# US 12,595,501 B2 · Expression of products from nucleic acid concatemers

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- Number: US 12,595,501 B2
- Document ID: US12595501B2
- Status: Granted
- Kind: B2
- Application number: 16/440,511
- Filed: 2019-06-13
- Published: 2020-12-17
- Granted: 2026-04-07
- Earliest claimed: 2019-06-13
- Assignees: Global Life Sciences Solutions USA LLC
- Applicants: General Electric Company
- Inventors: Brian Michael Davis; Erik Leeming Kvam; John Richard Nelson; Lisa Anne Lowery; Wei Gao
- Web page: https://patentgrep.com/patent/US12595501B2

## CPC

- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/67: General methods for enhancing the expression
- C12N 15/86: Viral vectors
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 2015/8518: {expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles}
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N 2740/16051: Methods of production or purification of viral material
- C12N 2750/14123: Virus like particles [VLP]
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2750/14151: Methods of production or purification of viral material
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided are techniques for generating expression products using one or more nucleic acid concatemers that include tandem repeats of a nucleic acid sequence encoding the expression product or products. In one embodiment, different expression products may be co-expressed using a concatemer mixture of a first nucleic acid concatemer and a second nucleic acid concatemer having a predefined ratio to one another.

## Claims

### Claim 1

A method comprising:
formulating a concatemer mixture comprising at least a first nucleic acid concatemer and a second nucleic acid concatemer having a predefined ratio to one another, wherein the first nucleic acid concatemer comprises tandem repeats of a first nucleic acid sequence and wherein the second nucleic acid concatemer comprises tandem repeats of a second nucleic acid sequence; and
co-expressing the concatemer mixture to generate a first expression product from the first nucleic acid sequence and a second expression product from the second nucleic acid sequence,
wherein the first nucleic acid concatemer, the second nucleic acid concatemer, or both are over 10 kb in length,
wherein the ratio of first expression product to the second expression product is proportional to the predefined ratio of the first nucleic acid concatemer to the second nucleic acid concatemer in the concatemer mixture, and
wherein the molar ratio of the first concatemer to the second concatemer is between 1:1 and 1:10.

### Claim 2 (depends on 1)

The method of claim 1, comprising generating the first nucleic acid concatemer and the second nucleic acid concatemer using rolling circle amplification.

### Claim 3 (depends on 2)

The method of claim 2, comprising using the rolling circle amplification to amplify circular or plasmid DNA to generate the first nucleic acid concatemer and/or the second nucleic acid concatemer.

### Claim 4 (depends on 3)

The method of claim 3, wherein the first tandem repeats and/or the second tandem repeats are tandem repeats of the circular or plasmid DNA.

### Claim 5 (depends on 1)

The method of claim 1, comprising transfecting cultured cells with the concatemer mixture to cause the co-expressing or wherein the co-expressing is in a cell-free expression system.

### Claim 6 (depends on 1)

The method of claim 1, comprising collecting the first expression product and the second expression product.

### Claim 7 (depends on 1)

The method of claim 1, wherein one or both of the first nucleic acid concatemer or the second nucleic acid concatemer is unprocessed before the co-expressing.

### Claim 8 (depends on 1)

The method of claim 1, wherein the first expression product is an envelope or packing protein of a virus and the second expression product comprises a transgene of the virus, wherein the first expression product and the second expression product form a viral vector for delivering the transgene.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first expression product is a nucleic acid product and the second expression product is a protein product.

### Claim 10 (depends on 9)

The method of claim 9, wherein the first expression product forms a complex with the second expression product.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first expression product acts on a third nucleic acid concatemer in the concatemer mixture.

### Claim 12

A method comprising:
formulating a mixture comprising at least a first nucleic acid concatemer, a second nucleic acid concatemer, and a third nucleic acid concatemer in a predefined ratio, wherein each of the nucleic acid concatemers comprises tandem repeats of two or more nucleic acid sequences; and
co-expressing the concatemer mixture to generate a first expression product, a second expression product, and a third expression product from each nucleic acid concatemer in the mixture,
wherein the first nucleic acid concatemer, the second nucleic acid concatemer, the third nucleic acid concatemer, or any combination thereof are over 10 kb in length,
wherein the molar ratio of the the first expression product, the second expression product, and the third expression product is proportional to the predefined molar ratio of the first nucleic acid concatemer, the second nucleic acid concatemer, and the third nucleic acid concatemer in the concatemer mixture, and
wherein the molar ratio of the first concatemer to the second concatemer to the third concatemer is C1:C2:Cn, where C1, C2, and Cn each is between 1 and 10.

### Claim 13 (depends on 12)

The method of claim 12, wherein the nucleic acid sequence of an individual concatemer in the concatemer mixture comprises a plurality of expression sequences that, when expressed to generate the two or more expression products, generate a plurality of proteins.

### Claim 14 (depends on 12)

The method of claim 12, wherein the nucleic acid sequence of an individual concatemer in the concatemer mixture comprises a plurality of expression sequences that, when expressed to generate the two or more expression products, generate a mix comprising at least one protein expression product and at least one nucleic acid expression product.

### Claim 15

A method, comprising:
amplifying at least one template comprising a first nucleic acid sequence using strand-displacement rolling circle amplification to a generate a first concatemer comprising tandem repeats of the first nucleic acid sequence;
contacting the first concatemer with a second concatemer comprising tandem repeats of a second nucleic acid sequence to form a concatemer mixture having a predefined ratio of the first nucleic acid concatemer to the second nucleic acid concatemer; and
co-expressing the concatemer mixture to generate a first expression product from the first nucleic acid sequence and a second expression product from the second nucleic acid sequence, wherein a ratio of the first expression product to the second expression product is proportional to the predefined ratio of the first nucleic acid concatemer to the second nucleic acid concatemer in the concatemer mixture,
wherein the molar ratio of the first concatemer to the second concatemer is between 1:1 and 1:10, and
wherein the first concatemer, the second concatemer, or both are over 10 kb in length.

### Claim 16 (depends on 15)

The method of claim 15, comprising allowing the first expression product to form a complex with the second expression product.

### Claim 17 (depends on 15)

The method of claim 15, wherein the first expression product and the second expression product comprise different viral products from a same virus.

### Claim 18 (depends on 17)

The method of claim 17, wherein the same virus is an adenovirus or a lentivirus.

### Claim 19 (depends on 15)

The method of claim 15, wherein the first expression product comprises a viral mRNA and the second expression product comprises a plurality of viral packaging proteins.

### Claim 20

A method comprising:
formulating a mixture comprising at least one nucleic acid concatemer and at least one plasmid having a predefined ratio to one another, wherein the at least one nucleic acid concatemer comprises tandem repeats of a first nucleic acid sequence and wherein the at least one plasmid comprises a second nucleic acid sequence; and
co-expressing the mixture to generate a first expression product from the first nucleic acid sequence and a second expression product from the second nucleic acid sequence,
wherein the molar ratio of the first concatemer to the plasmid is between 1:1 and 1:10,
wherein the first nucleic acid concatemer is over 10 kb in length, and
wherein the co-expression of the first expression product and the second expression product is ratiometric based on the predefined ratio.

### Claim 21 (depends on 1)

The method of claim 1, wherein the co-expression of the first expression product and the second expression product is ratiometric based on the predefined ratio.

### Claim 22 (depends on 12)

The method of claim 12, wherein the co-expression of the first expression product, the second expression product, and the third expression product is ratiometric based on the predefined ratio.