# US 2010/0167358 A1 · Compositions and processes for improved plasmid dna production

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- Number: US 2010/0167358 A1
- Document ID: US20100167358A1
- Status: Published application
- Kind: A1
- Application number: 11/814,294
- Filed: 2006-01-27
- Published: 2010-07-01
- Earliest claimed: 2005-01-28
- Assignees: Nature Technology Corporation
- Inventors: James A. Williams
- Web page: https://patentgrep.com/patent/US20100167358A1

## CPC

- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
- C12N 15/69: Increasing the copy number of the vector
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Improvements in plasmid DNA production technology are needed to insure the economic feasibility of future DNA vaccines and DNA therapeutics. General methods are described, by means of which it is possible to dramatically increase plasmid DNA productivity. These processes feature RNA based inducers of plasmid copy number.

## Claims

### Claim 1

A method for production of covalently closed super-coiled plasmid DNA comprising the steps of: A) growing the bacterial cells containing a plasmid, cosmid, or bacterial artificial chromosome replicon; and B) increasing plasmid replication by induction of anti-RNAI; and C) continuing growth to accumulate plasmid product; whereby said method increases plasmid yield.

### Claim 2 (depends on 1)

The method of claim 1 wherein said plasmid contains a ColE1-derived replication origin.

### Claim 3 (depends on 1)

The method of claim 1 wherein said plasmid contains a pMB1-derived replication origin

### Claim 4 (depends on 1)

The method of claim 1 wherein said plasmid contains a pMB1 replication origin containing the pUC G to A mutation.

### Claim 5 (depends on 1)

The method of claim 1 wherein said plasmid contains a pBR322-derived replication origin.

### Claim 6 (depends on 1)

The method of claim 1 wherein plasmid production is constitutively induced

### Claim 7 (depends on 1)

The method of claim 1 wherein said anti-RNAI is a molecule selected from the group consisting of: antisense RNA; ribozyme; protein; peptide; small molecule; chemical; aptamer; single stranded RNA; double stranded RNA; hairpin RNA.

### Claim 8 (depends on 1)

The method of claim 1 wherein said anti-RNAI is an antisense RNA selected from the group consisting of: antiRNAI(1-108); antiRNAI(10-108); antiRNAI(10-108)2x; antiRNAI(10-108)3x; antiRNAI(10-108)3.5x; antiRNAI(10-108)4x.

### Claim 9

9) A composition of matter comprising one or more strains of bacteria containing: A) at least one anti-RNAI antisense inducer; and B) at least one plasmid; wherein the anti-RNAI antisense inducer is used to improve the yield of the plasmid obtained from culture.

### Claim 10 (depends on 9)

10) The composition of claim 9 wherein said anti-RNAI antisense inducer encodes one or more copies of anti-RNAI(10-108).

### Claim 11 (depends on 9)

11) The composition of claim 9 wherein said anti-RNAI antisense inducer encodes one or more copies of anti-RNAI(1-108).