, and a maximum corona-to-core ratio (CCR) of about 1 to about 4.

### Claim 30

A method of preparing a library of RAFT block copolymers comprising:
i. providing an array of reaction mixtures in a multiwell plate, wherein each well comprises a mixture of a first chain transfer agent, a first initiator, one or more monomer units, and optionally a solvent or solvent mixture;
ii. reacting the mixture in each well under conditions that promote RAFT copolymerization to provide a series of first block copolymers in the wells of the multiwell plate;
iii. quenching the reactions in the wells of the multiwell plate;
iv. purifying the first block copolymer in each well of the multiwell plate;
v. optionally characterizing the first block copolymer in each well of the multiwell plate;
vi. optionally purifying the first block copolymer in each well of the multiwell plate;
vii. contacting to the first block copolymer in each well of the multiwell plate with a second array of reaction mixtures comprising a second chain transfer agent, a second initiator, and one or more monomer units, and optionally a solvent or solvent mixture;
viii. reacting the components of step (vii) under conditions that promote RAFT copolymerization to provide a series of RAFT block copolymers in the wells of the multiwell plate;
ix. quenching the reactions in the wells of the multiwell plate;
x. purifying the RAFT block copolymer in each well of the multiwell plate; and
xi. optionally characterizing the RAFT block copolymer in each well of the multiwell plate.