Regulated genetic suicide mechanism compositions and methods

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.

1 . 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,   wherein the chromosome of the minicell-producing bacteria comprises one or more recognition sites of the endonuclease.   
     
     
         2 . The minicell-producing bacteria of  claim 1 , wherein the minicell-producing gene is a transgene. 
     
     
         3 . The minicell-producing bacteria of  claim 1 , wherein the endonuclease gene is a transgene. 
     
     
         4 . The minicell-producing bacteria of  claim 1 , wherein the minicell-producing gene is a cell division gene. 
     
     
         5 . The minicell-producing bacteria of  claim 4 , wherein the cell division gene is selected from the group consisting of ftsZ, sulA, ccdB, and sfiC. 
     
     
         6 . The minicell-producing bacteria of  claim 5 , wherein the cell division gene is ftsZ. 
     
     
         7 . The minicell-producing bacteria of  claim 6 , wherein the ftsZ comprises a nucleic acid sequence of SEQ ID NO:3. 
     
     
         8 . The minicell-producing bacteria of  claim 1 , wherein the minicell-producing gene is expressed under the control of an inducible promoter. 
     
     
         9 . The minicell-producing bacteria of  claim 8 , wherein the promoter is a temperature-sensitive promoter. 
     
     
         10 . The minicell-producing bacteria of  claim 8 , wherein the promoter is inducible by the presence of one or more chemical compounds. 
     
     
         11 . The minicell-producing bacteria of  claim 1 , wherein the endonuclease gene is located on the chromosome of the minicell-producing bacteria. 
     
     
         12 . The minicell-producing bacteria of  claim 1 , wherein the endonuclease is a homing endonuclease. 
     
     
         13 . The minicell-producing bacteria of  claim 12 , wherein the endonuclease is selected from the group consisting of I-CeuI, PI-SceI, I-ChuI, I-CpaI, I-SceIII, I-CreI, I-MsoI, I-SceII, I-SceIV, I-CsmI, I-DomI, I-PorI, PI-TliI, PI-TliII, and PI-ScpI. 
     
     
         14 . The minicell-producing bacteria of  claim 13 , wherein the endonuclease is I-CeuI. 
     
     
         15 . The minicell-producing bacteria of  claim 14 , wherein the I-CeuI comprises an amino acid sequence of SEQ ID NO:4. 
     
     
         16 . The minicell-producing bacteria of  claim 1 , wherein the endonuclease is expressed under the control of an inducible promoter. 
     
     
         17 . The minicell-producing bacteria of  claim 16 , wherein the promoter is a temperature-sensitive promoter. 
     
     
         18 . The minicell-producing bacteria of  claim 16 , wherein the promoter is a inducible by the presence of one or more chemical compounds. 
     
     
         19 . The minicell-producing bacteria of  claim 1 , wherein the minicell-producing bacteria is a Gram-negative bacteria. 
     
     
         20 . The minicell-producing bacteria of  claim 19 , wherein the Gram-negative bacteria is selected from the group consisting of  Campylobacter jejuni, Lactobacillus  spp.,  Neisseria gonorrhoeae, Legionella pneumophila, Salmonella  spp.,  Shigella  spp.,  Pseudomonas aeruginosa , and  Escherichia coli.    
     
     
         21 . The minicell-producing bacteria of  claim 19 , comprising a gene encoding a gene product that is involved in lipopolysaccharide synthesis, wherein the gene is genetically modified compared to a corresponding wild-type gene. 
     
     
         22 . The minicell-producing bacteria of  claim 21 , wherein 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. 
     
     
         23 . The minicell-producing bacteria of  claim 22 , wherein 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. 
     
     
         24 . The minicell-producing bacteria of  claim 1 , wherein the minicell-producing bacteria is a Gram-positive bacteria. 
     
     
         25 . The minicell-producing bacteria of  claim 24 , wherein the Gram-positive bacteria is selected from the group consisting of  Staphylococcus  spp.,  Streptococcus  spp.,  Bacillus subtilis  and  Bacillus cereus.    
     
     
         26 . The minicell-producing bacteria of  claim 1 , comprising a gene that is involved in homologous recombination, wherein the gene is genetically modified compared to a corresponding wild-type gene, wherein the minicell-producing bacteria is deficient in DNA damage repair. 
     
     
         27 . A method of making minicells, comprising
 culturing the minicell-producing bacteria of  claim 1 ; and   substantially separating minicells from the minicell-producing parent cells, thereby generating a composition comprising minicells.   
     
     
         28 . The method of  claim 27 , further comprising
 inducing minicell formation from the minicell-producing parent cell.   
     
     
         29 . The method of  claim 27 , further comprising
 inducing expression of the gene encoding the endonuclease.   
     
     
         30 . The method of  claim 28 , wherein minicell formation is induced by the presence of one or more chemical compound selected from the group consisting of isopropyl β-D-1-thiogalactopyranoside (IPTG), rhamnose, arabinose, xylose, fructose, melbiose and tetracycline. 
     
     
         31 . The method of  claim 29 , wherein the expression of the gene encoding the endonuclease is induced by a change in temperature. 
     
     
         32 . The method of  claim 29 , further comprising
 purifying the minicells from the composition.   
     
     
         33 . The method of  claim 27 , wherein the minicells are substantially separated from the parent cells by a process selected from the group consisting of centrifugation, ultracentrifugation, density gradation, immunoaffinity and immunoprecipitation. 
     
     
         34 . A method of making minicells, comprising
 culturing the minicell-producing bacteria of  claim 21 ; and   substantially separating the minicells from the minicell-producing parent cells, thereby generating a composition comprising minicells.   
     
     
         35 . The method of  claim 34 , further comprising
 inducing minicell formation from the minicell-producing parent cell.   
     
     
         36 . The method of  claim 34 , further comprising
 inducing expression of the gene encoding the endonuclease.   
     
     
         37 . The method of  claim 35 , wherein minicell formation is induced by the presence of one or more chemical compound selected from the group consisting of isopropyl β-D-1-thiogalactopyranoside (IPTG), rhamnose, arabinose, xylose, fructose, melbiose and tetracycline. 
     
     
         38 . The method of  claim 36 , wherein the expression of the gene encoding the endonuclease is induced by a change in temperature. 
     
     
         39 . The method of  claim 35 , further comprising
 purifying the minicells from the composition.   
     
     
         40 . The method of  claim 34 , wherein the minicells are substantially separated from the parent cells by a process selected from the group consisting of centrifugation, ultracentrifugation, density gradation, immunoaffinity and immunoprecipitation. 
     
     
         41 . A eubacterial minicell comprising an outer membrane, wherein the outer membrane comprises Lipid A molecules having no myristolic acid moiety. 
     
     
         42 . The eubacterial minicell of  claim 41 , wherein the outer membrane has a composition that results in the reduction of pro-inflammatory immune responses in a mammalian host compared to the outer membrane of eubacterial minicells that are derived from a corresponding wild-type bacteria. 
     
     
         43 . The eubacterial minicell of  claim 41  further comprising one or more biologically active compounds. 
     
     
         44 . The eubacterial minicell of  claim 43 , wherein at least one of the biologically active compounds is selected from the group consisting of a radioisotope, a polypeptide, a nucleic acid, and a small molecule. 
     
     
         45 . The eubacterial minicell of  claim 43 , wherein at least one of the biologically active compounds is a small molecule drug. 
     
     
         46 . The eubacterial minicell of  claim 43 , wherein at least one of the biologically active compounds is a small molecule imaging agent. 
     
     
         47 . The eubacterial minicell of  claim 43 , wherein at least one of the biologically active compounds is a chemotherapeutic agent. 
     
     
         48 . The eubacterial minicell of  claim 43 , wherein at least one of the biologically active compounds is a nucleic acid. 
     
     
         49 . The eubacterial minicell of  claim 43 , wherein at least one of the biologically active compounds is a polypeptide. 
     
     
         50 . The eubacterial minicell of  claim 43 , wherein at least one of the biologically active compounds is a pro-drug converting enzyme. 
     
     
         51 . The eubacterial minicell of  claim 43 , wherein at least one of the biologically active compounds is a combination of a nucleic acid and a small molecule. 
     
     
         52 . The eubacterial minicell of  claim 43 , wherein at least one of the biologically active compounds is a combination of a small molecule imaging agent and a small molecule drug. 
     
     
         53 . The eubacterial minicell of  claim 43  wherein at least one of the biologically active compounds is a combination of a small molecule drug, a small molecule imaging agent, and a nucleic acid. 
     
     
         54 . The eubacterial minicell of  claim 43  wherein at least one of the biologically active compounds is a combination of a nucleic acid and a polypeptide. 
     
     
         55 . The eubacterial minicell of  claim 43 , further comprising a cell-surface localized targeting moiety. 
     
     
         56 . The eubacterial minicell of  claim 55 , wherein the cell-surface localized targeting moiety is a fusion protein, wherein the fusion protein is a fusion of a eubacterial outer membrane anchoring domain and an antibody fragment. 
     
     
         57 . The eubacterial minicell of  claim 56 , wherein the cell-surface localized targeting moiety is a fusion protein, wherein the fusion protein is a fusion of  Neisserria gonorrheae  IgAP and an antibody fragment that recognizes a mammalian cell surface antigen. 
     
     
         58 . The eubacterial minicell of  claim 57 , wherein the mammalian cell surface antigens is selected from the group consisting of adipophilin, AIM-2, BCLX (L), BING-4, CPSF, Cyclin D1, DKK1, ENAH, Ep-CAM, EphA3, FGF5, G250/MN/CAIX, HER-2/neu, IL-13R alpha 2, Intestinal carboxyl esterase, alpha-foetoprotein, M-CSF, MCSP, mdm-2, MMP-2, MUC-1, p53, PBF, FRAME, PSMA, RAGE-1, RGS5, RNF43, RU2AS, secernin 1, SOX10, STEAP1, survivin, Telomerase, WT1, Cdc27, CDK4, CDKN2α, BCR-ABL, BAGE-1, GAGE1-8, GnTV, HERV-K-MEL, KK-LC-1, KM-HN-1, LAGE-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE A6, MAGE-A9, MAGE-A9, mucin, NA-88, NY-ESO-1, LAGE-2, SAGE, Sp17, SSX-2, SSX-4, TRAG-3, CD-166, and TRP2-INT2. 
     
     
         59 . A eubacterial minicell produced by the method of  claim 34 .