U.S. patent application number 12/490273 was filed with the patent office on 2010-05-06 for regulated genetic suicide mechanism compositions and methods.
This patent application is currently assigned to Vaxiion Therapeutics, Inc.. Invention is credited to Matthew J. Giacalone, Stanley Maloy, Shingo Tsuji.
Application Number | 20100112670 12/490273 |
Document ID | / |
Family ID | 41137324 |
Filed Date | 2010-05-06 |
United States Patent
Application |
20100112670 |
Kind Code |
A1 |
Giacalone; Matthew J. ; et
al. |
May 6, 2010 |
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.
Inventors: |
Giacalone; Matthew J.; (San
Diego, CA) ; Maloy; Stanley; (San Diego, CA) ;
Tsuji; Shingo; (National City, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Assignee: |
Vaxiion Therapeutics, Inc.
San Diego
CA
|
Family ID: |
41137324 |
Appl. No.: |
12/490273 |
Filed: |
June 23, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61075687 |
Jun 25, 2008 |
|
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|
61168457 |
Apr 10, 2009 |
|
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Current U.S.
Class: |
435/252.5 ;
435/252.1; 435/252.8; 435/252.9; 435/253.3; 435/253.4 |
Current CPC
Class: |
A61P 31/04 20180101;
Y02A 50/473 20180101; C12N 15/70 20130101; A61K 38/43 20130101;
Y02A 50/478 20180101; C12N 1/08 20130101; A61K 9/5068 20130101;
A61K 31/7088 20130101; Y02A 50/30 20180101; C12N 15/74 20130101;
Y02A 50/475 20180101; A61K 38/00 20130101; Y02A 50/47 20180101;
Y02A 50/481 20180101; A61K 9/5052 20130101; C12N 9/22 20130101 |
Class at
Publication: |
435/252.5 ;
435/252.1; 435/252.9; 435/252.8; 435/253.3; 435/253.4 |
International
Class: |
C12N 1/20 20060101
C12N001/20 |
Claims
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 .beta.-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 .beta.-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, CDKN2.alpha., 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.
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 electronically available information mentioned
or cited in this application, are hereby incorporated by reference
in their entirety to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference. Applicants reserve the right to
physically incorporate into this application any and all materials
and information from any such articles, patents, patent
applications, or other documents.
[0006] Minicells are achromosomal, membrane-encapsulated biological
nano-particles (.ltoreq.400 nm) that are formed by bacteria
following a disruption in the normal division apparatus of
bacterial cells. 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. Although minicells do not contain chromosomal DNA,
plasmid DNA molecules, RNA molecules, native and/or recombinantly
expressed proteins, and other metabolites have all been shown to
segregate into minicells.
[0007] Throughout the last century, minicells have been exploited
as tools for research scientists studying cell division, plasmid
replication, plasmid segregation, RNA production, protein
production, plasmid isolation, plasmid characterization, and
plasmid-borne virulence factor production in prokaryotes.
[0008] As a result of advances in the fields of microbiology,
microbial genetics, and molecular biology, any given minicell,
regardless of the parental cell species from which it was derived,
can now be engineered and subsequently used as in vivo or in vitro
targeted delivery or imaging vehicles.
[0009] Minicells are uniquely suited as in vivo delivery and
imaging vehicles because they combine many of the singular
advantages of other delivery technologies into a single, versatile
delivery vehicle. Minicells can be "engineered" to preferentially
encapsulate, be coupled to, or absorb biologically active
molecules, including various nucleic acids, proteins, and small
molecule drugs for subsequent delivery in both therapeutic and
prophylactic medicinal applications. As described in much more
detail below, minicells have the added advantage in that they can
be targeted to specific cell, tissue, and organ types, through the
use of several different antibody or affinity-based approaches.
SUMMARY OF THE INVENTION
[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, minicell formation is induced by the presence of one
or more chemical compound selected from isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG), rhamnose, arabinose,
xylose, fructose, melbiose, and tetracycline. In some embodiments,
the expression of the gene encoding the endonuclease is induced by
a change in temperature. In some embodiments, the method further
comprises purifying the minicells from the composition. In some
embodiments, 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.
[0012] Some other embodiments provide a eubacterial minicell
comprising an outer membrane, where the outer membrane comprises
Lipid A molecules having no myristolic acid moiety. In some
embodiments, the outer membrane of the eubacterial minicell
disclosed herein 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. In some embodiments, the
eubacterial minicell further comprises one or more biologically
active compounds. In some embodiments, 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. The biologically active compound can be a small drug
molecule, a small molecule imaging agent, a chemotherapeutic agent,
or a pro-drug converting enzyme. The biologically active compound
can also be a combination of a nucleic acid and a small molecule; a
combination of a small molecule imaging agent and a small molecule
drug; a combination of a small molecule drug, a small molecule
imaging agent, and a nucleic acid; or a combination of a nucleic
acid and a polypeptide. In some embodiments, the eubacterial
minicell disclosed herein further comprises a cell-surface
localized targeting moiety. In some embodiments, 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. In some embodiments, 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 mammanlian cell
surface antigen. In some embodiments, 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, p. 53, PBF, PRAME, PSMA, RAGE-1, RGS5, RNF43, RU2AS,
secernin 1, SOX10, STEAP1, survivin, Telomerase, WT1, Cdc27, CDK4,
CDKN2a, 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a graph showing effects of I-CeuI on E. coli
culture growth.
[0014] FIG. 2 is a graph showing effects of I-CeuI on E. coli
viability.
[0015] FIGS. 3A and 3B are bar graphs showing simultaneous
overexpression of ftsZ and induction of I-CeuI leads to higher
minicell yields compared to minCDE-mutants or the overexpression of
ftsZ alone.
[0016] FIGS. 4A-D are images showing overexpression of ftsZ and
induction of I-CeuI based suicide system causes increased parent
cell filamentation.
[0017] FIG. 5 is a bar graph showing I-CeuI based suicide system
introduces irreparable double-stranded chromosomal breaks.
[0018] FIG. 6 is a bar graph showing I-CeuI based suicide system
reduces parental cell contaminations among purified minicells.
[0019] FIG. 7 is a silver stained SDS-PAGE gel showing deletion of
the msbb gene in S. typhimurium changes LPS profiles.
[0020] FIG. 8 is a bar graph showing deletion of msbb causes
J774.A1 mouse macrophage like cells to produce lower levels of
tumor necrosis factor alpha (TNF.alpha.) against S. typhimurium
LPS.
[0021] FIG. 9 shows the single stranded I-CeuI DNA recognition
sequence and the double stranded I-CeuI DNA cleavage site.
DETAILED DESCRIPTION
Definitions
[0022] The term "cell division gene" used herein refer to a gene
that encodes a gene product that participates in the cell division
process. Many cell division genes have been discovered and
characterized in the art. Examples of cell division genes include,
but are not limited to, zipA, sulA, secA, dicA, dicB, dicC, dicF,
ftsA, ftsI, ftsN, ftsK, ftsL, ftsQ, ftsW, ftsZ, minC, minD, minE,
seqA, ccdB, sfiC, and ddlB.
[0023] The term "transgene" used herein refers to a gene or genetic
material that has been transferred naturally or by any of a number
of genetic engineering techniques from one organism to another. In
some embodiments, the transgene is a segment of DNA containing a
gene sequence that has been isolated from one organism and is
introduced into a different organism. This non-native segment of
DNA may retain the ability to produce RNA or protein in the
transgenic organism, or it may alter the normal function of the
transgenic organism's genetic code. In some embodiments, the
transgene is an artificially constructed DNA sequence, regardless
of whether it contains a gene coding sequence, which is introduced
into an organism in which the transgene was previously not
found.
[0024] As used herein, an agent is said to have been "purified" if
its concentration is increased, and/or the concentration of one or
more undesirable contaminants is decreased, in a composition
relative to the composition from which the agent has been purified.
Purification thus encompasses enrichment of an agent in a
composition and/or isolation of an agent thereform.
[0025] The term "domain" or "protein domain" used herein refers to
a region of a molecule or structure that shares common physical
and/or chemical features. Non-limiting examples of protein domains
include hydrophobic transmembrane or peripheral membrane binding
regions, globular enzymatic or receptor regions, protein-protein
interaction domains, and/or nucleic acid binding domains.
[0026] The terms "Eubacteria" and "prokaryote" are used herein as
these terms are used by those in the art. The term "eubacterial"
and "prokaryotic" used herein encompass Eubacteria, including both
Gram-negative and Gram-positive bacteria, prokaryotic viruses
(e.g., bacteriophage), and obligate intracellular parasites (e.g.,
Richettsia, Chlamydia, etc.).
[0027] The term "nucleic acid" used herein refers to any collection
of diverse nucleic acid molecules. A nucleic acid may be a ssDNA, a
dsDNA, a ssRNA, a dsRNA, a tRNA (including a rare codon usage
tRNA), a mRNA, a ribosomal RNA (rRNA), a peptide nucleic acid
(PNA), a DNA:RNA hybrid, an antisense oligonucleotide, a ribozyme,
or an aptamer.
[0028] The term "overexpression" used herein refers to the
expression of a polypeptide or protein encoded by a DNA in a host
cell, wherein the polypeptide or protein is either not normally
present in the host cell, or wherein the polypeptide or protein is
present in the host cell at a higher level than that normally
expressed from the endogenous gene encoding the polypeptide or
protein.
[0029] The term "modulate" as used herein means to interact with a
target either directly or indirectly so as to alter the activity of
the target to regulate a biological process. The mode of "modulate"
includes, but is not limited to, enhancing the activity of the
target, inhibiting the activity of the target, limiting the
activity of the target, or extending the activity of the
target.
DESCRIPTION
[0030] Eubacterial minicells are very well suited to serve as
targeted delivery and imaging vectors. Because they are derived
from bacteria that are often times inherently pathogenic or at
least opportunistically pathogenic, it is advantageous that any
contaminating parental cells be functionally eliminated from a
given population before systemic in vivo administration,
particularly if given intravenously. Consequently, the desired
minicell formulation would be one in which the residual live
parental cell count would be as low as possible as minicells are
processed and purified. One way to accomplish this is to introduce
a suicide mechanism to kill residual parental cells after the
physical separation step has been completed. The enhanced safety
profile reduces the risks of infection and sepsis, decrease the
possibility of genetic reversions through recombination events with
other bacteria and minimize the risks of insertion events in the
host. It is preferred that antibiotic resistance markers be
eliminated from the bacterial chromosome of the minicell-producing
parental cell strain. The elimination of antibiotic resistance gene
markers in minicell-producing strains of bacteria is desirable to
overcome regulatory hurdles imposed by the U.S. Food and Drug
Administration (FDA) for use in humans. The FDA will only tolerate
the use of the Kanamycin resistance gene marker for selection
purposes for bacteria or bacterial production strains wherein the
final product is intended for use in humans. Further, the FDA
requires certain standards for the certification of analysis of
drug product and the minicell final formulation would have to meet
USP and ICH guidelines for purity, absence of aggregates and,
absence of particular matter. Thus, upstream, and downstream
processing of drug product would have to fall under the company's
Chemistry, Manufacturing, and Control (CMC) drug product production
activities.
[0031] The need for better purification methodologies is a
roadblock to the development of minicells derived from pathogenic
bacteria. Embodiments of the present invention relate to the
incorporation and use of a regulated genetic suicide mechanism that
upon exposure to the appropriate signals, introduces irreparable
double-stranded breaks to the chromosomes of minicell-producing
parental cells resulting in parent cell death. The activation of
the suicide mechanism also increases minicell yields compared to
other minicell-producing strains, and simultaneously converts all
of the minicell-producing parent cells into an irreversible
filamentous phenotype. Thus, the suicide mechanism disclosed herein
is not limited to promoting death of chromosome-bearing parental
bacterial cells but can have other multifunctional actions that act
in consort to improve minicell production. In some embodiments, the
multifunctional suicide mechanism, the "MSM" system, disclosed
herein functions to kill chromosomal-bearing parental cells. In
some embodiments, the "MSM" system disclosed herein functions to
increase minicell yield. In some embodiments, the "MSM" system
disclosed herein functions to induce an irreversible filamentous
phenotype exclusively by parental cells to aid in parental cell
separation from minicells. In some embodiments, the "MSM" system
disclosed herein functions simultaneously to (i) kill
chromosomal-bearing parental cells, (ii) increase minicell yield
and (iii) induce an irreversible filamentous phenotype exclusively
by parental cells to aid in parental cell separation from
minicells. The multifunctional actions of the MSM system can
improve minicell production and purity using techniques described
herein.
[0032] Some embodiments relate to compositions and methods for
optimizing the yield and purity of minicells produced while
reducing or eliminating the number of viable contaminating
minicell-producing live eubacterial parent cells by the
introduction of a multifunctional genetic suicide mechanism, the
"MSM" system, to a minicell-producing parental cell line. Some
embodiments also relate to the use of the MSM system for use in
synthetic biology applications.
[0033] The presence of contaminating live parental cells in a final
preparation of minicells is problematic especially for minicells
produced from live pathogenic and opportunistically pathogenic
bacteria. Safety and CMC issues related to contaminating parental
bacterial are of concern when producing biologics or medicaments
from bacteria that are intended for use in humans or other mammals
because of their ability to cause disease, profound inflammation,
and in some cases, death. The compositions and methods of producing
bacterial minicells described herein not only improve minicell
production and purity but simultaneously improve the safety profile
of minicell preparations for in vivo and other uses. Without being
limited to the following examples, in vivo applications of high
purity and safe minicell preparations can be used in targeted
bio-imaging and the therapeutic prevention and treatment of
cancer(s), genetic disorders, and infectious diseases. Some
embodiments of the present disclosure relates to the incorporation
and use of a regulated genetic MSM mechanism that, upon exposure to
the appropriate signals, introduces irreparable damage to the
chromosomes of minicell-producing parental cells. The suicide
mechanism simultaneously facilitates purification techniques
designed to better eliminate viable parental cells from
preparations of minicells intended for use in a multitude of
targeted delivery applications.
[0034] The term "regulated genetic suicide mechanism" used herein
refer to a mechanism in where a cell or a group of cells is/are
stimulated by a known and external source to produce a gene product
or products that is/are capable of irreversibly damaging a
biologically essential component or cellular process of a cell such
that said cell(s) are no longer viable nor able to recover from
said event. The term, multifunctional suicide mechanism, MSM,
refers to the use of the regulated genetic suicide mechanism to
simultaneously induce high level minicell production, parental cell
death, and a filamentous phenotype exclusively in the parental
cells during induction of the suicide element.
[0035] The term "targeted minicell" or "targeted delivery" used
herein refers to a minicell composition in which said minicell
encapsulates one or more bioactive molecule(s) of choice and
displays targeting moieties on the external surfaces of the
minicells whether the minicells are (i) fully intact, (ii)
protoplasts (outer membrane and cell wall removed) or, (iii)
poroplasts (outer membrane removed or permeabilized) such that said
moieties specifically bind to, are bound by, or in some other way
specifically recognized and thereby deliver, localize to, or
aggregate within a specific cell, organ, or tissue type to deliver
the molecular contents of said minicell to said target cell,
tissue, and organ type in vitro or in vivo. This specific targeting
is intended to use minicells to deliver a payload to the targeted
cell or tissue.
[0036] The in vivo delivery applications using minicells include
but are not limited to the targeted delivery of bioactive
(synonymous with biologically active) small molecule drugs,
bioactive nucleic acids, bioactive proteins, and bioactive
lipopolysaccharides to produce a "biological effect" (synonymous
with biological response) in an animal. Biological effects include
but are not limited to the an effect that kills the target cell
(e.g. a cancer cell), replaces a gene that might be deficient or
dysfunctional within a particular cell type that is targeted,
reduces the expression and/or activity of a protein or signaling
molecule that is dysregulated in a particular target cell(s),
reduces or increases the secretion of hormone from a particular
cell(s), reduces or increases the secretion of proteins from a
particular cell(s), stimulates an adaptive cellular immune response
to one or more antigens, stimulates an adaptive humoral response
against one or more antigens, stimulates both adaptive humoral and
cellular immune responses from one or more antigens, stimulates or
represses one or more innate immune responses; an effect that
positively or negatively impacts a biological process in an animal;
and an effect that impacts a biological process in a pathogenic
parasite, bacterium, virus, or other pathogenic microbe to treat or
prevent a disease in said animal. The biologically active element
does not necessarily have to be immunogenic itself in order to
induce an immune reaction in the host animal, but can indirectly
elicit an immune response as a consequence of its biological
activity.
[0037] Eubacterial minicells have a distinct advantage in delivery
as they can be engineered to target and deliver bioactive molecules
to specific cell types in vivo. Targeting can be achieved by
coupling to the surface of minicells antibodies or antibody
derivatives specific for target cell surface molecules.
Alternatively, targeting can be achieved through the genetic
engineering of minicell-producing parental strains such that the
minicells they produce express and display on the minicell outer
membrane antibody fragments or other polypeptides with affinity for
target cell specific surface molecules. In this later case, the
targeting moiety that is decorated on the minicell surface can be
tethered to the membrane by making a chimeric fusion protein
between a cell-surface localizing targeting moiety and a
transmembrane protein sequence, e.g. IgAP from Neisserria gonorrhea
(see below). Minicells displaying said antibodies and targeting
moieties on their surfaces are used to target specific cell types
in vivo to preferentially deliver their bioactive payloads to the
targeted tissue, organ, and cell type.
[0038] Antibodies, or any portion thereof, intended to aid in the
targeting of minicells to a specific tissue, organ, and cell type
may be derived from or be part of any immunoglobulin or
immunoglobulin subclass, including but not limited to IgA, IgM,
IgD, IgG, or IgE. Antibodies of any subclass intended for
facilitating the targeting function of minicells may be
"humanized", although any antibody of any subclass against a cell
specific antigen can be raised in any animal known to generate
antibody responses through adaptive immunity to achieve the same
goal. In nature, antibodies are generated such that they contain
two separate arms with distinct specificities for their respective
antigens. Without being limited by the following, a targeting
moiety decorating the surface of the minicells could be derived
from a phage display library or could be a chimeric fusion protein
derived from an extracellular receptor fragment that recognizes a
ligand on the target cell.
[0039] Antibodies can be engineered to be independently specific
for different antigens, such that a single antibody targets two
separate antigens simultaneously. This is referred to as a
`bispecific` antibody or `bispecific` targeting moiety. By way of
non-limiting example, antibodies could be engineered to recognize
putative surface components of a given eubacterial minicell (e.g.,
LPS O-antigens) on one Fab' and the other Fab' of the bispecific
antibody can be engineered to recognize a cell-specific surface
antigen such as those listed below. Additionally, those skilled in
the art readily recognize that two separate antibodies, with
separate specificities, can be non-covalently attached by coupling
them to Protein AIG to form a bispecific antibody derivative
capable of adhering to the surface of minicells wherein one
antibody within the complex specifically adheres to the surface of
said minicell and the other antibody is displayed to specifically
recognize and thereby "target" a specific cell, tissue, or organ
type in vivo. Similarly, one skilled in the art will recognize that
two separate antibodies, with separate specificities, could be
covalently link using myriad cross-linking techniques to achieve
the same effect.
[0040] In some embodiments, minicells are genetically "engineered"
to express and display recombinant targeting proteins on their
surfaces. This has been successfully accomplished in Salmonella
enterica by using fusion proteins that contain an Antigen
43-.alpha. outer membrane anchoring domain fused to a single chain
Fv (scFv) antibody fragment with specificity for Chlam 12 or CTP3.
In a similar study, E. coli cells expressing and displaying single
chain Fv antibody fragments directed towards Coronavirus epitopes
fused with the outer membrane localized IgA protease of Neisseria
gonorhoeae were shown to neutralize Coronavirus and prevent
infection in vitro. The same types of strategies could be employed
to generate and display targeted fusion proteins on the surfaces of
minicells. Other native outer membrane proteins including LamB,
OmpF, OmpC, OmpA, OmpD, PhoE, PAL, and various Flagellins have been
used as membrane anchoring and display domains in gram negative
Enterobacteriacea family members. Generally, the same approach
could be used to express and display antibody fragments on the
surface of minicells derived from any Enterobacteriaceae or
Bacillaceae family member such that said minicells become
"specific" targeted delivery vehicles for antigens present on the
surface of cell, tissue, or organ types involved in various
clinical indications. Achieving this goal is a matter of creating a
nucleic acid sequence encoding for a fusion protein between a
putative or predicted outer membrane protein or outer membrane
localization sequence and an antibody, antibody derivative, or
other polypeptide sequence with affinity for a surface molecule
present in a given cell, tissue, or organ type.
[0041] Another advantage in the use of minicells as delivery
vehicles (targeted or non-targeted) is that bioactive molecules may
be delivered in combination. For example, it has been demonstrated
that minicells can successfully generate humoral immune responses
against a heterologous antigen when used as a delivery vehicle for
plasmid DNA vaccines. When minicells were used to simultaneously
deliver both a DNA vaccine and the corresponding protein, humoral
responses were greatly improved upon, illustrating the benefits of
the flexibility of minicells with respect to delivery options. In
similar fashion, minicells are used to encapsulate and deliver
combinations of different nucleic acid types such as plasmid DNA or
various antisense interference RNA (e.g., shRNA, siRNA) molecules
specific for different mRNA transcripts such that several genes are
silenced in a single delivery event. Minicells are also used to
deliver two or more small molecule drugs simultaneously such that
several intracellular targets are addressed in a single delivery
event.
[0042] Some embodiments disclosed herein describe a targeted
eubacterial minicell capable of delivering several classes of
bioactive payload in concert or singularly wherein the final
preparation of minicells is substantially free of any remaining
viable contaminating parent cells by virtue of the combined effects
of an inducible genetic suicide mechanism applied to conventional
separation techniques.
1. Minicell Production
[0043] Minicells are achromosomal, membrane-encapsulated biological
nano-particles (.ltoreq.400 nm) that are formed by bacteria
following a disruption in the normal division apparatus of
bacterial cells. 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. Although minicells do not contain chromosomal DNA, the
ability of plasmids, RNA, native and/or recombinantly expressed
proteins, and other metabolites have all been shown to segregate
into minicells. Some methods of construction of minicell-producing
bacterial strains are discussed in detail in U.S. patent
application Ser. No. 10/154,951, filed May 24, 2002, which is
hereby incorporated by reference in its entirety.
[0044] Disruptions in the coordination between chromosome
replication and cell division lead to minicell formation from the
polar region of most rod-shaped prokaryotes. Disruption of the
coordination between chromosome replication and cell division can
be facilitated through the overexpression of some of the genes
involved in septum formation and binary fission. Alternatively,
minicells can be produced in strains that harbor mutations in genes
that modulate septum formation and binary fission. Impaired
chromosome segregation mechanisms can also lead to minicell
formation as has been shown in many different prokaryotes.
[0045] Similarly, minicell production can be achieved by the
overexpression or mutation of genes involved in the segregation of
nascent chromosomes into daughter cells. For example, mutations in
the parC or mukB loci of E. coli have been demonstrated to produce
minicells. Both affect separate requisite steps in the chromosome
segregation process in Enterobacteriacea. Like the cell division
genes described herein, manipulation of wild type levels of any
given gene involved in the chromosome segregation process that
result in minicell production will have similar effects in other
family members.
[0046] Because the cell division and chromosome replication
processes are so critical to survival, there exists a high level of
genetic and functional conservancy amongst prokaryotic family
members with respect to genes responsible for these processes. The
overexpression or mutation of a cell division gene capable of
driving minicell production in one family member, can be used to
produce minicells in another. For example, it has been shown that
the overexpression E. coli FtsZ gene in other Enterobacteriacea
family members such as Salmonella spp. and Shigella spp as well as
other class members such as Pseudomonas spp. will result in similar
levels of minicell production.
[0047] The same can be demonstrated in the mutation-based minicell
producing strains of the family Enterobacteriacea. For example,
deletion of the min locus in any of Enterobacteriacea family
members results in minicell production. Cell division genes from
the Enterobacteriacea in which mutation can lead to minicell
formation include but are not limited to the min genes (MinCDE).
While minicell production from the min mutant strains is possible,
these strains have limited commercial value in terms of being
production strains. The reason for this is that strains with
deletions or mutations within the min genes make minicells at
constitutively low levels. This presents two problems in terms of
commercialization and economies of scale. The first is that
minicell yields from these strains are low, which increases
production cost. The second is that minicell yields are highly
variable with the mutant strains and lot-to-lot variability has
enormous impacts on variable production costs associated with
manufacturing quality control and regulatory assurances. Using the
mutant strains to produce minicells that have encapsulated
biologically active molecules such as proteins, RNA, DNA, and other
metabolites for delivery made first by the parental cells so that
the minicells produced encapsulate said biologically active
molecules is problematic. The onset of minicell production in the
mutant strains cannot be controlled and occurs at a low level so
that the end result is that some minicells will contain no
biologically active molecules while others will contain widely
variable amounts of biologically active molecules. These
shortcomings when taken together or separately greatly restricts
the possibility of using these mutant strains to produce minicell
at commercially viable yields and/or quality.
[0048] Minicell-producing strains that overexpress cell division
genes ("overexpressers") are preferred over mutation-based strains
because the minicell-production phenotype is controllable when the
cell division genes to be overexpressed are placed under the
control of an inducible or other conditionally active eubacterial
promoter system. Minicell production from strains overexpressing
the cell division gene ftsZ was discovered by researchers who were
identifying essential cell division genes in E. coli using
plasmid-based complementation studies. In these studies, the ftsZ
gene was present in over 10 copies per cell. The presence of
multiple gene copies of ftsZ was demonstrated to produce minicells
and extremely long filamented cells. Ultimately, this transition
into the irreversible filamentous phenotype negatively impacts
minicell yields from strains overexpressing ftsZ from multi-copy
plasmids, although the number of minicells produced is still higher
than that of any mutant strain. It has since been demonstrated that
by reducing the number of ftsZ gene copies to a single, chromosomal
duplication, the number of minicells produced increases over those
strains where ftsZ is located on multi-copy plasmids and that the
filamentous phenotype is less profound. Thus, some preferred
composition(s) are inducible minicell-producing strains that
overexpress the ftsZ gene from a duplicate, chromosomally
integrated copy of ftsZ. The duplicate ftsZ gene used can be
derived directly from the species of bacteria in which the
minicell-production phenotype is being engineered and can also be
derived from the ftsZ gene sequence from other species of bacteria.
By way of non-limiting example, overexpression of the ftsZ gene of
Escherichia coli can be used to generate minicells from Escherichia
coli and Salmonella typhimurium. Resulting strains are comprised of
the wild type ftsZ gene and a separate, duplicative, and inducible
copy of the ftsZ gene on the chromosome and the inducible genetic
suicide mechanism(s) described in greater detail below.
[0049] This inducible phenotype approach to minicell production has
several distinct advantages over the mutant systems. The first is
that because there are no genetic mutations in these strains, there
exists no selective pressure during normal growth and the cells of
the culture maintain a very stable and normal physiology until the
minicell phenotype is induced. The end result is that inducible
minicell producing strains are healthier and more stable, which
ultimately results in higher yields of minicells as shown in FIG.
3. Another distinct advantage of using the inducible phenotype
approach to minicell production is in cases where minicells are to
be used to deliver biologically active molecules such as proteins,
RNA, DNA, and other metabolites that can be made by the
minicell-producing parent cells themselves such that the minicells
that are produced encapsulate those biologically active molecules.
In these cases, a preferred method is to induce the formation of
the biologically active molecule(s) within the parental cells prior
to inducing the minicell phenotype so that all of the minicells
produced will contain sufficient amounts of the desired molecule(s)
to be encapsulated for delivery. These advantages, when used in
combination, result in a higher quality and quantity of minicells.
By way of non-limiting example, division genes that can be
over-expressed to produce minicells in the family
Enterobacteriaceae include but are not limited to FtsZ, MinE, SulA,
CcdB, and SfiC. A preferred composition is to have a duplicate
copy(s) of a cell division gene(s) under the control of an
inducible promoter that is stably integrated into the chromosome of
a given eubacterial strain. This same strategy could be carried out
if the inducible cell division gene cassette were present on a
plasmid, cosmid, bacterial artificial chromosome (BAC), recombinant
bacteriophage or other episomal DNA molecule present in the cell.
Homologs of these gene or gene products from other organisms may
also be used.
[0050] The novel, inducible MSM system described herein increases
minicell yields of the inducible minicell strains even further. The
activation of the MSM system results in greater than 10-fold
increases in minicell yields compared to other strains that merely
over produce ftsZ to promote minicell formation (EXAMPLE 3). It is
possible to combine the MSM system with minicell-producing strains
that harbor a mutation(s) or deletion(s) in the MinCDE. One
preferred embodiment is one in which the MSM system controls both
the inducible minicell-producing phenotype, increases minicell
yields, results in irreparable cell damage, and promotes a
filamentous phenotype amongst the parent cell population. A
preferred MSM gene combination is comprised of an inducible
minicell-producing strain that over expresses ftsZ or any
functional homolog thereof and the inducible expression of a homing
endonuclease, preferably the I-CeuI gene from the algae
Chlamydomonas moewusii as described in more detail below.
[0051] The minicell producing and parental cell
suicide/filamentation phenotypes that result from activation of the
inducible MSM system is not limited to the family Enterobacteriacea
but can be reproduced in any rod-shaped bacilli including those
from either Gram-negative or Gram-positive origin. For example,
minicell producing strains of Bacillus subtilis and other members
of the Bacillaceae have been studied in great detail. Similar to
the minicell producing strains within the family Enterobacteriacea,
all of the family Bacillaceae minicell-producing strains are a
result of mutations in or overexpression of genes involved in the
cell division or chromosome segregation process. Therefore,
sufficient evidence exists to support the idea that the
manipulation of conserved genes involved in the cell division or
chromosome segregation processes of any rod-shaped bacilli family
or genera, can be useful in creating minicell producing strains
amongst other members of the same family or genera of organism(s).
Similarly, and as demonstrated by Table 1 (below), the class of
genes useful in creating the MSM system can recognize and destroy
the chromosomes of many different rod-shaped gram-negative and
gram-positive bacterial species.
Inducible Promoter
[0052] Inducible promoter can be used to regulate gene expression
by turning on or off gene transcription at certain stages of
development of an organism. The activity of these promoters can be
induced by the presence or absence of biotic or abiotic
factors.
[0053] Inducible promoters include, but are not limited to,
chemically-regulated promoters and physically-regulated promoters.
The transcriptional activity of chemically-regulated promoters
including promoters can be regulated by the presence or absence of
one or more chemical compounds. The chemical compounds include, but
are not limited to, small molecules, nucleic acids, polypeptides,
and proteins. Non-limiting examples of the chemical compound are
isopropyl .beta.-D-1-thiogalactopyranoside (IPTG), rhamnose,
arabinose, xylose, fructose, melbiose, tetracycline, alcohol,
steroids, metal, and other compounds. Physically-regulated
promoters include promoters whose transcriptional activity
regulated by the presence or absence of one or more physical
factors, such as water or salt stress, illumination, light or
darkness, radiation, low or high temperatures, oxygen, and
nitrogen.
2. Separation of Minicells from Parent Cells and Minicell
purification
[0054] Because minicells are derived from bacteria that are often
times inherently pathogenic or at least opportunistically
pathogenic, it is advantageous that any contaminating parental
cells be functionally eliminated from a given population before
administration. Conventionally, live parental cells have been
eliminated through either physical means or biological means.
[0055] Physical means include the use of centrifugation-based
separation procedures, filtration methodologies, chromatography
methodologies, density gradation, immunoaffinity,
immunoprecipitation, or any combination thereof. While effective,
each has its drawbacks and no one physical separation methodology
has been fully adapted to eliminate viable parental cells from
minicells. Ultimately for commercial production, filtration
methodologies or a combination thereof is the most preferable
technique because of its simplicity, practicality, low cost, and
scalability. However, current filtration schemes are limited
because many contaminating parental cells make it through the
filters; and while this can be avoided, there is a compromise in
reducing final minicell yield. Ultimately, the design and use of
biological factors that influence parent cell size and viability in
conjunction with conventional filtration methodologies will result
in the best elimination of live cells. As shown below, the MSM
system disclosed herein allows for the inducible development of
elongated, filamentous parental cells that can be more easily
separated from the minicells during production.
[0056] Biological elimination is achieved by, but not limited to,
the preferential lysis of parental cells, the use of auxotrophic
parental strains, treatment with antibiotics, treatment with UV
radiation, diaminopimelic acid (DAP) deprivation, selective
adsorption of parental cells, and treatment with other DNA damaging
agents.
[0057] Preferential lysis of parental cells is typically mediated
by inducing the lytic cycle of a lysogenic prophage. In the case of
minicell producing strains, it is most useful to use a prophage
that is lysis competent but defective at re-infection, such that
minicells are not subsequently infected and lysed during activation
of the lytic phenotype. Alternatively and by way of non-limiting
example, individual genes such as those classified as members of
the holin gene family, can be expressed to achieve similar levels
of lysis without the concerns over re-infection inherent to the use
of lysogenic prophages. Both approaches are limited by the fact
that the lysis event, regardless of the method used to achieve it,
expels unacceptable amounts of free endotoxin into the media.
Removal of such large amounts of free endotoxin is time consuming,
suffers from lot to lot variability, and is ultimately cost
prohibitive.
[0058] The use of auxotrophic strains raises concerns over
reversion and as such can only be used in cases where minicells are
to be produced from commensal or non-pathogenic strains of
bacteria. Thus, their application is limited with respect to being
used as a method for elimination of live parental cells in
minicells production.
[0059] Treating minicell preparations with antibiotics raise
concerns about the development of antibiotic resistance, especially
when making minicells from pathogenic or opportunistically
pathogenic parental strains. Regulatory concerns and cost can also
be of great concern when using antibiotics to eliminate parental
cells from a given minicell production run.
[0060] Treatment with UV irradiation can be useful in the
elimination of live parental cells on a minicell production run
with the exception of the fact that UV irradiation is random and
results are highly variable from lot to lot. In addition, this
method is not preferred when using minicells to deliver therapeutic
or prophylactic nucleic acids as UV irradiation does not
discriminate when it randomly damages nucleic acids. For instance,
plasmid DNA would also be highly susceptible to DNA damage by UV
irradiation and may be rendered ineffective although still
effectively delivered by minicells.
[0061] DAP deprivation can be useful in the elimination of live
parental cells with the exception that this approach is limited by
the number of species it can be used for. In other words, not all
parent cell species capable of producing minicells require DAP for
survival, in which case this approach is of no consequence.
Reversion of DAP dependent strains is also a concern with this
approach.
[0062] Selective adsorption methodologies have yet to be explored
with respect to purifying minicells from viable parental cells.
Selective adsorption is defined as any process in where parental
cells are preferentially adsorbed to a substrate by virtue of their
affinity for a substrate. By way of non-limiting example, high
affinity protein-protein interactions can be exploited for this
use. By way of non-limiting example, the outer membrane protein
Invasin from the gram-negative species Yersinia pseudotuberculosis
has a high affinity for RGD motifs embedded in the protein sequence
of Beta-integrins. The gene encoding for invasin under the control
an inducible promoter could easily be introduced into a minicell
producing strain. Minicells can be produced from this strain prior
to the activation of expression of the invasin gene such that the
minicells produced do not express or display invasin on their cell
surface. Once the desired quantity of minicells is produced from
said strain, the viable cells within the culture could be given the
signal to produce the invasin protein such that invasin is only
expressed and displayed upon viable cells. Once invasin is
expressed on the surface of viable parental cells, they can be
easily adsorbed to a substrate coated with Beta-integrins or RGD
motifs embedded into a synthetic polypeptide or other recombinant
protein. Once absorbed, minicells can be selectively purified away
from viable parental cells by a number of different means dependent
upon the substrate type used. Substrates include but are not
limited to solid-phase chromatographic columns used in gravity
filtration applications, magnetic beads, ion exchange columns, or
HPLC columns. This approach is limited by the disadvantage that no
single protein-protein interaction will work for all species of
minicell producing parent cells. For instance, the invasin-integrin
approach described above would be useful for most Gram-negative
Enterobacteriacea family members but not for use with minicell
producing Gram-positive Bacillaceae family members.
[0063] The use of the previously mentioned filamentous phenotype of
minicell-producing parent strains presents a very distinct
advantage in terms of aiding in conventional, size-based physical
separation technologies such as filtration because it
preferentially increases the size of contaminating live cells from
a length of .about.1 .mu.M to lengths of .about.10-15 .mu.M.
Minicells however, remain their typical size of .about.400 nM. The
increased disparity in size between minicells and filamentous
parental cells greatly simplifies and obviates filtration schemes
as a preferred method of viable parental cell elimination.
Filamentation can be induced in rod-shaped eubacteria by several
means and the most common include imparting physiological stress
upon cells by the addition of high concentrations of salts or by
increasing or decreasing the pH of the culture, the overexpression
of cell division genes (such as thefts genes as described above),
and the induction of the SOS response. The induction of the SOS
stress response in bacteria is typically induced by introduction of
significant chromosomal damage although other mechanisms have been
shown to work. The problem with applying physiological stress to a
culture of cells to induce filamentation is that not all cells
within the population respond equally to the stress which leads to
variations in size that range from parental cells that are not
affected at all to those cells that are partially filamented to
those that are completely filamented. This unequal response amongst
the population limits this approach with respect to reproducibility
between purification runs. The same is true for the induction of
the SOS response by the addition of an exogenous DNA-damaging agent
in that not all of the cells in the population will respond equally
to that agent and make filaments.
[0064] Given all of the limitations of the biological approaches
listed above, a great need remains to develop a universally
reliable and effective method of eliminating viable minicell
producing parental cells to improve the safety profile of minicells
for in vivo applications. To this end, embodiments of the present
invention addresses this need and provide methods capable of
irreparably damaging the chromosomes of viable minicell producing
parental cells by use of a previously undescribed regulated genetic
suicide mechanism. The activation of the genetic suicide mechanism
simultaneously and irreversibly kills cells while inducing a
filamentous phenotype useful for aiding in conventional,
filtration-based separation techniques of minicells from live
contaminating parental cells.
[0065] A preferred and novel way to ensure that all viable parental
minicell-producing cells of a population will become uniformly
filamentous is to genetically engineer onto the chromosome of
minicell-producing strains a gene or set of genes under inducible
promoter control that upon activation with inducer will cause
filamentation as is achieved with the MSM system described herein
(EXAMPLE 4). It was determined that the activation of the genetic
suicide (MSM) mechanism described herein causes profound
filamentation (EXAMPLE 4). Thus, embodiments of the present
invention overcomes the uniformity issues present with other
approaches to filamentation by ensuring that any viable cell (a
cell with a chromosome) will become filamented on command. It is
desirable to circumvent suicide mechanism expression problems
associated with inducer uptake and other physiological factors that
affect promoter activities to ensure that all cells in a given
population will commit suicide when given the appropriate signals.
To eliminate insufficient promoter activities and ensure that each
cell within the population will become filamented, one preferred
promoter system for activating the genetic suicide mechanism is
thermoregulated, such as that used by the CI857ts promoter system.
Some embodiments comprises a gram-negative or gram positive
bacterial strain that contains a nucleic acid further comprising a
gene that encodes for a minicell-producing gene (preferably ftsZ)
which is operably linked to inducible prokaryotic expression
signals, and a second nucleic acid comprising a gene that encodes
for a suicide gene that does not lyse the parental cells
(preferably the homing endonuclease I-CeuI) which is operably
linked to inducible prokaryotic expression signals (preferably
CI857ts). The prokaryotic expression signals linked to the minicell
producing gene and the suicide gene may be under the control o the
same prokaryotic expression signals or different prokaryotic
expression signals. Further, the minicell producing gene and the
suicide gene may be located on the same or different nucleic acids
within a cell, one of which may be an episomal nucleic acid (e.g.
plasmid). In some other embodiments, the minicell producing gene
and the suicide gene are operably linked in a transcriptional
fusion (i.e. on the same mRNA transcript) and under the control of
common inducible prokaryotic expression signals. Both the minicell
producing gene and the suicide gene may be located in more than one
gene copy per cell.
[0066] In some embodiments, minicells are substantially separated
from the minicell-producing parent cells in a composition
comprising minicells. After separation, the compositions comprising
the minicells is less than about 99.9%, 99.5%, 99%, 98%, 97%, 96%,
95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%,
82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%,
65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% free of minicell-producing
parent cells.
3. Methods of Inducing Irreparable Chromosomal Damage
[0067] The concept that irreparable amounts of damage to the
chromosome of a given cell will result in irreversible cell death
has been best illustrated by the use of UV irradiation. UV
irradiation causes the formation of thymine dimers between adjacent
thymine nucleotides in a given DNA molecule. If the number of
thymine dimers reaches a threshold level in where insufficient
amounts of the proteins involved in the DNA repair of these adducts
are available, the cell will effectively die. However, as mentioned
above, this approach is severely limited because of its lack of
specificity amongst adduct formation sites within the chromosome,
its unbiased effects on all nucleic acid types, and variability
independent of exposure time.
[0068] Irreparable damage to the chromosome can also be achieved by
the overexpression of endonucleases. Endonucleases can cleave
double stranded DNA at sequence specific cleavage sites. Cleavage
can result in blunt-ended or staggered cleavage products dependent
upon the restriction enzyme employed.
4. I-CeuI Gene of Chlamydomonas moewusii
[0069] The I-CeuI restriction enzyme encoded by the chloroplast DNA
(SEQ ID NO:1) of the algae Chlamydomonas moewusii is particularly
useful for introducing irreparable damage to the chromosome of a
broad range of eubacterial minicell producing parental strains.
I-CeuI belongs to a unique family of intron encoded Type I
restriction enzymes commonly known as homing endonucleases. The
I-CeuI homing restriction enzyme specifically cleaves within the
15-19 base pair conserved sequence of the 23S ribosomal RNA (rRNA)
rrn operon sites (SEQ ID NO:2). Because 23S rRNA sequences are so
conserved amongst eubacteria, I-CeuI may be used to introduce
irreparable chromosome damage amongst a wide range of minicell
producing parental cell species. The 23S rRNA sites are located at
any where from 4-10 distinct positions in most eubacteria (see
Table 1), a range of sites that will support irreparable damage.
Typically, no 23S rRNA sites are located within the sequence of
common plasmid DNA molecules and as such I-CeuI can be used to
eliminate parental cells while still allowing for the propagation
and segregation of plasmids into minicells with the intent to
deliver them as a therapeutic or prophylactic payload. Furthermore,
the I-CeuI homing endonuclease operates most efficiently at
42-47.degree. C., thereby making it uniquely suited for use with a
thermoregulated promoter system such as the CI857ts promoter system
from phage lambda. The CI857ts promoter system is inactivated at
temperatures below 39.degree. C. and when shifted to 42-45.degree.
C. becomes extraordinarily highly active allowing for the rapid,
prolonged, and uniform exposure of each minicell-producing parental
cell within the culture to I-CeuI. Activation of this promoter
system is largely independent of many prohibitive physiological
factors such as inducer uptake.
TABLE-US-00001 TABLE 1 List of I-CeuI Recognition Sites Within
Different Eubacterial Genomes Recognition ATCC Bacterium Sites
Number Escherichia coli K12 MG1655 7 ATCC 47076 Escherichia coli
W3110 7 ATCC 27325 Escherichia coli O157:H7 str. Sakai 7 ATCC BAA-
460 Shigella dysenteriae Sd197 7 N/A Shigella flexneri 2a str.
2457T 7 ATCC 700930 Shigella boydii Sb227 7 N/A Shigella sonnei
Ss046 7 N/A Salmonella enterica serovar Typhi Ty2 7 ATCC 700931
Salmonella enterica serovar Typhimurium LT2 7 ATCC 700720
Salmonella enterica subsp. Enterica serovar Choleraesuis str. 7 N/A
SC-B67 Salmonella enterica subsp. enterica serovar Paratyphi A 7
ATCC 9150 Salmonella enterica subsp. Enterica serovar Paratyphi B
str. 7 ATCC BAA- SPB7 1250 Pseudomonas aeruginosa PA7 4 N/A
Pseudomonas aeruginosa PAO1 4 ATCC 15692 Pseudomonas aeruginosa
UCBPP-PA14 4 N/A Pseudomonas entomophila str. L48 7 N/A Pseudomonas
putida F1 6 ATCC 700007 Pseudomonas stutzeri A1501 4 N/A Vibrio
cholerae O395 chromosome 2 8 ATCC 39541 Vibrio cholerae O1 biovar
eltor str. N16961 chromosome I 7 ATCC 39315 Yersinia pestis Angola
7 N/A Neisseria meningitidis MC58 4 ATCC BAA- 335 Neisseria
meningitidis serogroup C FAM18 4 ATCC 700532 Neisseria gonorrhoeae
FA1090 4 ATCC 700825 Listeria monocytogenes strain EGDe 6 ATCC BAA-
679 Legionella pneumophila subsp. pneumophila str. Philadelphia 1 3
ATCC 33152 Staphylococcus aureus subsp. aureus strain MRSA252 5 N/A
Staphylococcus aureus subsp. aureus Mu3 5 ATCC 700698
Staphylococcus epidermidis FDA strain PCI 1200 5 ATCC 12228
Streptococcus pyogenes M1 GAS 6 ATCC 700294 Streptococcus
pneumoniae R6 4 ATCC BAA- 255 Enterococcus faecalis V583 4 ATCC
700802 Clostridium botulinum A str. ATCC 19397 8 ATCC 19397
Clostridium botulinum A str. ATCC 3502 9 ATCC 3502 Clostridium
difficile 630 11 ATCC BAA- 1382
[0070] Originally, I-CeuI was isolated from Chlamydomonas moewusii
to digest purified eubacterial chromosomal DNA molecules for pulse
field gel electrophoretic genome analysis. It is commercially
available for research use and has been exploited for years by
microbiologists studying genome arrangements, genomic
rearrangements, performing BAC cloning, and performing other
analyses of eubacterial chromosomal DNA. I-CeuI and its Type I
family members are unique in that they are not affected by
different DNA methylation patterns which can vary greatly amongst
the eubacteria. Thus, I-CeuI can be used in a broad range of
different minicell producing parent strains. By way of non-limiting
example, I-CeuI has been used in vitro to analyze the genomes of
Salmonella, Shigella, E. coli, Pseudomonas spp., Aeromonas spp.,
Clostridium spp., Staphylococcus spp., Bacillus spp., and Neisseria
spp. all with equivalent efficiency.
[0071] Further, I-CeuI is a member of the subfamily of homing
endonucleases known as the LAGLIDADG family. Members of this family
number over 100 and all contain the conserved amino acid sequence
motif LAGLIDADG that serves as the homodimer interaction interface
as well as active site formation and function.
[0072] I-CeuI and the other Type I restriction enzymes are not as
stringent as the more typical Type II restriction endonucleases
with respect to the sequence within which they recognize and
perform their respective cleavage reactions. While some bases
within the 15-19 base pair sequence are essential for cleavage,
others are dispensable. Thus, certain variations of the 15-19 base
pair sequence could be engineered such that they differ in sequence
at non-critical bases but are still functional cleavage sites. Such
sites could be engineered and introduced into the chromosome(s) of
minicell producing parent strains as such. These modified cleavage
sites would also serve as targets recognized by I-CeuI in vivo or
in vitro and could be used to introduce irreparable damage to the
chromosome.
5. The Use of Functional Equivalents of I-CeuI
[0073] As mentioned previously, the I-CeuI gene of Chlamydomonas
moewusii is a member of a subclass of homing endonucleases known as
the LAGLIDADG family. As such, some embodiments include
constructing and utilizing a similar genetic suicide mechanism by
utilizing one of the other LAGLIDADG homing endonuclease family
members. Members of the LAGLIDADG that can be substituted for
I-CeuI include but are not limited to PI-SceI, I-ChuI, I-CpaI,
I-SceIII, I-Crel, I-MsoI, I-SceII, I-SceIV, I-CsmI, I-DmoI, I-PorI,
PI-TliI, PI-TliII, and PI-ScpI.
[0074] Another subfamily of Type I homing endonucleases is termed
the GIY-YIG family. Members of this family include but are not
limited to the bacteriophage T4 endonuclease I-TevI, Am atpase-6,
and SegA.
[0075] Yet another subfamily of Type I homing endonucleases is
termed the H--N--H family and its members include but are not
limited to Eco CoE8, Eco CoE9, Eco CoE2, Eco Mcr, I-HmuI, I-TevIII,
Cpc1 gpII, Cpc2 gpII, Avi gpII, Sob gpII, and See gpII.
[0076] The last subfamily of Type I homing endonucleases is termed
the His-Cys box family and its members include but are not limited
to I-DirI, I-NaaI, and I-PpoI.
[0077] Any or all of the 4 classes of homing endonucleases and
their respective DNA target sequences or functional variants
thereof can be used to construct a regulated genetic suicide system
described herein for use in the elimination of contaminating viable
parental cells from minicells.
6. Overexpression of ftsZ in Combination with Activation of the
Genetic Suicide Mechanism Generates High Yield Minicell Producing
Strains
[0078] As shown in FIG. 3, the simultaneous activation of I-CeuI
and induction of the minicell-producing phenotype worked in concert
to have positive effects on minicell production and yield as well
as decreased viability and filamentation of contaminating parent
cells. EXAMPLE 3 shows high yield minicell-producing strains as the
number of minicells produced increased 10-fold when ftsZ and I-CeuI
were simultaneously overexpressed. High yield minicell producing
strains were defined to be those that generate 10.sup.9 or greater
minicells from a 100 mL starting culture. In addition to generating
high yields of minicells, the parental cells became uniformly
filamentous upon activation of the I-CeuI and ftsZ genes as shown
in FIG. 4. The resulting filamentous phenotype is a factor which is
exploited to better facilitate filtration-based minicell
purification schemes.
7. Homologous Recombination and Other DNA Damage Repair
Pathways
[0079] Homologous recombination pathways in eubacteria are highly
conserved in terms of function and mechanism of action.
Essentially, homologous DNA recombination is mediated by the
introduction of a double stranded break in duplex DNA followed by
5' to 3' exonuclease activity that creates single stranded DNA
overhangs to be used in an enzyme dependent process known as strand
invasion. During strand invasion, homologous regions of two
separate duplex DNA molecules base pair with each other. The
synthesis of new DNA replaces the region(s) degraded by
exonucleases and the result is a short-lived 4-armed heteroduplex
DNA structure termed a Holliday junction. The Holliday junction is
subject to a helicase dependent process known as branch migration
in where the center of the heteroduplex DNA structure can shift
from its original position to any other position along the length
of any of the 4 arms of the Holliday junction. Once stabilized, the
heteroduplex Holliday junction is "resolved" by enzymes termed
resolvases and two separate duplex DNA molecules result. In some
cases, significant amounts of DNA are transferred from one DNA
molecule to the other, hence the term recombination.
[0080] It is known that in some instances, double stranded break
repair is mediated through the homologous recombination pathway(s)
of eubacteria. As stated previously, I-CeuI and the other Type I
homing endonuclease family members introduce double stranded breaks
and said breaks are subject to repair by homologous recombination.
Thus, eliminating the ability of the cell to perform homologous
recombination also eliminates the possibility that the double
stranded breaks introduced by a Type I homing endonuclease family
member will be repaired. Repair of the chromosome is essential to
recovery in the case of double stranded breaks and as such, the use
of homologous recombination pathway null or conditional mutants in
conjunction with said genetic suicide mechanism would be of great
benefit in reducing viable contaminating minicell producing parent
cells. Some embodiments provide the use of DNA recombination and
damage repair pathways to prevent cells containing the genetic
suicide mechanism from repairing any chromosomal lesions introduced
as a result of the activation of the suicide mechanism.
[0081] In the eubacterial family Enterobacteriacea, genes involved
in homologous recombination or any step of the process described
herein that can be mutated, inactivated, made to be expressed
conditionally, or modified in any way as to aid in the elimination
of viable contaminating minicell producing parental cells upon the
activation of the genetic suicide mechanism include, but are not
limited to, recA, recBCD, uvrABC, lexA, recN, recQ, recR, ruv,
gyrAB, helD, lig, polA, ssb, recO, mutH, mutL, mutS, topA, uvrD,
xseA, srfA, recF, recJ, recE, recT, rusA, dam, dut, xth, or rdgB.
Any homologs of said genes can be disrupted in other genera and
separate eubacterial families.
[0082] Mutations that affect the expression of these genes may be
present singularly or in combination with each other. The
transcription levels of said gene(s) can be affected by chromosomal
deletion, promoter disruption, promoter replacement, promoter
modification, or RNA-mediated promoter interference. Translation of
said gene(s) can be affected by the expression of antisense mRNA,
shRNA, siRNA, or by modification of the Shine Dalgarno sequence.
Function of said gene(s) product(s) can be affected by the
overexpression of dominant negative version(s) of said gene(s) or
other suppressors of said gene(s) such that function is
impaired.
[0083] Some embodiments provide the replacement of any and all of
the genes involved in homologous recombination or double strand
break repair pathways listed above with an allele of said gene(s)
that is a well characterized temperature sensitive mutant. In an
illustrative example of this, the gene product, RecA for example,
would function normally at temperatures below 39.degree. C. so to
allow for normal growth, physiology, and minicell production but
would not function at temperatures higher than 39.degree. C. such
that when the temperature of the minicell-producing culture was
shifted to 42-45.degree. C. to activate the I-CeuI gene, the RecA
molecules present within the cell would be unable to perform their
necessary function(s) at a level sufficient enough to aid in the
repair of double stranded chromosomal lesions. This approach
expedites the effective killing of I-CeuI while providing another
level of assurance in the elimination of viable minicell-producing
parent cells by providing the cell with no repair mechanism(s).
[0084] In some embodiments, the wild type copy of the gene lexA is
replaced with a cleavage deficient mutant allele. The LexA protein
is a global regulator of the SOS response genes in eubacteria and
acts as a repressor to genes within that regulon by juxtaposed
occupation of the transcriptional start sites within the promoter
regions of SOS response genes. Thus, when the LexA repressor is
bound to the promoter regions of the SOS response genes, the genes
are inactivated as a result of transcription factor inaccessibility
due to LexA-mediated steric hindrance. In the event that cells are
subjected to stress such as that provided when double stranded
chromosomal DNA breaks are introduced, LexA is cleaved. As a result
of cleavage, LexA can no longer bind and repress the activity of
SOS response genes. Cleavage can occur through two mechanisms. The
first is RecA-mediated cleavage that is stimulated by the activity
of RecA proteins in the presence of single stranded DNA. Single
stranded DNA is produced by the RecBCD exonuclease complex as the
very next sequence of events immediately following the introduction
of double stranded chromosomal breaks. The second mechanism is
termed "autocleavage" and occurs spontaneously in an intramolecular
reaction in response to changes in temperature or pH. Both cleavage
mechanisms rely on serine protease activity mediated by the serine
residue at amino acid position 119 (S-119) and the lysine residue
at amino acid position 156 (L-156). Cleavage occurs between the
alanine residue at amino acid position 84 (A-84) and the adjacent
glycine residue at amino acid position 85 (G-85). The well
characterized cleavage deficient mutant allele of the lexA gene
termed lexA3 and its counterpart lexA33 may be used with some
embodiments of the present invention.
8. Targeting Minicells
[0085] Following production, activation of the genetic suicide
mechanism, and subsequent purification, minicells are used as
targeted delivery vehicles. Minicells displaying antibodies,
antibody derivatives, and other targeting moieties on their
surfaces are used to target specific cell types in vivo to
preferentially deliver their bioactive payloads to the targeted
tissue, organ, and cell type.
[0086] Antibodies, or any portion thereof, intended to aid in the
targeting of minicells to a specific tissue, organ, and cell type
may be derived from or be part of any immunoglobulin subclass,
including but not limited to IgA, IgM, IgD, IgG, or IgE. Antibodies
of any subclass intended for facilitating the targeting function of
minicells may be "humanized", although any antibody of any subclass
against a cell specific antigen can be raised in any animal known
to generate antibody responses through adaptive immunity to achieve
the same goal. In nature, antibodies are generated such that they
contain two separate arms with distinct specificities for their
respective antigens.
[0087] Antibodies can be engineered to be independently specific
for different antigens, such that a single antibody targets two
separate antigens simultaneously. By way of non-limiting example,
antibodies could be engineered to recognize putative surface
components of a given eubacterial minicell (e.g., LPS O-antigens)
on one arm and the other arm be engineered to recognize a
cell-specific surface antigen. In this approach, minicell surface
molecules that would be of use include but are not limited to
naturally occurring molecules such as lipopolysaccharides (LPS),
outer membrane proteins (OMPs), flagellar proteins, pilus proteins,
and porins. Alternatively, minicell-producing parental strains can
be engineered to express and display protein or LPS molecules on
their surfaces that are not naturally occurring or occur in other
organisms such that said molecule is recognized by one or more arms
of an antibody used to couple targeting antibodies or other
targeting moieties to the surfaces of minicells. For example, a
protein engineered to express and display the FLAG epitope could be
designed and utilized such that one arm or antibody recognizes the
FLAG epitope and that the other can recognize a specific cell
selective antigen of choice. Additionally, those skilled in the art
readily recognize that two separate antibodies, with separate
specificities, can be non-covalently attached by coupling them to
Protein AIG to form a bi-specific antibody derivative capable of
adhering to the surface of minicells wherein one antibody within
the complex specifically adheres to the surface of said minicell
and the other antibody is displayed to specifically recognize and
thereby "target" a specific cell, tissue, or organ type in vivo.
Similarly, one skilled in the art will recognize that two separate
antibodies, with separate specificities, could be covalently linked
using myriad cross-linking techniques to achieve the same effect.
All of these potential approaches to targeting are readily
recognized by those skilled in the art.
[0088] Alternative and preferable to the exogenous addition of
antibodies and antibody derivatives, minicells can be "engineered"
to express and display recombinant targeting proteins on their
surfaces by creating outer membrane fusion proteins that display
polypeptide-based targeting moieties. This can be accomplished
using any of the outer membrane proteins from Gram-negative
bacteria although some outer membrane proteins or regions therein
are more suitable for display. This has been successfully
accomplished in Salmonella enterica by using fusion proteins that
contain an Antigen 43-.alpha. outer membrane anchoring domain fused
to a single chain FcV antibody fragment with specificity for Chlam
12 or CTP3. In a similar study, E. coli cells expressing and
displaying single chain FcV antibody fragments directed towards
Coronavirus epitopes fused with the outer membrane localized,
autotransporter IgA protease (IgAP) of Neisseria gonorrhoeae were
shown to neutralize Coronavirus and prevent infection in vitro. The
same types of strategies could be employed to generate and display
targeted fusion proteins on the surfaces of minicells. Other native
outer membrane proteins including LamB, OmpF, OmpC, OmpA, OmpD,
PhoE, PAL, and various Flagellins have been used as membrane
anchoring and display domains in gram negative Enterobacteriacea
family members. Generally, the same approach could be used to
express and display antibody fragments on the surface of minicells
derived from any Enterobacteriacea or Bacillaceae family member
such that said minicells become "specific" targeted delivery
vehicles for antigens present on the surface of cell, tissue, or
organ types involved in various clinical indications. One skilled
in the art will recognize that achieving this goal is a matter of
creating a nucleic acid sequence encoding for a fusion protein
between a putative or predicted outer membrane protein or outer
membrane localization sequence and an antibody, antibody
derivative, or other polypeptide sequence with affinity for a
surface molecule present in a given cell, tissue, or organ
type.
[0089] A preferred embodiment to displaying antibodies, antibody
fragments, and any of the other polypeptide-based targeting
moieties described herein on the surface of minicells is by fusion
with an outer membrane of the "autotransporter" family. The
monomeric autotransporters belonging to the sub-class type 5
secretion system of autotransporters (commonly classified as type
5a) are most preferred. Of those autotransporters classified as
type 5a, the IgA protease (IgAP) of Neisseria gonorrhoeae is
preferred. The IgAP autoransporter passenger domain is easily
replaced by variable light and heavy antibody chains that are
spaced by a short 8-10 repeat proline linker sequence. Sequences
from variable heavy (VH) and light (VL) chains are easily
identified, isolated, sequenced, and cloned from B-cell hybridomas
or any other conventional recombinant DNA or RNA sources of
variable light and heavy chain sequence as one ordinarily skilled
in the art will readily recognize. Several different antibody
fragments and antibody fragment types have been displayed and
characterized using the IgAP system in E. coli although this
approach is entirely novel with respect to their use to target
tissues, organs, or cell types in conjunction with use in
minicells. Thus, by identifying antibody
[0090] One skilled in the art will recognize that there are other
methods by which targeting of minicells to specific cell, organ, or
tissue types could be achieved in addition to the display of
antibodies or antibody derivatives that have specificity for
cell-specific surface antigens on the surface of minicells. One
such method is to express and display on the outer most surface of
minicells, non-antibody derived polypeptides that target
cell-specific antigens. These polypeptides can be derived but are
not limited to, naturally occurring sequences or useful portions
thereof and synthetically derived sequences.
[0091] Naturally-occurring sequences include those that are known
in the art to interact with a cell-specific surface antigen.
Examples of these types of interactions include but are not limited
to naturally occurring ligand and receptor interactions such as the
well-characterized VEGF and the VEGF receptor interaction. For
example, VEGF receptors displayed on the surfaces of endothelial or
other cells could be targeted by decorating minicells with receptor
binding domains of the VEGF protein, thus providing a targetings
moiety for delivering minicells to endothelial cells. This would be
an alternative to using a scFv fragment of an anti-VEGF receptor as
the targeting moiety. In some embodiments, the same naturally
occurring surface-localized molecules of the minicell listed above
can be engineered using standard molecular biological techniques to
create fusion proteins that display the binding portion(s) of these
ligands such that said minicells are now capable of specifically
recognizing, localizing, and delivering their respective payloads
to specific cell types, tissues, and organs of interest.
[0092] Synthetic molecules that selectively bind to cell-specific
surface antigens, such as mammalian cell surface antigens, may be
identified and incorporated into some embodiments of the present
invention to serve as targeting moieties. For instance, peptide
sequences identified by phage display library can easily be cloned
as fusions with any of the native minicell outer membrane proteins
as described above to serve as targeting molecules. Similarly,
synthetic targeting molecules can be coupled to the surface of
minicells using standard chemical conjugation or cross-linking
techniques.
[0093] Cancer cells, in particular, are highly sought after cell
types that can be targeted using minicells. Many cancers display
cell surface protein variants or other immunologically
distinguishable cell surface markers known collectively as
tumor-specific antigens or sometimes referred to as tumor-selective
antigens (TSAs). Many antibodies that specifically recognize TSAs,
and nucleic acid sequences of the variable regions therefore, are
already known in the art. Any of these antibodies can be used in
exogenous fashion with invention or alternatively, expressed as a
membrane-bound fusion protein and displayed on the surface of the
minicell as described above. Many TSAs have been identified to
which there no antibodies and therefore nucleic acid sequences of
the variable regions of those antibodies currently available.
However, methods to produce antibodies to TSAs are well known in
the art and the methods disclosed herein are designed such that any
and all antibodies to TSAs, or any other cell-specific surface
antigen, can be incorporated in to the composition as
described.
[0094] Tumor-selective antigens include, but are not limited to,
adipophilin, AIM-2, BCLX (L), BING-4, CPSF, Cyclin D1, DKK1, ENAH,
Ep-CAM, EphA3, FGF5, G250/MN/CALX, HER-2/neu, IL-13R alpha 2,
Intestinal carboxyl esterase, alpha-foetoprotein, M-CSF, MCSP,
MMP-2, MUC-1, p53, PBF, FRAME, PSMA, RAGE-1, RGS5, RNF43, RU2AS,
secernin I, SOX10, STEAP1, survivin, Telomerase, WT1, Cdc27, CDK4,
CDKN2.alpha., 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, and TRP2-INT2.
[0095] In addition to targeting cancer cells and tumors derived
therefrom, embodiments of the present invention also encompass any
cell type that displays a selective cell surface antigen(s). For
instance, targeting minicells to the pancreas to deliver diabetes
drugs, or targeting minicells to dendritic cells or any subclass
thereof to deliver protein, carbohydrate, or nucleic acids encoding
for antigens for use in vaccine development or innate immune
regulation are desirable. A VEGF-based targeting system for
endothelial cells is described above. Similarly, targeting
minicells to specific cell types of the mucosal epithelium, such as
the Peyer's patches of the small intestine, are desirable.
9. Payload Types
[0096] Eubacterial minicells are capable of encapsulating and
delivering several classes of biologically active compounds that
have therapeutic, prophylactic, or diagnostic benefit to an animal.
Types of the biologically active compounds (payloads) that can be
delivered by minicells include but are not limited to small
molecules, nucleic acids, polypeptides, radioisotope, lipids,
lipopolysaccharides, and any combination thereof.
[0097] The term "small molecule" used herein includes any chemical
or other moiety that can act to affect biological processes in a
positive or a negative sense. Small molecules can include any
number of therapeutic agents presently known and used, or can be
small molecules synthesized in a library of such molecules for the
purpose of screening for biological function(s). Small molecules
are distinguished from macromolecules by size. The small molecules
as disclosed herein usually have molecular weight less than about
5,000 daltons (Da), preferably less than about 2,500 Da, more
preferably less than 1,000 Da, most preferably less than about 500
Da.
[0098] Small molecules include without limitation organic
compounds, peptidomimetics and conjugates thereof. As used herein,
the term "organic compound" refers to any carbon-based compound
other than the macromolecules nucleic acids and polypeptides. In
addition to carbon, organic compounds may contain calcium,
chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen,
oxygen, sulfur and other elements. An organic compound may be in an
aromatic or aliphatic form. Non-limiting examples of organic
compounds include acetones, alcohols, anilines, carbohydrates,
monosaccharides, oligosaccharides, polysaccharides, amino acids,
nucleosides, nucleotides, lipids, retinoids, steroids,
proteoglycans, ketones, aldehydes, saturated, unsaturated and
polyunsaturated fats, oils and waxes, alkenes, esters, ethers,
thiols, sulfides, cyclic compounds, heterocylcic compounds,
imidizoles and phenols. An organic compound as used herein also
includes nitrated organic compounds and halogenated (e.g.,
chlorinated) organic compounds.
[0099] "Small molecules" can be synthetic, naturally occurring, and
purified from a natural source. Small molecules include, but are
not limited to, small molecule drugs and small molecule imaging
agents. Types of small molecule drugs include those that prevent,
inhibit, stimulate, mimic, or modify a biological or biochemical
process within a cell, tissue type, or organ to the benefit of an
animal suffering from a disease, whether somatic, germinal,
infectious, or otherwise. Examples of drugs include
chemotherapeutic agents (cancer drugs), antibiotics, antivirals,
antidepressants, antihistamines, anticoagulants, and any other
class or subclass thereof as listed in the Physicians Desk
Reference. Small molecules also include the class of molecules
collectively known as fluorophores. Minicells encapsulating
fluorophores and displaying cell-specific targeting moieties can be
used for in vivo imaging of cell types, tissues, organs, or tumors
in an animal. Small molecule fluorophores include but are not
limited to DAN, Cybr Gold, Cybr Green, Ethidium Bromide, Alexa
Flour, Texas Red, CFSE, and the like. Small molecule
chemotherapeutic agents can be targeted and delivered to tissues,
cells, and organs using minicells displaying targeting molecules.
The term "chemotherapeutic agent" used herein refers to
anti-cancer, anti-metastatic, anti-angiogenic, and other
anti-hyperproliferative agents. Put simply, a "chemotherapeutic
agent" refers to a chemical intended to destroy cells and tissues.
Such agents include, but are not limited to: (I) DNA damaging
agents and agents that inhibit DNA synthesis such as anthracyclines
(doxorubicin, donorubicin, epirubicin), alkylating agents
(bendamustine, busulfan, carboplatin, carmustine, cisplatin,
chlorambucil, cyclophosphamide, dacarbazine, hexamethylmelamine,
ifosphamide, lomustine, mechlorethamine, melphalan, mitotane,
mytomycin, pipobroman, procarbazine, streptozocin, thiotepa, and
triethylenemelamine), platinum derivatives (cisplatin, carboplatin,
cis diamminedichloroplatinum), telomerase and topoisomerase
inhibitors (Camptosar), (2) tubulin-depolymerizing agents such as
taxoids (Paclitaxel, docetaxel, BAY 59-8862), (3) anti-metabolites
such as capecitabine, chlorodeoxyadenosine, cytarabine (and its
activated form, ara-CMP), cytosine arabinoside, dacabazine,
floxuridine, fludarabine, 5-fluorouracil, 5-DFUR, gemcitibine,
hydroxyurea, 6-mercaptopurine, methotrexate, pentostatin,
trimetrexate, and 6-thioguanine (4) anti-angiogenics (Avastin,
thalidomide, sunitinib, lenalidomide), vascular disrupting agents
(flavonoids/flavones, DMXAA, combretastatin derivatives such as
CA4DP, ZD6126, AVE8062A, etc.), (5) biologics such as antibodies or
antibody fragments (Herceptin, Avastin, Panorex, Rituxan, Zevalin,
Mylotarg, Campath, Bexar, Erbitux, Lucentis), and (6) endocrine
therapy such as aromatase inhibitors (4-hydroandrostendione,
exemestane, aminoglutehimide, anastrozole, letozole),
anti-estrogens (Tamoxifen, Toremifine, Raoxifene, Faslodex),
steroids such as dexamethasone, (7) immuno-modulators: cytokines
such as IFN-beta and IL2), inhibitors to integrins, other adhesion
proteins and matrix metalloproteinases), (8) histone deacetylase
inhibitors, (9) inhibitors of signal transduction such as
inhibitors of tyrosine kinases like imatinib (Gleevec), (10)
inhibitors of heat shock proteins, (11) retinoids such as all trans
retinoic acid, (12) inhibitors of growth factor receptors or the
growth factors themselves, (13) anti-mitotic compounds such as
navelbine, Paclitaxel, taxotere, vinblastine, vincristine,
vindesine, and vinorelbine, (14) anti-inflammatories such as COX
inhibitors and (15) cell cycle regulators such as check point
regulators and telomerase inhibitors.
[0100] Nucleic acids include DNA and RNA and their structural
equivalents such as RNA molecules or DNA molecules that utilize
phosphothiolate backbones as opposed to the naturally occurring
phosphodiester backbones. DNA molecules include episomal DNA (not
located on or part of the host cell chromosome) and include plasmid
DNA, cosmid DNA, bacteriophage DNA, and bacterial artificial
chromosomes (BACs). DNA molecules encode for proteins as described
by the central dogma of molecular biology. Thus DNA may encode for
proteins of any origin, naturally occurring or synthetic. Likewise,
DNA can be engineered to contain "promoter sequences" that are
recognized by host cell machinery to activate expression of said
encoded proteins. Promoter sequences can be cell specific, tissue
specific, or inducer specific. Inducers are exogenously applied
signals that help to activate said promoters to produce said
proteins. Inducers can be chemical or physical in nature. Many
promoter systems are known to those skilled in the art as are the
sequences that render them functional. Preferred prokaryotic
expression sequences include but are not limited to the pRHA
system, the pBAD system, the T7 polymerase system, the pLac system
and its myriad derivatives, the pTet system, and the CI857ts
system. Preferred eukaryotic promoter systems include but are not
limited to the CMV promoter, the SV40 promoter system, and the BGH
promoter system. RNAs include but are not limited to messenger RNA
(mRNA), transfer RNA (tRNA), and small nuclear RNAs. Many RNAs,
classified as antisense RNAs, include but are not limited to
small-interfering RNAs (siRNA), short hairpin RNAs (shRNAs), and
full length antisense RNAs. Micro RNAs are also included.
[0101] Proteins are comprised of polypeptides and are encoded for
by DNA. Proteins can be biologically functional, such as enzymes or
signaling proteins. Proteins can be structural, such as is the case
for actin and the like. Proteins can serve as immunogens or serve
other therapeutic purposes (such as supplying or restoring enzyme
in a target cell, tissue, organ, or animal). Proteins can aid in
the post-endocytosis intracellular transfer of other payload types.
For example, proteins such as listeriolysin 0 from Listeria
monocytogenes can be employed to facilitate the transfer of the
minicell payload(s) from the endocytotic compartment(s) of a target
cell to the cytosol of a target cell. Proteins can also be pro-drug
converting enzymes.
[0102] Any and all of these payload types may be used in
combination or singular at the discretion of the user. One skilled
in the art will appreciate and recognize which combinations are to
be used for which purposes.
10. Reducing Toxicity of LPS
[0103] Safety concerns surrounding the immunogenicity and pyrogenic
effects of lipopolysaccharides (LPS), a constitutive component of
the minicell outer membrane commonly referred to as endotoxin, is
advantageously addressed to further the commercial viability of
minicell-based targeted delivery compositions. These safety
concerns are advantageously addressed in addition to addressing
safety issues that revolve around the possible contamination of
minicell-based targeted delivery compositions for use in vivo with
viable minicell-producing parent cells. The LPS molecule(s) is
essentially comprised of three parts. The first part is the pair of
hydrocarbon chains that anchor the molecule into the outer leaflet
of the outer membrane which are collectively called the "Lipid A"
portion of the molecule. The second is a series of sugar residues
commonly referred to the "inner core". The inner core is different
from genera to genera but is identical amongst inter-genera
members. For example, Salmonella and Shigella have different inner
core structures because they are not members of the same genera
while Salmonella typhi and Salmonella typhimurium share the same
inner core structures because they are both members of the genera
Salmonella. The third component of the LPS molecule, commonly
called the "O-antigen" is a series of sugar molecules, the chain
length, branch structure, sequence, and composition of varies
greatly amongst bacteria, even amongst genera members. Many genes
involved in lipopolysaccharide synthesis have been identified and
sequenced. For instance, the rfa gene clusters contains many of the
genes for LPS core synthesis, includes at least 17 genes.
[0104] While the LPS molecule as a whole is very pyrogenic, the
major contributor to pyrogenicity with respect to the three
components described above is the Lipid A component. The Lipid A
component has been shown to bind to and activate Toll-like
receptors, a family of signaling molecule present on the surface of
mammalian cells that help to recognize specific pathogen associated
molecular patterns (PAMP's) of which LPS is a classic member. The
potent pyrogenic effects of Lipid A are mediated by a specific
portion of the Lipid A molecule that comprising a myristolic acid
group attached to one of the hydrocarbon chains. In gram-negative
bacteria, this myristolic acid group is added by a single,
non-essential gene commonly referred to as msbB. By eliminating the
msbB gene, the myristolic acid component is eliminated, and the
pyrogenicity of the LPS molecule(s) is drastically reduced. This
approach has been exploited to reduce toxicity of LPS in
attenuated, living Salmonella serovars that happen to colonize
hypoxic regions within tumors as an experimental cancer therapy
used in human clinical trials. The same msbB gene or its functional
equivalent, leads to reduced toxicity of the LPS incorporated into
minicells, when deleted from the parental minicell-producing
strain(s). The reduced toxicity of the LPS has been shown to result
in the reduction of pro-inflammatory immune responses in a
mammalian host.
[0105] As shown in FIGS. 7 and 8, successful deletion of msbB in a
minicell-producing Salmonella strain gave rise to minicells with
reduced toxicity as measured by the production of TNF-.alpha. by
cultured murine macrophages exposed to minicells produced from
strains harboring msbB mutations versus those minicells produced
from wild type strains.
11. Free Endotoxin Removal
[0106] In most in vivo applications, it is desirable to remove any
free endotoxin, primarily in the form of free LPS, from the
composition. By and large endotoxin removal can be facilitated by
the filtration technologies and methodologies employed. As an
example, a dead-end filtration step captures minicells and allows
smaller molecules such as LPS to pass through the membrane filter,
thereby effectively eliminating a large majority of free endotoxin.
It is desirable to achieve endotoxin levels for in vivo
applications that are at or below the levels mandated by the United
States Food and Drug Administration (www.fda.gov). Other
conventional and well described approaches that can be used in lieu
of or in conjunction with filtration-based endotoxin removal are
different chromatographic, immuno-chromatographic, and
immuno-precipitation methodologies. In the case of immuno-based
methods, a typical method is to use an antibody or other moiety
that specifically recognizes and binds to the Lipid A portion of
the LPS molecule. The advantage in targeting this segment of the
LPS molecule is two-fold. The first advantage is that Lipid A is
only exposed when LPS is liberated from the outer membrane of the
minicells and thus creates a selective bias towards the removal of
only free endotoxin versus the removal of intact minicells.
Secondly, many commercially available anti-Lipid A antibodies are
available. Coupling antibodies to a solid or semi-solid matrix such
as a column or magnetic beads has further advantage in that free
endotoxin can be readily and selectively absorbed or adsorbed to
the matrix to better facilitate endotoxin removal from minicell
compositions. Endotoxin levels in final preparations can be
determined by pelleting minicells and analyzing the supernatant for
endotoxin levels using the quantitative limulus amoebocyte lysate
(LAL) test.
12. Pharmaceutical Compositions.
[0107] Another aspect of the present invention relates to
compositions, including but not limited to pharmaceutical
compositions. The term "composition" used herein refers to a
mixture comprising at least one carrier, preferably a
physiologically acceptable carrier, and one or more minicell
compositions. The term "carrier" used herein refers to a chemical
compound that does not inhibit or prevent the incorporation of the
biologically active peptide(s) into cells or tissues. A carrier
typically is an inert substance that allows an active ingredient to
be formulated or compounded into a suitable dosage form (e.g., a
pill, a capsule, a gel, a film, a tablet, a microparticle (e.g., a
microsphere), a solution; an ointment; a paste, an aerosol, a
droplet, a colloid or an emulsion etc.). A "physiologically
acceptable carrier" is a carrier suitable for use under
physiological conditions that does not abrogate (reduce, inhibit,
or prevent) the biological activity and properties of the compound.
For example, dimethyl sulfoxide (DMSO) is a carrier as it
facilitates the uptake of many organic compounds into the cells or
tissues of an organism. Preferably, the carrier is a
physiologically acceptable carrier, preferably a pharmaceutically
or veterinarily acceptable carrier, in which the minicell
composition is disposed.
[0108] A "pharmaceutical composition" refers to a composition
wherein the carrier is a pharmaceutically acceptable carrier, while
a "veterinary composition" is one wherein the carrier is a
veterinarily acceptable carrier. The term "pharmaceutically
acceptable carrier" or "veterinarily acceptable carrier" used
herein includes any medium or material that is not biologically or
otherwise undesirable, i.e., the carrier may be administered to an
organism along with a minicell composition without causing any
undesirable biological effects or interacting in a deleterious
manner with the complex or any of its components or the organism.
Examples of pharmaceutically acceptable reagents are provided in
The United States Pharmacopeia, The National Formulary, United
States Pharmacopeial Convention, Inc., Rockville, Md. 1990, hereby
incorporated by reference herein into the present application. The
terms "therapeutically effective amount" or "pharmaceutically
effective amount" mean an amount sufficient to induce or effectuate
a measurable response in the target cell, tissue, or body of an
organism. What constitutes a therapeutically effective amount will
depend on a variety of factors, which the knowledgeable
practitioner will take into account in arriving at the desired
dosage regimen.
[0109] The compositions can further comprise other chemical
components, such as diluents and excipients. A "diluent" is a
chemical compound diluted in a solvent, preferably an aqueous
solvent, that facilitates dissolution of the composition in the
solvent, and it may also serve to stabilize the biologically active
form of the composition or one or more of its components. Salts
dissolved in buffered solutions are utilized as diluents in the
art. For example, preferred diluents are buffered solutions
containing one or more different salts. A preferred buffered
solution is phosphate buffered saline (particularly in conjunction
with compositions intended for pharmaceutical administration), as
it mimics the salt conditions of human blood. Since buffer salts
can control the pH of a solution at low concentrations, a buffered
diluent rarely modifies the biological activity of a biologically
active peptide.
[0110] An "excipient" is any more or less inert substance that can
be added to a composition in order to confer a suitable property,
for example, a suitable consistency or to form a drug. Suitable
excipients and carriers include, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, agar, pectin, xanthan gum, guar gum, locust bean gum,
hyaluronic acid, casein potato starch, gelatin, gum tragacanth,
methyl cellulose, hydroxypropylmethyl-cellulose, polyacrylate,
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP),
If desired, disintegrating agents can also be included, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Other suitable excipients and
carriers include hydrogels, gellable hydrocolloids, and chitosan.
Chitosan microspheres and microcapsules can be used as carriers.
See WO 98/52547 (which describes microsphere formulations for
targeting compounds to the stomach, the formulations comprising an
inner core (optionally including a gelled hydrocolloid) containing
one or more active ingredients, a membrane comprised of a water
insoluble polymer (e.g., ethylcellulose) to control the release
rate of the active ingredient(s), and an outer layer comprised of a
bioadhesive cationic polymer, for example, a cationic
polysaccharide, a cationic protein, and/or a synthetic cationic
polymer; U.S. Pat. No. 4,895,724. Typically, chitosan is
cross-linked using a suitable agent, for example, glutaraldehyde,
glyoxal, epichlorohydrin, and succinaldehyde. Compositions
employing chitosan as a carrier can be formulated into a variety of
dosage forms, including pills, tablets, microparticles, and
microspheres, including those providing for controlled release of
the active ingredient(s). Other suitable bioadhesive cationic
polymers include acidic gelatin, polygalactosamine, polyamino acids
such as polylysine, polyhistidine, polyornithine, polyquaternary
compounds, prolamine, polyimine, diethylaminoethyldextran (DEAE),
DEAE-imine, DEAE-methacrylate, DEAE-acrylamide, DEAE-dextran,
DEAE-cellulose, poly-p-aminostyrene, polyoxethane,
copolymethacrylates, polyamidoamines, cationic starches,
polyvinylpyridine, and polythiodiethylaminomethylethylene.
[0111] The compositions can be formulated in any suitable manner.
Minicell compositions may be uniformly (homogeneously) or
non-uniformly (heterogenously) dispersed in the carrier. Suitable
formulations include dry and liquid formulations. Dry formulations
include freeze dried and lyophilized powders, which are
particularly well suited for aerosol delivery to the sinuses or
lung, or for long term storage followed by reconstitution in a
suitable diluent prior to administration. Other preferred dry
formulations include those wherein a composition disclosed herein
is compressed into tablet or pill form suitable for oral
administration or compounded into a sustained release formulation.
When the composition is intended for oral administration but is to
be delivered to epithelium in the intestines, it is preferred that
the formulation be encapsulated with an enteric coating to protect
the formulation and prevent premature release of the minicell
compositions included therein. As those in the art will appreciate,
the compositions of the invention can be placed into any suitable
dosage form. Pills and tablets represent some of such dosage forms.
The compositions can also be encapsulated into any suitable capsule
or other coating material, for example, by compression, dipping,
pan coating, spray drying, etc. Suitable capsules include those
made from gelatin and starch. In turn, such capsules can be coated
with one or more additional materials, for example, and enteric
coating, if desired. Liquid formulations include aqueous
formulations, gels, and emulsions.
[0112] Some preferred embodiments concern compositions that
comprise a bioadhesive, preferably a mucoadhesive, coating. A
"bioadhesive coating" is a coating that allows a substance (e.g., a
minicell composition) to adhere to a biological surface or
substance better than occurs absent the coating. A "mucoadhesive
coating" is a preferred bioadhesive coating that allows a
substance, for example, a composition to adhere better to mucosa
occurs absent the coating. For example, micronized particles (e.g.,
particles having a mean diameter of about 5, 10, 25, 50, or 100
.mu.m) can be coated with a mucoadhesive. The coated particles can
then be assembled into a dosage form suitable for delivery to an
organism. Preferably, and depending upon the location where the
cell surface transport moiety to be targeted is expressed, the
dosage form is then coated with another coating to protect the
formulation until it reaches the desired location, where the
mucoadhesive enables the formulation to be retained while the
composition interacts with the target cell surface transport
moiety.
[0113] Compositions disclosed herein may be administered to any
organism, preferably an animal, preferably a mammal, bird, fish,
insect, or arachnid. Preferred mammals include bovine, canine,
equine, feline, ovine, and porcine animals, and non-human primates.
Humans are particularly preferred. Multiple techniques of
administering or delivering a compound exist in the art including,
but not limited to, oral, rectal (e.g. an enema or suppository)
aerosol (e.g., for nasal or pulmonary delivery), parenteral, and
topical administration. Preferably, sufficient quantities of the
biologically active peptide are delivered to achieve the intended
effect. The particular amount of composition to be delivered will
depend on many factors, including the effect to be achieved, the
type of organism to which the composition is delivered, delivery
route, dosage regimen, and the age, health, and sex of the
organism. As such, the particular dosage of a composition
incorporated into a given formulation is left to the ordinarily
skilled artisan's discretion.
[0114] Those skilled in the art will appreciate that when the
compositions of the present invention are administered as agents to
achieve a particular desired biological result, which may include a
therapeutic or protective effect(s) (including vaccination), it may
be possible to combine the fusion proteins with a suitable
pharmaceutical carrier. The choice of pharmaceutical carrier and
the preparation of the fusion protein as a therapeutic or
protective agent will depend on the intended use and mode of
administration. Suitable formulations and methods of administration
of therapeutic agents include those for oral, pulmonary, nasal,
buccal, ocular, dermal, rectal, or vaginal delivery.
[0115] Depending on the mode of delivery employed, the
context-dependent functional entity can be delivered in a variety
of pharmaceutically acceptable forms. For example, the
context-dependent functional entity can be delivered in the form of
a solid, solution, emulsion, dispersion, micelle, liposome, and the
like, incorporated into a pill, capsule, tablet, suppository,
aerosol, droplet, or spray. Pills, tablets, suppositories,
aerosols, powders, droplets, and sprays may have complex,
multilayer structures and have a large range of sizes. Aerosols,
powders, droplets, and sprays may range from small (1 micron) to
large (200 micron) in size.
[0116] Pharmaceutical compositions disclosed herein can be used in
the form of a solid, a lyophilized powder, a solution, an emulsion,
a dispersion, a micelle, a liposome, and the like, wherein the
resulting composition contains one or more of the compounds of the
present invention, as an active ingredient, in admixture with an
organic or inorganic carrier or excipient suitable for enteral or
parenteral applications. The active ingredient may be compounded,
for example, with the usual non-toxic, pharmaceutically acceptable
carriers for tablets, pellets, capsules, suppositories, solutions,
emulsions, suspensions, and any other form suitable for use. The
carriers which can be used include glucose, lactose, mannose, gum
acacia, gelatin, mannitol, starch paste, magnesium trisilicate,
talc, corn starch, keratin, colloidal silica, potato starch, urea,
medium chain length triglycerides, dextrans, and other carriers
suitable for use in manufacturing preparations, in solid,
semisolid, or liquid form. In addition auxiliary, stabilizing,
thickening and coloring agents and perfumes may be used. Examples
of a stabilizing dry agent includes triulose, preferably at
concentrations of 0.1% or greater (See, e.g., U.S. Pat. No.
5,314,695). The active compound is included in the pharmaceutical
composition in an amount sufficient to produce the desired effect
upon the process or condition of diseases.
13. Production of Other Cell-Based Biologics
[0117] Although the disclosure thus far has been to the better
purification of minicells with respect to the ratio of minicells to
viable parental cells, it is certainly not limited solely to this
application. Additionally, the present disclosure can be used in
the production of other cell-based biologics as a means of
eliminating viable production cells. By way of non-limiting
example, the present disclosure can be useful in the production and
preparation of enzymes and other proteins, nucleic acids, bacterial
ghosts, lipids, biofilms, sugars, and small molecules.
[0118] To this end, the present disclosure addresses this need as
it provides a method capable of irreparably damaging the
chromosomes of viable parental cells by use of a regulated genetic
suicide mechanism that has not been previously described.
14. Use of the MSM System in Synthetic Biology
[0119] Another aspect of the present invention relates to the use
of the MSM system in the field of synthetic biology. As used
herein, "synthetic biology" includes the construction and use of a
replication competent nucleic acid, a "synthetic genome" or a
"synthetic chromosome", wherein said nucleic acid comprises a
minimal gene set required for sustained growth in defined media.
Synthetic genomes may include one or more genes than are required
to constitute a minimal gene set any or all of which may or may not
be found together in nature. Synthetic genomes may be created using
a transposon-mediated subtractive approach wherein a starting
genome has its non-essential genes removed or replaced through a
combination of transposon-mediated disruption(s) and homologous
recombination(s). Homologous recombination(s) can occur naturally,
or may be facilitated by myriad recombination systems including but
not limited to the Red recombinase system, the loxP system, the cre
recombinase system and the like. Alternatively, the synthetic
genome may be created using an additive approach wherein said
synthetic genome is rationally designed and constructed de novo.
Synthetic genomes constructed using the additive de novo approach
may but need not be first constructed in silico. It is desirable to
have the synthetic genome further comprising a gene or set of genes
that result in a discreet and desired phenotype. For instance, a
new organism that can metabolize hydrocarbons to produce biofuels
such as hydrogen, ethanol, or bio-diesel(s) could be created and
commercialized by the introduction of a synthetic genome containing
a gene or set of genes capable of said metabolism in a surrogate
microorganism. Other examples include but are not limited to the
creation of microorganisms that can fix carbon dioxide directly
from the atmosphere, produce industrially relevant by-products or
precursors thereto (e.g. sulfite for the production of sulfuric
acid), or capable of adding beneficial molecules or removing toxic
molecules from the environment.
[0120] Once constructed, the synthetic genome may be introduced
into a cell derived from a microorganism, including but not limited
to a bacterium, using standard transformation techniques, wherein
the synthetic genome replaces the original genome of the surrogate
microorganism. One method to ensure that the synthetic genome has
replaced the original genome is through the incorporation of a
selective genetic marker, including but not limited to an
antibiotic resistance gene, and selecting for stable transformants.
Other selection methodologies known to those skilled in the art
will be readily recognized and applicable as alternative selection
strategies. Selection strategies can be applied in singular or in
plurality. The order of selection is at the sole discretion of the
user and can be imparted in any order, temperature, and growth
condition.
[0121] To ensure the elimination of the original genome from the
surrogate microorganism, it is preferred that the chromosome(s) of
said surrogate microorganism be destroyed or irreparably damaged at
some point during the transformation of the synthetic genome.
Preferably, the irreparable destruction of the chromosome(s) would
be inducible and would occur prior to the introduction of the
synthetic genome into the surrogate microorganism. The MSM system
described herein facilitates the inducible and irreparable
destruction of the chromosome(s) of bacteria and is easily utilized
as a mechanism by which to destroy the original chromosome(s) of
the surrogate cell prior to the introduction of the synthetic
genome. The modular nature of the MSM system is advantageous
because it allows the system to be employed in numerous strains of
bacteria including but not limited to those listed in Table 1.
15. The Use of Bacterial Minicells in Synthetic Biology
[0122] Another aspect of the present invention relates to use
bacterial minicells as the surrogate cell for use in synthetic
biology applications as opposed to a bacterium with a chromosome
that has been irreparably damaged by the MSM system. Minicells are
anucleated cells derived directly from parental bacterial cells.
Because bacterial cells are not compartmentalized as compared to
eukaryotic cells, all of the DNA synthesis and replication
machinery required to replicate a synthetic genome is also present
within the minicell. The advantage is that the minicell, by
definition, has already "lost" the parental chromosome. Minicells,
just as whole cell bacterium, are transformed with synthetic
genomes and other nucleic acid types using standard transformation
and selection procedures readily recognized by those ordinarily
skilled in the art. Selection of transformants can be performed as
described above.
[0123] Overexpression of DNA synthesis and replication machinery
proteins by the minicell-producing parent cell prior to induction
of minicell formation will ensure that the synthetic genome is
readily synthesized by the minicell upon transformation by
providing an abundance of said components by way of segregation
into the minicells. Thus, said minicells are enriched with the DNA
synthesis and replication machinery prior to transformation. For
example, in E. coli, genes involved in the replication of the
chromosome include but are not limited to dnaA, dnaB, dnaC, ssb,
dnaG, polA, dnaE, dnaQ, hole, dnaX, dnaN, dnaX, holA, holB, holC,
hold, lig, gyrA, and gyrB. These genes and their functional
equivalents can be overexpressed by the minicell-producing parental
cell prior to the induction of the minicell phenotype such that
they are encapsulated in minicells. Replication and synthesis genes
can be overexpressed in any combination and can be present on the
chromosome of the parent cell line or on an episomal nucleic acid
element such as a plasmid, cosmid, BAC, and the like.
[0124] Similarly, genes involved in chromosome partitioning,
segregation, and cell division per se may be overexpressed and
packaged into the minicell such that said minicells are capable of
chromosome partitioning, segregation and cell division as a final
requirement in completing the construction of a synthetic
organism.
[0125] Synthesis of the synthetic genome requires energy in the
form of adenosine tri-phosphate (ATPs) molecules and free
nucleotides (e.g., adenine, cytosine, guanine, thymine, and uracil
or any of their nucleoside or nucleotide derivatives). These
molecules passively diffuse across the lipid-bilayers of bacterial
minicells and can be added back to supplement transformed minicells
until the synthetic genome is stabilized and replicating
independently. Polypeptide(s) production from the newly introduced
requires free amino acids to incorporate into nascent polypeptide
chains. Free amino acids are added back to newly transformed
minicells to supplement said minicells with enough free amino acids
such as to support nascent protein synthesis from the synthetic
genome. Once sufficient levels of metabolic proteins have been
synthesized from the synthetic genome by the newly transformed
minicells, amino acids may be removed as said minicells are now
capable of producing their own stores of amino acids for protein
synthesis.
[0126] Minicells derived from any prokaryotic source may be used
for construction of a synthetic organism using the methods
described herein.
16. Minicell Preparations
[0127] Some embodiments provide a method of reducing the number of
viable eubacterial minicell producing parental cells to improve the
safety of minicell preparations intended for in vivo delivery
applications with respect to the number of infectious particles
administered. Some embodiments comprise a gram-negative or gram
positive bacterial strain that contains a nucleic acid encoding for
a minicell-producing gene (for example, ftsZ) which is operably
linked to inducible prokaryotic expression signals, and a second
nucleic acid comprising a gene that encodes for a suicide gene that
does not lyse the parental cells (for example, the homing
endonuclease I-CeuI) which is operably linked to inducible
prokaryotic expression signals (for example, CI857ts). The
prokaryotic expression signals linked to the minicell producing
gene and the suicide gene may be under the control of the same
prokaryotic expression signals or different prokaryotic expression
signals. Further, the minicell producing gene and the suicide gene
may be located on the same or different nucleic acids within a
cell, one of which may be an episomal nucleic acid (e.g. plasmid).
Further yet, the minicell producing gene and the suicide gene may
be operably linked in a transcriptional fusion (i.e. on the same
mRNA transcript) and under the control of common inducible
prokaryotic expression signals. Both the minicell producing gene
and the suicide gene may be located in more than one gene copy per
cell. Eubacterial strains containing the MSM system have the
ability to (i) produce high yields of minicells (greater than
10.sup.9 per 100 mL of culture grown in normal shake flasks, FIG.
3), (ii) introduce irreparable cellular damage that does not lyse
cells (FIGS. 1-2, 5-6), and (iii) enter into an irreversible
filamentous phenotype (FIG. 4).
[0128] Minicells intended for use in in vivo delivery applications
are produced from a eubacterial strain that contains said regulated
MSM genetic suicide mechanism. Once the desired number of minicells
is produced as needed per said application, the genetic suicide
mechanism (MSM) would be activated by the exposure to a known
stimulus, preferably a shift in temperature, and allowed sufficient
time to introduce irreparable damage to the chromosomes of said
cells, thereby rendering said cells unviable.
[0129] Some embodiments of the present invention relate to induce
the minicell production phenotype using the MSM system from an
eubacterial minicell-producing strain preferably from, but not
limited to, the family Enterobacteriaceae that contains a DNA
molecule encoding for a therapeutic or deleterious gene or gene
product, such that the resulting minicell contains said DNA
molecule by way of encapsulation. Following production of the
desired quantity of minicells from a given culture and condition,
activation of the genetic suicide mechanism would be stimulated
following exposure of said culture or cells to a known signal. The
signal would be applied in each step of the purification process to
ensure maximal killing of viable cells in the final
preparation.
[0130] Some embodiments of the present invention relate to induce
the minicell production phenotype from an optimized eubacterial
minicell-producing strain from, but not limited to, the family
Enterobacteriaceae that contains any subclass of RNA, including but
not limited to siRNA, antisense RNA, ribozymes, shRNA, and miRNA
such that the resulting minicell contains an enriched amount of
said RNA molecules by way of encapsulation. Following production of
the desired quantity of minicells from said culture and condition,
activation of the genetic suicide mechanism (MSM) would be
stimulated following exposure of said culture or cells to a known
signal. The signal would be applied in each step of the
purification process to ensure maximal killing of viable cells in
the final preparation.
[0131] Some embodiments of the present invention relate to induce
the minicell production phenotype from an optimized eubacterial
minicell-producing strain from, but not limited to, the family
Enterobacteriaceae that contains a protein molecule, such that the
resulting minicell contains said protein molecule by way of
encapsulation. Following production of the desired quantity of
minicells from a given culture and condition, activation of the
genetic suicide mechanism would be stimulated following exposure of
said culture or cells to a known signal. The signal would be
applied in each step of the purification process to ensure maximal
killing of viable cells in the final preparation.
[0132] Some embodiments of the present invention relate to induce
the minicell production phenotype from an optimized eubacterial
minicell-producing strain from, but not limited to, the family
Enterobacteriaceae that contains a predetermined and deliberate
combination of DNA molecules encoding for a therapeutic or
deleterious gene or gene product, any subclass of RNA, and/or
proteins, such that the resulting minicell contains said
combination of molecules by way of encapsulation. Following
production of the desired quantity of minicells from a given
culture and condition, activation of the genetic suicide mechanism
would be stimulated following exposure of said culture or cells to
a known signal. The signal would be applied in each step of the
purification process to ensure maximal killing of viable cells in
the final preparation.
[0133] Some embodiments of the present invention relate to induce
the minicell production phenotype from an optimized eubacterial
minicell-producing strain from, but not limited to, the family
Enterobacteriaceae such that it may be "loaded" with small
molecules that comprise but are not limited to a drug, a pro-drug,
or a hormone following purification. Following production of the
desired quantity of "empty" minicells from a given culture and
condition, activation of the genetic suicide mechanism would be
stimulated following exposure of said culture or cells to a known
signal. The signal would be applied in each step of the
purification process to ensure maximal killing of viable cells in
the final preparation. Following purification, minicells would be
"loaded" with said small molecule(s) by incubation in a high
concentration of said small molecule at a temperature ranging from
4 to 65.degree. C.
[0134] Some embodiments of the present invention relate to induce
the minicell production phenotype from an optimized eubacterial
minicell-producing strain from, but not limited to, the family
Bacillaceae that contains a DNA molecule encoding for a therapeutic
or deleterious gene or gene product, such that the resulting
minicell contains said DNA molecule by way of encapsulation.
Following production of the desired quantity of minicells from a
given culture and condition, activation of the genetic suicide
mechanism would be stimulated following exposure of said culture or
cells to a known signal. The signal would be applied in each step
of the purification process to ensure maximal killing of viable
cells in the final preparation.
[0135] Some embodiments of the present invention relate to induce
the minicell production phenotype from an optimized eubacterial
minicell-producing strain from, but not limited to, the family
Bacillaceae that contains any subclass of RNA, including but not
limited to siRNA, antisense RNA, ribozymes, shRNA, and miRNA such
that the resulting minicell contains an enriched amount of said RNA
molecules by way of encapsulation. Following production of the
desired quantity of minicells from said culture and condition,
activation of the genetic suicide mechanism would be stimulated
following exposure of said culture or cells to a known signal. The
signal would be applied in each step of the purification process to
ensure maximal killing of viable cells in the final
preparation.
[0136] Some embodiments of the present invention relate to induce
the minicell production phenotype from an optimized eubacterial
minicell-producing strain from, but not limited to, the family
Bacillaceae that contains a protein molecule, such that the
resulting minicell contains said protein molecule by way of
encapsulation. Following production of the desired quantity of
minicells from a given culture and condition, activation of the
genetic suicide mechanism would be stimulated following exposure of
said culture or cells to a known signal. The signal would be
applied in each step of the purification process to ensure maximal
killing of viable cells in the final preparation.
[0137] Some embodiments of the present invention relate to induce
the minicell production phenotype from an optimized eubacterial
minicell-producing strain from, but not limited to, the family
Bacillaceae that contains a predetermined and deliberate
combination of DNA molecules encoding for a therapeutic or
deleterious gene or gene product, any subclass of RNA, and/or
proteins, such that the resulting minicell contains said
combination of molecules by way of encapsulation. Following
production of the desired quantity of minicells from a given
culture and condition, activation of the genetic suicide mechanism
would be stimulated following exposure of said culture or cells to
a known signal. The signal would be applied in each step of the
purification process to ensure maximal killing of viable cells in
the final preparation.
[0138] Some embodiments of the present invention relate to induce
the minicell production phenotype from an optimized eubacterial
minicell-producing strain from, but not limited to, the family
Bacillaceae such that it may be "loaded" with small molecules that
comprise but are not limited to a drug, a pro-drug, or a hormone
following purification. Following production of the desired
quantity of "empty" minicells from a given culture and condition,
activation of the genetic suicide mechanism would be stimulated
following exposure of said culture or cells to a known signal. The
signal would be applied in each step of the purification process to
ensure maximal killing of viable cells in the final preparation.
Following purification, minicells would be "loaded" with said small
molecule(s) by incubation in a high concentration of said small
molecule at a predetermined temperature.
[0139] In one embodiment, the level of minicell producing parental
cell contamination is 1 in 10.sup.7 minicells.
[0140] In another embodiment the level of minicell producing
parental cell contamination is 1 in 10.sup.8 minicells.
[0141] In another embodiment the level of minicell producing
parental cell contamination is 1 in 10.sup.9 minicells.
[0142] In another embodiment the level of minicell producing
parental cell contamination is 1 in 10.sup.10 minicells.
[0143] In another embodiment the level of minicell producing
parental cell contamination is 1 in 10.sup.11 minicells.
[0144] In another embodiment the level of minicell producing
parental cell contamination is 1 in 10.sup.12 minicells.
[0145] In another embodiment the level of minicell producing
parental cell contamination is 1 in 10.sup.13 minicells.
[0146] In another embodiment the level of minicell producing
parental cell contamination is in 10.sup.14 minicells.
[0147] In another embodiment the level of minicell producing
parental cell contamination is in 10.sup.15 minicells.
[0148] In another embodiment the level of minicell producing
parental cell contamination is 1 in 10.sup.16 minicells.
[0149] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. Although the present invention has been
described with reference to embodiments and examples, it should be
understood that various modifications can be made without departing
from the spirit of the invention. All references cited herein are
expressly incorporated herein by reference in their entirety.
EXAMPLES
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 .mu.g/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 .mu.g/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::ftsZ.OMEGA.CI857ts:I-CeuI) were
integrated into the .alpha.ttB.lamda. site on the chromosome of the
minicell-producing E. coli cells, respectively. Minicell
productions of the Ptac::ftsZ strain was conducted at 37.degree. C.
and minicell products of the Ptac::ftsZ.OMEGA.CI857ts::I-CeuI
strain was conducted at 42.degree. 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 overexpressed in the mini-cell
producing cells, the number of minicells produced increased 36-fold
compared to the minCDE-strain and increased 10-fold compared to the
overexpression of ftsZ alone.
Example 4
Overexpression of ftsZ and Induction of I-CeuI Based Suicide System
(MSM) Caused Cell Filamentation
[0153] E. coli strain VAX8I3 with the inducible ftsZ minicell
production system and heat inducible I-CeuI suicide system (pVX-66
(SEQ ID NO:5); Ptac::ftsZ.OMEGA.CI857ts::I-CeuI) were grown in LB
medium. At O.D. A600 of 0.1, FtsZ and I-CeuI protein production
were induced by elevating temperature to 42.degree. C. After 24
hours of induction, cells were Gram-stained. FIG. 4A shows the E.
coli strain grown at 30.degree. C. in presence of glucose (0.2%) to
suppress I-CeuI and ftsZ overexpression. In FIG. 4B, IPTG (20
.mu.g/ml) was added to overexpress ftsZ, but the expression of
I-CeuI was suppressed by incubating at 30.degree. C. In FIG. 4C,
the expression of I-CeuI was induced at 42.degree. C., but the
overexpression of ftsZ was suppressed by glucose. FIG. 4D shows
that simultaneous overexpression of ftsZ and induction of I-CeuI
cause more extensive filamentation of cells compared to
overexpression of ftsZ in FIG. 48 and induction of I-CeuI
expression alone in FIG. 4C. Accordingly, in addition to generating
high yields of minicells, simultaneous overexpression of ftsZ and
I-CeuI has the advantage of enabling the minicell-producing parent
cells to become uniformly filamentous, which can better facilitate
filtration-based minicell purification schemes.
Example 5
Induction of I-CeuI Based Suicide System (MSM) Caused Accumulation
of Cells with TUNEL Labeled 3'OH DNA Ends which Indicates
Double-Stranded Chromosomal Breaks
[0154] E. coli strain VAX813, which contains the MSM system under
the control of the CI857ts and pTac promoter systems (pVX-66;
Ptac::ftsZQCI857ts::I-CeuI, controlling the expression of I-CeuI
and ftsZ, respectively) was grown at either 30.degree. C. or
42.degree. C. in LB medium supplemented with IPTG for 24 hours.
Cells were TUNEL (FITC) stained at the indicated time points. As a
comparative control, cells were also counter-stained with FM-464
(stains all cells) such that the percentage of TUNEL positive cells
amongst the total population were quantified via FACS. FIG. 5 shows
that the I-CeuI based suicide system successfully introduced
irreparable double-stranded chromosomal breaks in the
minicell-producing parent cells, and resulted in the death of over
70% of the cell population within 12 hours after the induction of
I-CeuI.
Example 6
I-CeuI Based Suicide System Reduced Parental Cell Contaminations
Among Purified Minicells
[0155] IPTG inducible ftsZ was integrated into attB.lamda. site on
E. coli chromosome to make a minicell-producing E. coli strain
(Ptac::ftsZ) via overexpression of ftsZ. Heat-inducible I-CeuI
based suicide system was also integrated into the .alpha.ttB.lamda.
site together with the IPTG inducible ftsZ using the integration
plasmid pVX-66 (pVX-66; Ptac::ftsZ.OMEGA.CI857ts::I-CeuI) to make a
suicidal minicell-producing E. coli strain
(Ptac::ftsZ.OMEGA.CI857ts::I-CeuI). The I-CeuI suicide system was
activated by incubating at 42.degree. C. Minicells were produced in
LB medium supplemented with IPTG and purified via differential
purifications. Purified minicells were spread onto LB agar plates
supplemented with glucose (0.2%) to examine the presence of live
parental E. coli cells. After 48 hours of incubation at 30.degree.
C., colonies were counted and concentrations of contaminating
parental cells were calculated as colony forming unit (CFU) in
10.sup.10 minicells. FIG. 6 shows that the activation of the I-CeuI
based suicide system reduced parental cell contamination by over
800 fold.
Example 7
Deletion of msbB in S. typhimurium Changed LPS Profiles
[0156] LPS was purified from S. typhimurium strains with wild type
msbB (WT) and deleted msbB (msbB-). Deletion of msbB was conducted
by substitution of msbB with FRT-cat-FRT via .lamda. Red
recombinase system (Red Swap). Acetone dried cells were first
treated with DNase I and RNase A followed by Proteinase K
treatments. LPS was then extracted via hot water-phenol extraction.
LPS was purified via dialysis against water. Purified LPS were
separated with SDS-PAGE gel electrophoresis and silver stained. LPS
of MsbB mutants have lipid A without myristoyl group. The lack of
the myristoyl group reduces molecular weight that can be visualized
by shifts in LPS band patterns. FIG. 7 shows that deletion of msbB
gene resulted in altered LPS profiles in the S. typhimurium mutant
strain as compared to the wild-type S. typhimurium strain.
Example 8
Deletion of msbB Causes J774.A1 Mouse Macrophage Like Cells to
Produce Less Amounts of Tumor Necrosis Factor .alpha. (TNF.alpha.)
Against S. typhimurium LPS
[0157] LPS was purified from S. typhimurium strains with wild type
(WT) msbB and S. typhimurium strains harboring msbB deletion
mutation, respectively. J774.A1 mouse macrophage-like cells
(10.sup.6 cells) were incubated with 0.1 ng of each type of
purified LPS for 12 hours. TNF.alpha. concentration was determined
via Enzyme Linked Immuno Sorbent Assay (ELISA). FIG. 8 shows that
deletion of msbB in a minicell-producing Salmonella strain gave
rise to minicells with reduced toxicity as measured by the
production of TNF-.alpha. by cultured murine macrophages.
Sequence CWU 1
1
61657DNAChlamydomonas moewusii 1atgtcaaact ttatacttaa accgggcgaa
aaactacccc aagacaaact agaagaatta 60aaaaaaatta atgatgctgt taaaaaaacg
aaaaatttct caaaatactt gattgactta 120agaaaacttt ttcaaattga
cgaagtccaa gtaacttctg aatcaaaact ctttttagct 180ggttttttag
aaggtgaagc ttctctaaat attagcacta aaaagctcgc tacttctaaa
240tttggtttgg tggttgatcc tgaattcaat gtgactcaac atgtcaatgg
ggttaaagtg 300ctttatttag cattagaagt atttaaaaca gggcgtattc
gtcataaaag tggtagtaat 360gcaactttag ttttaactat tgacaatcgt
caaagtttgg aagaaaaagt aattcctttt 420tatgaacaat atgttgttgc
cttcagttct ccagaaaaag tcaaacgtgt agctaatttt 480aaagctttgt
tagaattatt taataatgac gctcaccaag atttagaaca attggtaaac
540aaaatcctac caatttggga tcaaatgcgt aaacaacaag gacaaagtaa
cgaaggcttt 600cctaatttag aagcagctca agactttgct cgtaattata
aaaaaggtat aaagtag 657229DNAArtificial SequenceSingle-Stranded
I-CeuI DNA recognition site 2cgtaactata acggtcctaa ggtagcgaa
293383PRTEscherichia coli 3Met Phe Glu Pro Met Glu Leu Thr Asn Asp
Ala Val Ile Lys Val Ile1 5 10 15Gly Val Gly Gly Gly Gly Gly Asn Ala
Val Glu His Met Val Arg Glu 20 25 30Arg Ile Glu Gly Val Glu Phe Phe
Ala Val Asn Thr Asp Ala Gln Ala 35 40 45Leu Arg Lys Thr Ala Val Gly
Gln Thr Ile Gln Ile Gly Ser Gly Ile 50 55 60Thr Lys Gly Leu Gly Ala
Gly Ala Asn Pro Glu Val Gly Arg Asn Ala65 70 75 80Ala Asp Glu Asp
Arg Asp Ala Leu Arg Ala Ala Leu Glu Gly Ala Asp 85 90 95Met Val Phe
Ile Ala Ala Gly Met Gly Gly Gly Thr Gly Thr Gly Ala 100 105 110Ala
Pro Val Val Ala Glu Val Ala Lys Asp Leu Gly Ile Leu Thr Val 115 120
125Ala Val Val Thr Lys Pro Phe Asn Phe Glu Gly Lys Lys Arg Met Ala
130 135 140Phe Ala Glu Gln Gly Ile Thr Glu Leu Ser Lys His Val Asp
Ser Leu145 150 155 160Ile Thr Ile Pro Asn Asp Lys Leu Leu Lys Val
Leu Gly Arg Gly Ile 165 170 175Ser Leu Leu Asp Ala Phe Gly Ala Ala
Asn Asp Val Leu Lys Gly Ala 180 185 190Val Gln Gly Ile Ala Glu Leu
Ile Thr Arg Pro Gly Leu Met Asn Val 195 200 205Asp Phe Ala Asp Val
Arg Thr Val Met Ser Glu Met Gly Tyr Ala Met 210 215 220Met Gly Ser
Gly Val Ala Ser Gly Glu Asp Arg Ala Glu Glu Ala Ala225 230 235
240Glu Met Ala Ile Ser Ser Pro Leu Leu Glu Asp Ile Asp Leu Ser Gly
245 250 255Ala Arg Gly Val Leu Val Asn Ile Thr Ala Gly Phe Asp Leu
Arg Leu 260 265 270Asp Glu Phe Glu Thr Val Gly Asn Thr Ile Arg Ala
Phe Ala Ser Asp 275 280 285Asn Ala Thr Val Val Ile Gly Thr Ser Leu
Asp Pro Asp Met Asn Asp 290 295 300Glu Leu Arg Val Thr Val Val Ala
Thr Gly Ile Gly Met Asp Lys Arg305 310 315 320Pro Glu Ile Thr Leu
Val Thr Asn Lys Gln Val Gln Gln Pro Val Met 325 330 335Asp Arg Tyr
Gln Gln His Gly Met Ala Pro Leu Thr Gln Glu Gln Lys 340 345 350Pro
Val Ala Lys Val Val Asn Asp Asn Ala Pro Gln Thr Ala Lys Glu 355 360
365Pro Asp Tyr Leu Asp Ile Pro Ala Phe Leu Arg Lys Gln Ala Asp 370
375 3804218PRTChlamydomonas moewusii 4Met Ser Asn Phe Ile Leu Lys
Pro Gly Glu Lys Leu Pro Gln Asp Lys1 5 10 15Leu Glu Glu Leu Lys Lys
Ile Asn Asp Ala Val Lys Lys Thr Lys Asn 20 25 30Phe Ser Lys Tyr Leu
Ile Asp Leu Arg Lys Leu Phe Gln Ile Asp Glu 35 40 45Val Gln Val Thr
Ser Glu Ser Lys Leu Phe Leu Ala Gly Phe Leu Glu 50 55 60Gly Glu Ala
Ser Leu Asn Ile Ser Thr Lys Lys Leu Ala Thr Ser Lys65 70 75 80Phe
Gly Leu Val Val Asp Pro Glu Phe Asn Val Thr Gln His Val Asn 85 90
95Gly Val Lys Val Leu Tyr Leu Ala Leu Glu Val Phe Lys Thr Gly Arg
100 105 110Ile Arg His Lys Ser Gly Ser Asn Ala Thr Leu Val Leu Thr
Ile Asp 115 120 125Asn Arg Gln Ser Leu Glu Glu Lys Val Ile Pro Phe
Tyr Glu Gln Tyr 130 135 140Val Val Ala Phe Ser Ser Pro Glu Lys Val
Lys Arg Val Ala Asn Phe145 150 155 160Lys Ala Leu Leu Glu Leu Phe
Asn Asn Asp Ala His Gln Asp Leu Glu 165 170 175Gln Leu Val Asn Lys
Ile Leu Pro Ile Trp Asp Gln Met Arg Lys Gln 180 185 190Gln Gly Gln
Ser Asn Glu Gly Phe Pro Asn Leu Glu Ala Ala Gln Asp 195 200 205Phe
Ala Arg Asn Tyr Lys Lys Gly Ile Lys 210 21559446DNAArtificial
SequencepVX-66 integration vector (RK6 origin of replication;
I-CeuI under c1857ts promoter control; FtsZ under IPTG inducible
promoter control; proBA; attpP) 5tctagaaacc atggaagcta gcgaacttac
tttatacctt ttttataatt acgagcaaag 60tcttgagctg cttctaaatt aggaaagcct
tcgttacttt gtccttgttg tttacgcatt 120tgatcccaaa ttggtaggat
tttgtttacc aattgttcta aatcttggtg agcgtcatta 180ttaaataatt
ctaacaaagc tttaaaatta gctacacgtt tgactttttc tggagaactg
240aaggcaacaa catattgttc ataaaaagga attacttttt cttccaaact
ttgacgattg 300tcaatagtta aaactaaagt tgcattacta ccacttttat
gacgaatacg ccctgtttta 360aatacttcta atgctaaata aagcacttta
accccattga catgttgagt cacattgaat 420tcaggatcaa ccaccaaacc
aaatttagaa gtagcgagct ttttagtgct aatatttaga 480gaagcttcac
cttctaaaaa accagctaaa aagagttttg attcagaagt tacttggact
540tcgtcaattt gaaaaagttt tcttaagtca atcaagtatt ttgagaaatt
tttcgttttt 600ttaacagcat cattaatttt ttttaattct tctagtttgt
cttggggtag tttttcgccc 660ggtttaagta taaagtttga catgagttat
ttcctcctaa aactcgaggc gccaatgctt 720cgtttcgtat cacacacccc
aaagccttct gctttgaatg ctgcccttct tcagggctta 780atttttaaga
gcgtcacctt catggtggtc agtgcgtcct gctgatgtgc tcagtatcac
840cgccagtggt atttatgtca acaccgccag agataattta tcaccgcaga
tggttatctg 900tatgtttttt atatgaattt attttttgca ggggggcatt
gtttggtagg tgagagatcc 960ccggggggca gaactcaaaa attccggtgc
aaaacagaca ggcgaaacac tgaagatcaa 1020cattcttgat ctttagctgt
cttggtttgc ccaaagcgca ttgcataatc tttcagggtt 1080atgcgttgtt
ccatacaacc tccttagtac atgcaaccat tatcaccgcc agaggtaaaa
1140tagtcaacac gcacggtgtt agatatttat cccttgcggt gatagattta
acgtatgagc 1200acaaaaaaga aaccattaac acaagagcag cttgaggacg
cacgtcgcct taaagcaatt 1260tatgaaaaaa agaaaaatga acttggctta
tcccaggaat ctgtcgcaga caagatgggg 1320atggggcagt caggcgttgg
tgctttattt aatggcatca atgcattaaa tgcttataac 1380gccgcattgc
ttgcaaaaat tctcaaagtt agcgttgaag aatttagccc ttcaatcgcc
1440agagaaatct acgagatgta tgaagcggtt agtatgcagc cgtcacttag
aagtgagtat 1500gagtaccctg ttttttctca tgttcaggca gggatgttct
cacctaagct tagaaccttt 1560accaaaggtg atgcggagag atgggtaagc
acaaccaaaa aagccagtga ttctgcattc 1620tggcttgagg ttgaaggtaa
ttccatgacc gcaccaacag gctccaagcc aagctttcct 1680gacggaatgt
taattctcgt tgaccctgag caggctgttg agccaggtga tttctgcata
1740gccagacttg ggggtgatga gtttaccttc aagaaactga tcagggatag
cggtcaggtg 1800tttttacaac cactaaaccc acagtaccca atgatcccat
gcaatgagag ttgttccgtt 1860gtggggaaag ttatcgctag tcagtggcct
gaagagacgt ttggctgatc ccacagccgc 1920cagttccgct ggcggcattt
tggatccact agtaacggcc gccagtgtgc tggaattcgc 1980ccttcaaggt
taaaactaag gtaccatgcg tcaatggcct tgtgaatcaa atggctactt
2040ttgcatcacc cggttttatt tacgcacgaa tggtgtaatc accaatgccg
atccacttgt 2100aagtggtcag tgcttccagc cccattgggc cacgcgcgtg
gagtttttgt gtgcttaccg 2160ccacttccgc acccagacca aactggccgc
cgtcggtaaa acgcgtagag gcgttaacgt 2220aaacagcgga cgaatccact
tcgttaacaa aacgctgggc gttgcgcata tcgcgggtca 2280ggatcgcatc
ggagtgttgt gtgccgtgtt cacgaatatg ggcgatggca tcgtcaagat
2340cgctgacgat tttgacgttc aaatctaatg acagaaactc atcgtcatac
tcttcggctt 2400taacagcaac caccttcgca gggcctgcct gcaactgcgc
cagtgcagct gcatctgcgt 2460gtaatgtcac gccgctttcc gccatttgtt
tgcttaatgc gggcaggaag ctatcggcga 2520tgtttttatt caccagcaac
gtttcaaccg tattacatgt gctcggacgc tgagttttcg 2580cgttgacgat
cacttttaat gcttcagcga tctctacact ttcatcaacg taaatatggc
2640atacgcctat accacctgtg atcaccggga ttgtcgactg ttcacggcac
agtttatgca 2700aaccagcgcc accacgcggg atcagcatgt cgatgtattt
atccatacgc agcatttcac 2760tgaccagcgc acggtcagga ttatcaatcg
cctgcacggc acccgccggt aagccgcagg 2820atttcagggc gtcctgaatc
accgccaccg ttgcagcgtt agtgcgacac gtttctttgc 2880caccgcgcag
gatcaccgca ttaccggttt tcaggcacag cgaagcgaca tcaaccgtca
2940cgttcgggcg cgcttcataa atcacgccaa taacccccag cggtacgcga
cgacgctcaa 3000gacgcaggcc gctgtccagt acgccgccat cgattacctg
ccccaccgga tcggcgaggt 3060tgcacacctg acgtacatcg tcggcaatgc
ctttcagccg tgcgggcgtc agtgccagac 3120ggtcaagcat cgcttcgcta
aggccattgg ctcgcgcgtc agcaacatcc tgggcgttag 3180cgttgaggat
gatttcgctt tgtgcttcca gttcatcggc gattttttcc agcacgcgat
3240ttttttcgcg gctggagagt tgcgctaatt tatacgaggc ttgcttcgcg
gcaatgccca 3300tttgttccag catcagcctg ctccttaacg ggtaatcatg
tcatcacggt gaacggcaac 3360cgggccgtat tcatatccca gtattgcatc
aatttcttgc gagtggtgtc cggcaatacg 3420gcgtaatgca tcgctgttgt
aacgactgac gccgtgggcg atatcgcggc cttcgaggtt 3480gcaaatgcgg
atgacttcac cacgcgagaa attgccagtc acgcttttaa tgcctttcgg
3540caacagggag ctgccgcgtt ccagaatggc ggcagttgcc ccttcatcta
ccgtgatttc 3600acccgccggc ggcgcaccga aaatccagcg tttacggttt
tcaagcggag tcgcctgggc 3660atggaacagc gtaccgacgg aaatgccttc
catcacatca ccaataacgc ccggcttgct 3720gcccgcggca ataatggtgt
cgatacccgc acggcaagcc acgtcagcgg cctgcaattt 3780ggtactcatg
ccgccagttc cgaggcctga aacgctgtca ccggcaatcg cgcgcagtgc
3840gtcatcaatg ccgtaaacat ctttaatcag ttctgcctgc ggattgctgc
gcgggtcagc 3900ggtatacaaa cctttttgat cggtcagcag caacagttta
tcggcacccg caagaatcgc 3960cgccagcgca gaaaggttat cgttatcgcc
gaccttaatc tctgccgtag cgacagcatc 4020gttctcattg attaccggaa
cgatattgtt atcgagcaac gctcgcaggg tgtcgcgggc 4080gttcaggaag
cgttcacggt cttccatatc agcacgggtc agcagcattt gcccgacgtg
4140aatgccataa atcgaaaaca gctgttccca cagttgaatc agtcgactct
gccctaccgc 4200cgccagcagt tgtttcgagg cgatggtcgc tggcagttcc
gggtaaccca ggtgctcacg 4260tccggcggcg atcgcgcccg acgtcacaat
aacaatccga tgcccggcgg catgtaactg 4320cgcgcactgg cgaacaagtt
caacgatatg ggcacggttc agacggcgcg atccgcctgt 4380tagcacactg
gtgccgagtt ttaccaccag cgtctggctg tcactcatga ttctctgcca
4440ttcaatttta ggaaaaatga tatcaaacga acgttttagc aggactgtcg
tcggttgcca 4500accatctgcg agcaaagcat ggcgttttgt tgcgcgatct
gtaataaaag cgtaaacgca 4560tgcgatatcg agctctcccg ggaattcttg
cgctaatgct ctgttacagg tcactaatac 4620catctaagta gttgattcat
agtgactgca tatgttgtgt tttacagtat tatgtagtct 4680gttttttatg
caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt
4740cagctttttt atactaagtt ggcattataa aaaagcattg cttatcaatt
tgttgcaacg 4800aacaggtcac tatcagtcaa aataaaatca ttatttgatt
tcaattttgt cccgaattcg 4860atcgctagtt tgttttgact ccatccatta
gggcttctaa aacgccttct aaggccatgt 4920cagccgttaa gtgttcctgt
gtcactgaaa attgctttga gaggctctaa gggcttctca 4980gtgcgttaca
tccctggctt gttgtccaca accgttaaac cttaaaagct ttaaaagcct
5040tatatattct tttttttctt ataaaactta aaaccttaga ggctatttaa
gttgctgatt 5100tatattaatt ttattgttca aacatgagag cttagtacgt
gaaacatgag agcttagtac 5160gttagccatg agagcttagt acgttagcca
tgagggttta gttcgttaaa catgagagct 5220tagtacgtta aacatgagag
cttagtacgt gaaacatgag agcttagtac gtactatcaa 5280caggttgaac
tgcggatctt gcggccgcat tcccaattcc aggcatcaaa taaaacgaaa
5340ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
acgctctcct 5400gagtaggaca aatccgccgg gagcggattt gaacgttgcg
aagcaacggc ccggagggtg 5460gcgggcagga cgcccgccat aaactgccag
gaattaattc caggcatcaa ataaaacgaa 5520aggctcagtc gaaagactgg
gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc 5580tgagtaggac
aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
5640ggcgggcagg acgcccgcca taaactgcca ggaattaatt ccaggcatca
aataaaacga 5700aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
gtttgtcggt gaacgctctc 5760ctgagtagga caaatccgcc gggagcggat
ttgaacgttg cgaagcaacg gcccggaggg 5820tggcgggcag gacgcccgcc
ataaactgcc aggaattaat tccaggcatc aaataaaacg 5880aaaggctcag
tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
5940cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
ggcccggagg 6000gtggcgggca ggacgcccgc cataaactgc caggaattgg
ggatcggaat tcgacgaacg 6060ccagcaagac gtagcccagc gcgtcggcca
gcttgcaatt cgcgctaact tacattaatt 6120gcgttgcgct cactgcccgc
tttccagtcg ggaaacctgt cgtgccagct gcattaatga 6180atcggccaac
gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
6240caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
agagttgcag 6300caagcggtcc acgctggttt gccccagcag gcgaaaatcc
tgtttgatgg tggttgacgg 6360cgggatataa catgagctgt cttcggtatc
gtcgtatccc actaccgaga tatccgcacc 6420aacgcgcagc ccggactcgg
taatggcgcg cattgcgccc agcgccatct gatcgttggc 6480aaccagcatc
gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
6540ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
tgcgagtgag 6600atatttatgc cagccagcca gacgcagacg cgccgagaca
gaacttaatg ggcccgctaa 6660cagcgcgatt tgctggtgac ccaatgcgac
cagatgctcc acgcccagtc gcgtaccgtc 6720ttcatgggag aaaataatac
tgttgatggg tgtctggtca gagacatcaa gaaataacgc 6780cggaacatta
gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
6840aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
tacaggcttc 6900gacgccgctt cgttctacca tcgacaccac cacgctggca
cccagttgat cggcgcgaga 6960tttaatcgcc gcgacaattt gcgacggcgc
gtgcagggcc agactggagg tggcaacgcc 7020aatcagcaac gactgtttgc
ccgccagttg ttgtgccacg cggttgggaa tgtaattcag 7080ctccgccatc
gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
7140caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
cgtataacgt 7200tactggtttc acattcacca ccctgaattg actctcttcc
gggcgctatc atgccatacc 7260gcgaaaggtt ttgcaccatt cgatggtgtc
aacgtaaatg ccgcttcgcc ttcgcgcgcg 7320aattgcaagc tgatccgggc
ttatcgactg cacggtgcac caatgcttct ggcgtcaggc 7380agccatcgga
agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc
7440tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac
ggttctggca 7500aatattctga aatgagctgt tgacaattaa tcatcggctc
gtataacgtg tggaattgtg 7560agcggataat aatttcacac aggaaacaga
attaattccc ggggatctca ggcgacaggc 7620acaaatcgga gagaaactat
gtttgaacca atggaactta ccaatgacgc ggtgattaaa 7680gtcatcggcg
tcggcggcgg cggcggtaat gctgttgaac acatggtgcg cgagcgcatt
7740gaaggtgttg aattcttcgc ggtaaatacc gatgcacaag cgctgcgtaa
aacagcggtt 7800ggacagacga ttcaaatcgg tagcggtatc accaaaggac
tgggcgctgg cgctaatcca 7860gaagttggcc gcaatgcggc tgatgaggat
cgcgatgcat tgcgtgcggc gctggaaggt 7920gcagacatgg tctttattgc
tgcgggtatg ggtggtggta ccggtacagg tgcagcacca 7980gtcgtcgctg
aagtggcaaa agatttgggt atcctgaccg ttgctgtcgt cactaagcct
8040ttcaactttg aaggcaagaa gcgtatggca ttcgcggagc aggggatcac
tgaactgtcc 8100aagcatgtgg actctctgat cactatcccg aacgacaaac
tgctgaaagt tctgggccgc 8160ggtatctccc tgctggatgc gtttggcgca
gcgaacgatg tactgaaagg cgctgtgcaa 8220ggtatcgctg aactgattac
tcgtccgggt ttgatgaacg tggactttgc agacgtacgc 8280accgtaatgt
ctgagatggg ctacgcaatg atgggttctg gcgtggcgag cggtgaagac
8340cgtgcggaag aagctgctga aatggctatc tcttctccgc tgctggaaga
tatcgacctg 8400tctggcgcgc gcggcgtgct ggttaacatc acggcgggct
tcgacctgcg tctggatgag 8460ttcgaaacgg taggtaacac catccgtgca
tttgcttccg acaacgcgac tgtggttatc 8520ggtacttctc ttgacccgga
tatgaatgac gagctgcgcg taaccgttgt tgcgacaggt 8580atcggcatgg
acaaacgtcc tgaaatcact ctggtgacca ataagcaggt tcagcagcca
8640gtgatggatc gctaccagca gcatgggatg gctccgctga cccaggagca
gaagccggtt 8700gctaaagtcg tgaatgacaa tgcgccgcaa actgcgaaag
agccggatta tctggatatc 8760ccagcattcc tgcgtaagca agctgattaa
gaattgactg gaatttgggt ttcgaggctc 8820tttgtgctaa actggcccgc
cgaatgtata gtacacttcg gttggatagg taatttggcg 8880agataatacg
atgatcaaac aaaggacact taaacgtatc gttcaggcga cgggtgtcgg
8940tttacatacc ggcaagaaag tcaccctgac gttacgccct gcgccggcca
acaccggggt 9000catctatcgt cgcaccgact tgaatccacc ggtagatttc
ccggccgatg ccaaatctgt 9060gcgtgatacc atgctctgta cgtgtctggt
caacgagcat gatgtacgga tttcaaccgt 9120agagcacctc aatgctgctc
tcgcgggctt gggcatcgat ggccccccga tggtagtgtg 9180gggtctcccc
atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
9240gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
tgagtaggac 9300aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg
cccggagggt ggcgggcagg 9360acgcccgcca taaactgcca ggcatcaaat
taagcagaag gccatcctga cggatggcct 9420ttttgcgtgg ccagtgccaa gcttct
944666251DNAArtificial SequencepVX-55 expression vector (contains
I-CeuI gene under the control of the rhamnose inducible pRHA
promoter system) 6aagggcgaat tctgcagata tccatcacac tggcggccgc
tcgagcatgc atctagaggg 60cccaattcgc cctatagtga gtcgtattac aattcactgg
ccgtcgtttt acaacgtcgt 120gactgggaaa accctggcgt tacccaactt
aatcgccttg cagcacatcc ccctttcgcc 180agctggcgta atagcgaaga
ggcccgcacc gatcgccctt cccaacagtt gcgcagccta 240tacgtacggc
agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
300gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc
cctggccagt 360gcacgtctgc tgtcagataa agtctcccgt gaactttacc
cggtggtgca tatcggggat 420gaaagctggc gcatgatgac caccgatatg
gccagtgtgc cggtctccgt tatcggggaa 480gaagtggctg atctcagcca
ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc 540tggggaatat
aaatgtcagg catgagatta tcaaaaagga tcttcaccta gatccttttc
600acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag
ctactgggct 660atctggacaa gggaaaacgc aagcgcaaag agaaagcagg
tagcttgcag tgggcttaca 720tggcgatagc tagactgggc ggttttatgg
acagcaagcg
aaccggaatt gccagctggg 780gcgccctctg gtaaggttgg gaagccctgc
aaagtaaact ggatggcttt ctcgccgcca 840aggatctgat ggcgcagggg
atcaagctct gatcaagaga caggatgagg atcgtttcgc 900atgattgaac
aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
960ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
ccggctgtca 1020gcgcaggggc gcccggttct ttttgtcaag accgacctgt
ccggtgccct gaatgaactg 1080caagacgagg cagcgcggct atcgtggctg
gccacgacgg gcgttccttg cgcagctgtg 1140ctcgacgttg tcactgaagc
gggaagggac tggctgctat tgggcgaagt gccggggcag 1200gatctcctgt
catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
1260cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
gaaacatcgc 1320atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
atcaggatga tctggacgaa 1380gagcatcagg ggctcgcgcc agccgaactg
ttcgccaggc tcaaggcgag catgcccgac 1440ggcgaggatc tcgtcgtgac
ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 1500ggccgctttt
ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
1560atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
tgaccgcttc 1620ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
tcgccttcta tcgccttctt 1680gacgagttct tctgaattat taacgcttac
aatttcctga tgcggtattt tctccttacg 1740catctgtgcg gtatttcaca
ccgcatacag gtggcacttt tcggggaaat gtgcgcggaa 1800cccctatttg
tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
1860cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag
ttgaccagtg 1920ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt
cgagttctgg accgaccggc 1980tcgggttctc ccgggacttc gtggaggacg
acttcgccgg tgtggtccgg gacgacgtga 2040ccctgttcat cagcgcggtc
caggaccagg tggtgccgga caacaccctg gcctgggtgt 2100gggtgcgcgg
cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
2160gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
cgggagttcg 2220ccctgcgcga cccggccggc aactgcgtgc acttcgtggc
cgaggagcag gactgacacg 2280tgctaaaact tcatttttaa tttaaaagga
tctaggtgaa gatccttttt gataatctca 2340tgaccaaaat cccttaacgt
gagttttcgt tccactgagc gtcagacccc gtagaaaaga 2400tcaaaggatc
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
2460aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
ctttttccga 2520aggtaactgg cttcagcaga gcgcagatac caaatactgt
ccttctagtg tagccgtagt 2580taggccacca cttcaagaac tctgtagcac
cgcctacata cctcgctctg ctaatcctgt 2640taccagtggc tgctgccagt
ggcgataagt cgtgtcttac cgggttggac tcaagacgat 2700agttaccgga
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
2760tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
gaaagcgcca 2820cgcttcccga agggagaaag gcggacaggt atccggtaag
cggcagggtc ggaacaggag 2880agcgcacgag ggagcttcca gggggaaacg
cctggtatct ttatagtcct gtcgggtttc 2940gccacctctg acttgagcgt
cgatttttgt gatgctcgtc aggggggcgg agcctatgga 3000aaaacgccag
caacgcggcc tttttacggt tcctgggctt ttgctggcct tttgctcaca
3060tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
tttgagtgag 3120ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
gtcagtgagc gaggaagcgg 3180aagagcgccc aatacgcaaa ccgcctctcc
ccgcgcgttg gccgattcat taatgcagct 3240ggcacgacag gtttcccgac
tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 3300agctcactca
ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
3360gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
tacgccaagc 3420tatttaggtg acactataga atactcaagc tatgcatcaa
gcttggtacc gagctcggat 3480ccactagtaa cggccgccag tgtgctggaa
ttcgcccttt ctagaattaa tctttctgcg 3540aattgagatg acgccactgg
ctgggcgtca tcccggtttc ccgggtaaac accaccgaaa 3600aatagttact
atcttcaaag ccacattcgg tcgaaatatc actgattaac aggcggctat
3660gctggagaag atattgcgca tgacacactc tgacctgtcg cagatattga
ttgatggtca 3720ttccagtctg ctggcgaaat tgctgacgca aaacgcgctc
actgcacgat gcctcatcac 3780aaaatttatc cagcgcaaag ggacttttca
ggctagccgc cagccgggta atcagcttat 3840ccagcaacgt ttcgctggat
gttggcggca acgaatcact ggtgtaacga tggcgattca 3900gcaacatcac
caactgcccg aacagcaact cagccatttc gttagcaaac ggcacatgct
3960gactactttc atgctcaagc tgaccgataa cctgccgcgc ctgcgccatc
cccatgctac 4020ctaagcgcca gtgtggttgc cctgcgctgg cgttaaatcc
cggaatcgcc ccctgccagt 4080caagattcag cttcagacgc tccgggcaat
aaataatatt ctgcaaaacc agatcgttaa 4140cggaagcgta ggagtgttta
tcgtcagcat gaatgtaaaa gagatcgcca cgggtaatgc 4200gataagggcg
atcgttgagt acatgcaggc cattaccgcg ccagacaatc accagctcac
4260aaaaatcatg tgtatgttca gcaaagacat cttgcggata acggtcagcc
acagcgactg 4320cctgctggtc gctggcaaaa aaatcatctt tgagaagttt
taactgatgc gccaccgtgg 4380ctacctcggc cagagaacga agttgattat
tcgcaatatg gcgtacaaat acgttgagaa 4440gattcgcgtt attgcagaaa
gccatcccgt ccctggcgaa tatcacgcgg tgaccagtta 4500aactctcggc
gaaaaagcgt cgaaaagtgg ttactgtcgc tgaatccaca gcgataggcg
4560atgtcagtaa cgctggcctc gctgtggcgt agcagatgtc gggctttcat
cagtcgcagg 4620cggttcaggt atcgctgagg cgtcagtccc gtttgctgct
taagctgccg atgtagcgta 4680cgcagtgaaa gagaaaattg atccgccacg
gcatcccaat tcacctcatc ggcaaaatgg 4740tcctccagcc aggccagaag
caagttgaga cgtgatgcgc tgttttccag gttctcctgc 4800aaactgcttt
tacgcagcaa gagcagtaat tgcataaaca agatctcgcg actggcggtc
4860gagggtaaat cattttcccc ttcctgctgt tccatctgtg caaccagctg
tcgcacctgc 4920tgcaatacgc tgtggttaac gcgccagtga gacggatact
gcccatccag ctcttgtggc 4980agcaactgat tcagcccggc gagaaactga
aatcgatccg gcgagcgata cagcacattg 5040gtcagacaca gattatcggt
atgttcatac agatgccgat catgatcgcg tacgaaacag 5100accgtgccac
cggtgatggt atagggctgc ccattaaaca catgaatacc cgtgccatgt
5160tcgacaatca caatttcatg aaaatcatga tgatgttcag gaaaatccgc
ctgcgggagc 5220cggggttcta tcgccacgga cgcgttacca gacggaaaaa
aatccacact atgtaatacg 5280gtcatactgg cctcctgatg tcgtcaacac
ggcgaaatag taatcacgag gtcaggttct 5340taccttaaat tttcgacgga
aaaccacgta aaaaacgtcg atttttcaag atacagcgtg 5400aattttcagg
aaatgcggtg agcatcacat caccacaatt cagcaaattg tgaacatcat
5460cacgttcatc tttccctggt tgccaatggc ccattttcct gtcagtaacg
agaaggtcgc 5520gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
gaggatggtc gacaggagga 5580cttcttttat gtcaaacttt atacttaaac
cgggcgaaaa actaccccaa gacaaactag 5640aagaattaaa aaaaattaat
gatgctgtta aaaaaacgaa aaatttctca aaatacttga 5700ttgacttaag
aaaacttttt caaattgacg aagtccaagt aacttctgaa tcaaaactct
5760ttttagctgg ttttttagaa ggtgaagctt ctctaaatat tagcactaaa
aagctcgcta 5820cttctaaatt tggtttggtg gttgatcctg aattcaatgt
gactcaacat gtcaatgggg 5880ttaaagtgct ttatttagca ttagaagtat
ttaaaacagg gcgtattcgt cataaaagtg 5940gtagtaatgc aactttagtt
ttaactattg acaatcgtca aagtttggaa gaaaaagtaa 6000ttccttttta
tgaacaatat gttgttgcct tcagttctcc agaaaaagtc aaacgtgtag
6060ctaattttaa agctttgtta gaattattta ataatgacgc tcaccaagat
ttagaacaat 6120tggtaaacaa aatcctacca atttgggatc aaatgcgtaa
acaacaagga caaagtaacg 6180aaggctttcc taatttagaa gcagctcaag
actttgctcg taattataaa aaaggtataa 6240agtaatctag a 6251
* * * * *