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Regulated genetic suicide mechanism compositions and methods

Inactive Publication Date: 2010-05-06
VAXIION THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]Some embodiments provide a minicell-producing bacteria comprising: an expressible gene encoding a minicell-producing gene product that modulates one or more of septum formation, binary fission, and chromosome segregation; and an expressible gene encoding an endonuclease, where the chromosome of the minicell-producing bacteria comprises one or more recognition sites of the endonuclease. In some embodiments, the minicell-producing gene is a cell division gene. The cell division gene includes, but is not limited to ftsZ, sulA, ccdB, and sfiC. In some embodiments, the minicell-producing gene is expressed under the control of an inducible promoter. In some embodiments, the endonuclease gene is located on the chromosome of the minicell-producing bacteria. In some embodiments, the endonuclease is a homing endonuclease. The homing endonuclease includes, but is not limited to, I-CeuI, PI-SceI, I-ChuI, I-CpaI, I-SceIII, I-CreI, I-MsoI, I-SceII, I-SceIV, I-CsmI, I-DmoI, I-PorI, PI-TliI, PI-TliII, and PI-ScpI. In some embodiments, the endonuclease is expressed under the control of an inducible promoter. In some embodiments, the minicell-producing bacteria is a Gram-negative bacteria. The Gram-negative bacteria includes, but is not limited to Campylobacter jejuni, Lactobacillus spp., Neisseria gonorrhoeae, Legionella pneumophila, Salmonella spp., Shigella spp Pseudomonas aeruginosa, and Escherichia coli. In some embodiments, the minicell-producing bacteria comprising a gene encoding a gene product that is involved in lipopolysaccharide synthesis, where the gene is genetically modified compared to a corresponding wild-type gene. In some embodiments, the gene is a msbB gene that encodes a gene product that causes the bacteria to produce an altered lipid A molecule compared to lipid A molecules in a corresponding wild-type bacteria. In some embodiments, the altered lipid A molecule is deficient with respect to the addition of myristolic acid to the lipid A portion of the lipopolysaccharide molecule compared to lipid A molecules in a corresponding wild-type bacteria. The minicell-producing bacteria can be a Gram-positive bacteria. The Gram-positive bacteria includes, but is not limited to, Staphylococcus spp., Streptococcus spp., Bacillus subtilis or Bacillus cereus. In some embodiments, the minicell-producing bacteria comprising a gene that is involved in homologous recombination, where the gene is genetically modified compared to a corresponding wild-type gene, where the minicell-producing bacteria is deficient in DNA damage repair.
[0011]Some other embodiments provides a method of making minicells, comprising culturing the minicell-producing bacteria disclosed herein and substantially separating minicells from the minicell-producing parent cells, thereby generating a composition comprising minicells. In some embodiments, the method further comprises inducing minicell formation from the minicell-producing parent cell. In some embodiments, the method further comprises inducing expression of the gene encoding the endonuclease. In some embodiments, minice

Problems solved by technology

In essence, minicells are small, metabolically active replicas of normal bacterial cells with the exception that they contain no chromosomal DNA and as such, are non-dividing and non-viable.

Method used

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  • Regulated genetic suicide mechanism compositions and methods
  • Regulated genetic suicide mechanism compositions and methods
  • Regulated genetic suicide mechanism compositions and methods

Examples

Experimental program
Comparison scheme
Effect test

example 1

Effects of I-CeuI on the Growth of E. coli

[0150]E. coli TOP10 cells were transformed with pVX-55 expression vector (SEQ ID NO:6). The pVX-55 expression vector contains an I-CeuI gene under the control of the rhamnose inducible pRHA promoter system. The transformed E. coli cell culture was grown in LB broth supplemented with Kanamycin (50 μg / ml). Glucose (0.2%) was added into the cell culture at 0 hours, and rhamnose (10 mM) were added into the cell culture at 2.22 hours and OD of 0.3. Growth of the bacterium was monitored by measuring absorbance at 600 nm. FIG. 1 shows that the growth of the E. coli cell culture was significantly reduced by the induction of I-CeuI homing endonuclease.

example 2

Effects of I-CeuI on Viability of E. coli

[0151]E. coli TOP10 cells were transformed with pVX-55 expression vector. The pVX-55 expression vector contains an I-CeuI gene under the control of the rhamnose inducible pRHA promoter system. The E. coli cells was cultured in LB broth with Kanamycin (50 μg / ml) supplemented with glucose (0.2%) or rhamnose (10 mM) before spotted on LB agar plates. Viable cell populations (CFU / ml) were determined via colony counts on LB agar plates. No recovery of colonies was observed. FIG. 2 shows that the number of viable E. coli cells was significantly reduced by the induction of I-CeuI homing endonuclease.

example 3

Simultaneous Overexpression of ftsZ and Induction of I-CeuI (MSM) Lead to Higher Minicell Yields

[0152]An E. coli strain containing IPTG inducible ftsZ and minCDE deletion mutation were grown in LB medium. A ftsZ construct (Ptac::ftsZ) and a ftsZ construct with the heat inducible I-CeuI based suicide system (Ptac::ftsZΩCI857ts:I-CeuI) were integrated into the αttBλ site on the chromosome of the minicell-producing E. coli cells, respectively. Minicell productions of the Ptac::ftsZ strain was conducted at 37° C. and minicell products of the Ptac::ftsZΩCI857ts::I-CeuI strain was conducted at 42° C. to induce the I-CeuI based suicide system. Minicells were purified via differential purifications. FIG. 3A shows the numbers of minicells purified from each ml of LB cultures used for minicell productions. FIG. 3B shows the ratios of minicell yields of the IPTG inducible ftsZ strains against the minCDE-strain. FIGS. 3A and 3B demonstrates that when ftsZ and I-CeuI were simultaneously overexpr...

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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.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims the benefit of U.S. Provisional Patent Applications 61 / 075,687 filed Jun. 25, 2008 and 61 / 168,457 filed Apr. 10, 2009. The contents of each of these related applications are hereby incorporated by reference in their entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]Embodiments of the present invention relate to compositions and methods for the production, purification, formulation, and use of eubacterial minicells as targeted delivery vehicles for in vivo and in vitro nucleic acid, protein, and small molecule drug delivery as well as a targeted in vivo imaging and diagnostic technology.[0004]2. Description of the Related Art[0005]The following description is provided to aid in understanding the present disclosure, but is not admitted to describe or constitute prior art to the present disclosure. The contents of the articles, patents, and patent applications, and all other documents and electro...

Claims

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Application Information

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IPC IPC(8): C12N1/20
CPCC12N1/08C12N9/22C12N15/70C12N15/74A61K9/5052A61K9/5068Y02A50/475A61K38/00A61K38/43Y02A50/481Y02A50/478Y02A50/47Y02A50/473A61K31/7088A61P31/04Y02A50/30
Inventor GIACALONE, MATTHEW J.MALOY, STANLEYTSUJI, SHINGO
Owner VAXIION THERAPEUTICS