Regulated genetic suicide mechanism compositions and methods
Inventors
Giacalone, Matthew J • Maloy, Stanley • Tsuji, Shingo
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9574196-B2
Publication Date
2017-02-21
Expiration Date
Abstract
Embodiments of the present invention relates to the incorporation and use of a regulated genetic suicide mechanism for use in the improved purification of biologics, including adjunct use in various eubacterial minicell production and purification methodologies. Described herein are high-yield eubacterial minicell-producing strains with genetic modifications that comprise a regulated genetic suicide mechanism that irreparably destroys the parent cell chromosome such that live parental cells in a culture can be functionally eliminated at any time during the course of a minicell production and purification run. Embodiments of the present invention also describe methods useful in the elimination of live parental cells during the production of other cell-based biologics.
Core Innovation
The invention provides an eubacterial minicell derived from minicell-producing bacteria in which an expressible cell division gene is present. The cell division gene is selected from a specified group including zipA, sulA, secA, dicA, dicB, dicC, dicF, ftsA, ftsI, ftsN, ftsK, ftsL, ftsQ, ftsW, ftsZ, minC, minD, minE, seqA, ccdB, sfiC, and ddlB. The minicell-producing bacteria further comprises an expressible transgene encoding a homing endonuclease.
The homing endonuclease gene is a transgene, and the chromosome of the minicell-producing bacteria comprises one or more recognition sites of the homing endonuclease. The document describes a regulated genetic suicide mechanism in which induction triggers irreparable double-stranded chromosomal breaks in parent cells, leading to irreversible filamentation and death to eliminate viable parental contamination during minicell purification.
In examples, induction associated with the homing endonuclease shows reduced parental growth and viability and increases minicell yield compared to a cell-division gene alone. The document further reports TUNEL evidence supporting double-strand chromosomal breaks and a high level of parent-cell death after induction, alongside greatly reduced parental contamination after minicell purification. The invention also includes characterizing and modifying lipopolysaccharide activity by deleting msbB to alter lipid A, including deficiency of myristolic acid addition.
Claims Coverage
The document contains two independent claims, an eubacterial minicell composition and a method of making minicells. Across these independent claims, the inventive coverage centers on combining a selected expressible cell division gene with an expressible transgene encoding a homing endonuclease, while placing one or more homing endonuclease recognition sites on the chromosome and substantially separating minicells from parent cells.
Eubacterial minicell with cell division gene and homing endonuclease recognition sites
An eubacterial minicell derived from minicell-producing bacteria, wherein the minicell-producing bacteria comprises an expressible cell division gene selected from the group consisting of zipA, sulA, secA, dicA, dicB, dicC, dicF, ftsA, ftsI, ftsN, ftsK, ftsL, ftsQ, ftsW, ftsZ, minC, minD, minE, seqA, ccdB, sfiC, and ddlB; and an expressible gene encoding a homing endonuclease, wherein the homing endonuclease gene is a transgene, and wherein the chromosome of the minicell-producing bacteria comprises one or more recognition sites of the homing endonuclease.
Method of making minicells with minicell separation
A method of making minicell comprising culturing a minicell-producing bacteria, wherein the minicell-producing bacteria comprises an expressible cell division gene selected from the group consisting of zipA, sulA, secA, dicA, dicB, dicC, dicF, ftsA, ftsI, ftsN, ftsK, ftsL, ftsQ, ftsW, ftsZ, minC, minD, minE, seqA, ccdB, sfiC, and ddlB; an expressible gene encoding a homing endonuclease, wherein the homing endonuclease gene is a transgene; and wherein the chromosome of the minicell-producing bacteria comprises one or more recognition sites of the homing endonuclease; and substantially separating minicells from the minicell-producing parent cells, thereby generating a composition comprising minicells.
Overall, the independent claims define minicell-producing bacteria that express a selected cell division gene and express a transgene encoding a homing endonuclease, with chromosome-encoded recognition sites, and they require substantially separating minicells from parent cells to generate a minicell composition.
Stated Advantages
Improves eubacterial minicell production and safety during minicell purification by eliminating viable parental contamination.
Greatly increases minicell yield compared to cell-division gene alone and compared to minCDE mutants.
Reduces parental contamination by greater than 800-fold.
Provides evidence of double-strand chromosomal breaks and parent-cell death upon induction.
Reduces LPS inflammatory or toxic activity by modifying lipid A through msbB deletion.
Documented Applications
Minicell purification workflow in which induced parent-cell death eliminates viable parental contamination while generating a composition comprising minicells.
Synthetic biology application or workflow integration of the regulated genetic suicide mechanism for minicell production.
Interested in licensing this patent?