# US 7,611,883 B2 · Plasmid maintenance > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,611,883 B2 - Document ID: US7611883B2 - Status: Granted - Kind: B2 - Application number: 10/579,981 - Filed: 2004-11-22 - Published: 2007-05-17 - Granted: 2009-11-03 - Earliest claimed: 2003-11-20 - Assignees: Cobra Biologics Limited - Inventors: Rocky Marc Cranenburgh - Web page: https://patentgrep.com/patent/US7611883B2 ## CPC - C12N 15/72: Expression systems using regulatory sequences derived from the lac-operon - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression - A61K 2035/11: {Medicinal preparations comprising living procariotic cells} - Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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) - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract The invention relates to a system for stable maintenance of a plasmid, to host cells for use in this system and to methods of using the system to obtain a plasmid useful in medical applications. In particular, the invention provides transformed host cell containing: i) a chromosomal gene which inhibits cell growth; and ii) a plasmid encoding an antisense sequence, wherein the antisense sequence encoded by the plasmid inhibits the action of the chromosomal gene, thereby permitting cell growth and a method for stable maintenance of a plasmid in a host cell in vivo. ## Claims ### Claim 1 An isolated transformed host cell comprising i) a chromosomal gene which inhibits cell growth operably linked to a regulatory sequence; and ii) a plasmid comprising an origin of replication encoding an antisense sequence which binds to mRNA transcribed from the regulatory sequence, wherein the binding of the antisense sequence encoded by the origin of replication of the plasmid to mRNA transcribed from the regulatory sequence inhibits the action of the chromosomal gene, thereby permitting cell growth. ### Claim 2 (depends on 1) An isolated transformed host cell according to claim 1 wherein the plasmid comprises a cloning site for insertion of a gene of interest. ### Claim 3 (depends on 2) An isolated transformed host cell according to claim 2, wherein the plasmid further comprises a gene of interest. ### Claim 4 (depends on 1) An isolated transformed host cell according to claim 1, wherein the antisense sequence encoded by the plasmid is RNAI or a portion thereof and the regulatory sequence operatively linked to the chromosomal gene encodes RNAII or a portion thereof. ### Claim 5 (depends on 1) An isolated transformed host cell according to claim 1, wherein the antisense sequence encoded by the plasmid is RNAII or a portion thereof and the regulatory sequence operatively linked to the chromosomal gene encodes RNAI or a portion thereof. ### Claim 6 (depends on 1) An isolated transformed host cell according to claim 1, wherein the cell is in culture in vitro. ### Claim 7 (depends on 1) An isolated transformed host cell according to claim 1 which is a prokaryotic cell. ### Claim 8 (depends on 7) An isolated transformed host cell according to claim 7 which is a bacterial cell. ### Claim 9 (depends on 8) An isolated transformed host cell according to claim 8, wherein the cell is a gram negative bacterial cell. ### Claim 10 (depends on 9) An isolated transformed host cell according to claim 9, wherein the cell is an E. coli cell or a Salmonella cell. ### Claim 11 (depends on 8) An isolated transformed host cell according to claim 8, wherein the cell is a gram positive bacterial cell. ### Claim 12 (depends on 11) An isolated transformed host cell according to claim 11, wherein the cell is a Bacillus cell. ### Claim 13 (depends on 8) An isolated transformed host cell according to claim 8 which is an attenuated cell. ### Claim 14 (depends on 1) An isolated transformed host cell according to claim 1 wherein the cell is a eukaryotic cell. ### Claim 15 (depends on 14) An isolated transformed host cell according to claim 14 wherein the cell is a fungi. ### Claim 16 (depends on 14) An isolated transformed host cell according to claim 14, wherein the cell is a plant cell. ### Claim 17 (depends on 14) An isolated transformed host cell according to claim 14 wherein the cell is an animal cell. ### Claim 18 (depends on 1) An isolated transformed host cell according to claim 1, wherein the chromosomal gene is a toxin gene. ### Claim 19 (depends on 18) An isolated transformed host cell according to claim 18, wherein the toxin gene is sacB. ### Claim 20 (depends on 1) An isolated transformed host cell according to claim 1, wherein the chromosomal gene encodes a repressor protein that inhibits expression of a second chromosomal gene essential for cell growth. ### Claim 21 (depends on 20) An isolated transformed host cell according to claim 20, wherein the second chromosomal gene is conditionally essential for cell growth. ### Claim 22 (depends on 20) An isolated transformed host cell according to claim 20 wherein the chromosomal gene encodes the repressor lacI and the second chromosomal gene is operatively linked to a lac operator and promoter. ### Claim 23 (depends on 20) An isolated transformed host cell according to claim 20 wherein the chromosomal gene is dapD or fabA. ### Claim 24 (depends on 1) An isolated transformed host cell according to claim 1, wherein the chromosomal gene encodes an antisense sequence that inhibits expression of a second chromosomal gene essential for cell growth. ### Claim 25 (depends on 24) An isolated transformed host cell according to claim 24, wherein the antisense sequence encoded by the chromosomal gene inhibits expression of the second chromosomal gene by binding to the chromosomal gene. ### Claim 26 (depends on 24) An isolated transformed host cell according to claim 24, wherein the antisense sequence encoded by the chromosomal gene inhibits expression of the second chromosomal gene by binding to mRNA transcribed from the second chromosomal gene. ### Claim 27 (depends on 24) An isolated transformed host cell according to claim 24 wherein the second chromosomal gene is conditionally essential for cell growth. ### Claim 28 (depends on 24) An isolated transformed host cell according to claim 24, wherein the second chromosomal gene is dapD or fabA. ### Claim 29 (depends on 1) An isolated transformed host cell according to claim 1 wherein the chromosomal gene operably linked to a regulatory sequence is under the control of a constitutive promoter. ### Claim 30 (depends on 1) An isolated transformed host cell according claim 1 wherein the chromosomal gene operably linked to a regulatory sequence is under the control of an inducible promoter. ### Claim 31 (depends on 1) A method of maintaining a plasmid in a host cell in vitro comprising the step of culturing an isolated transformed host cell according to claim 1 under conditions sufficient to permit said cell to grow. ### Claim 32 (depends on 31) A method of producing plasmid DNA comprising culturing an isolated transformed host cell according to the method of claim 31 and isolating the plasmid DNA. ### Claim 33 (depends on 31) A method of producing a recombinant protein comprising culturing an isolated transformed host cell comprising a plasmid encoding a protein of interest according to the method of claim 31 and isolating the protein from the cell. ### Claim 34 (depends on 1) A composition comprising an isolated transformed host cell according to claim 1 together with a pharmaceutically acceptable excipient, diluent or buffer. ### Claim 35 (depends on 3) A method of delivering a gene to a patient comprising administering to the patient an isolated transformed host cell according to claim 3, wherein the isolated transformed host cell is suitable for veterinary or human therapeutic use. ### Claim 36 (depends on 1) A method of maintaining a plasmid in a recipient organism comprising introducing an isolated transformed host cell according to claim 1 into said organism, wherein said chromosomal gene in said transformed host cell is essential for cell growth in vivo and the transformed host cell is suitable for veterinary or human therapeutic use. ### Claim 37 (depends on 1) A method of immunizing a patient against a disease caused by a pathogen comprising administering an isolated transformed host cell according to claim 1 to the patient, wherein the isolated transformed host cell is suitable for veterinary or human therapeutic use.