U.S. patent application number 15/868487 was filed with the patent office on 2018-09-27 for bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tighten gut mucosal barrier.
This patent application is currently assigned to Synlogic, Inc.. The applicant listed for this patent is Synlogic, Inc.. Invention is credited to Dean Falb, Vincent M. Isabella, Jonathan W. Kotula, Paul F. Miller.
Application Number | 20180273956 15/868487 |
Document ID | / |
Family ID | 56406992 |
Filed Date | 2018-09-27 |
United States Patent
Application |
20180273956 |
Kind Code |
A1 |
Falb; Dean ; et al. |
September 27, 2018 |
Bacteria Engineered to Treat Diseases that Benefit from Reduced Gut
Inflammation and/or Tighten Gut Mucosal Barrier
Abstract
Genetically engineered bacteria, pharmaceutical compositions
thereof, and methods of treating or preventing autoimmune
disorders, inhibiting inflammatory mechanisms in the gut, and/or
tightening gut mucosal barrier function are disclosed.
Inventors: |
Falb; Dean; (Sherborn,
MA) ; Isabella; Vincent M.; (Cambridge, MA) ;
Kotula; Jonathan W.; (Somerville, MA) ; Miller; Paul
F.; (Salem, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Synlogic, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Synlogic, Inc.
Cambridge
MA
|
Family ID: |
56406992 |
Appl. No.: |
15/868487 |
Filed: |
January 11, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14998376 |
Dec 22, 2015 |
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15868487 |
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62095415 |
Dec 22, 2014 |
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62095415 |
Dec 22, 2014 |
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62127131 |
Mar 2, 2015 |
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62248825 |
Oct 30, 2015 |
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62256044 |
Nov 16, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2035/115 20130101;
Y02A 50/473 20180101; Y02A 50/30 20180101; A61K 38/2066 20130101;
C12N 15/70 20130101; C12Y 207/02007 20130101; C12N 9/001 20130101;
C12Y 115/01001 20130101; A61K 38/446 20130101; Y02A 50/414
20180101; C12Y 103/08001 20130101; A61K 38/20 20130101; A61K 38/26
20130101; A61K 31/19 20130101; C12N 9/1217 20130101; A61K 35/741
20130101; A61K 38/2013 20130101; Y02A 50/401 20180101; A61K 31/198
20130101 |
International
Class: |
C12N 15/70 20060101
C12N015/70; C12N 9/12 20060101 C12N009/12; C12N 9/02 20060101
C12N009/02; A61K 38/44 20060101 A61K038/44; A61K 31/19 20060101
A61K031/19; A61K 38/26 20060101 A61K038/26; A61K 38/20 20060101
A61K038/20; A61K 35/741 20150101 A61K035/741; A61K 31/198 20060101
A61K031/198 |
Claims
1. A genetically engineered bacterium comprising: a) at least one
non-native copy of a first gene that encodes a transcription factor
protein that is regulated by a reactive nitrogen species (RNS),
wherein the first gene is operatively linked to a promoter; and b)
at least one of: i. a second gene encoding a non-native,
anti-inflammation molecule; ii. a second gene encoding a non-native
gut barrier function enhancer molecule; iii. a gene cassette
encoding a biosynthetic pathway, wherein a final product of the
biosynthetic pathway is an anti-inflammation molecule; iv. a gene
cassette encoding a biosynthetic pathway, wherein a final product
of the biosynthetic pathway is a gut barrier function enhancer
molecule, wherein the second gene or gene cassette in b) is
expressed under the control of a tunable regulatory region
heterologous to the gene or gene cassette, wherein induction of the
tunable regulatory region is directly or indirectly controlled by
the transcription factor.
2. The bacterium of claim 1, wherein the transcription factor is a
transcription activator.
3. The bacterium of claim 2, wherein induction of the tunable
regulatory region is directly controlled by the transcription
activator.
4. The bacterium of claim 1, wherein the transcription factor is a
transcription repressor.
5. The bacterium of claim 4, wherein derepression of the tunable
regulatory region is directly controlled by the transcription
repressor.
6. The bacterium of claim 1, wherein: the transcription factor is a
first transcription repressor; the bacterium further comprises a
third gene encoding a second transcription repressor, wherein
expression of the second transcription repressor is repressed by
the first repressor and the tunable regulatory region is repressed
by the second transcription repressor.
7. The bacterium of claim 1, wherein at least one of the one or
more non-native copies of the gene that encodes the transcription
factor is located on a plasmid in the bacterium.
8. The bacterium of claim 1, wherein at least one of the one or
more non-native copies of the gene that encodes the transcription
factor is located on a chromosome in the bacterium.
9. The bacterium of claim 1, wherein the promoter that controls
expression of at least one of the one or more non-native copies of
the gene that encodes the transcription factor is a constitutive
promoter.
10. The bacterium of claim 1, wherein the promoter that controls
expression of at least one of the one or more non-native copies of
the gene that encodes the transcription factor is an inducible
promoter.
11. The bacterium of claim 1, wherein the gene encoding the
anti-inflammation molecule, the gut barrier enhancer molecule, or
the gene cassette encoding the biosynthetic pathway is located on a
plasmid in the bacterium.
12. The bacterium of claim 1, wherein the gene encoding the
anti-inflammation molecule, the gut barrier enhancer molecule, or
the gene cassette encoding the biosynthetic pathway is located on a
chromosome in the bacterium.
13. The bacterium of claim 1, wherein the gene that encodes the
transcription factor protein is nitric oxide sensing repressor
NsrR.
14. The bacterium of claim 1, wherein the tunable regulatory region
that controls expression of the anti-inflammation molecule, the gut
barrier enhancer molecule, or the biosynthetic pathway is selected
from a native or a modified functional form of a regulatory region
from any one of nitric oxide reductase (norB), aniA, nsrR, hmpA,
ytfE, ygbA, hcp, hcr, nrfA, and alternative oxidase (aox).
15. The bacterium of claim 1, wherein the molecule of b) is
selected from propionate, butyrate, acetate, interleukin 10
(IL-10), interleukin 27 (IL-27), transforming growth factor 2 (TGF-
2), transforming growth factor 1 (TGF- 1), glucagon-like peptide
(GLP-2), N-acylphosphatidylethanolamines (NAPEs), elafin, trefoil
factor, and single-chain variable fragment (scFv), antisense RNA,
short interfering RNA (siRNA), or short hairpin RNA (shRNA)
directed against a pro-inflammatory molecule.
16. The bacterium of claim 1, wherein the bacterium is a
non-pathogenic bacterium.
17. The bacterium of claim 16, wherein the bacterium is a probiotic
bacterium.
18. The bacterium of claim 17, wherein the bacterium is selected
from the group consisting of Bacteroides, Bifidobacterium,
Clostridium, Escherichia, Lactobacillus, and Lactococcus.
19. The bacterium of claim 18, wherein the bacterium is Escherichia
coli strain Nissle.
20. The bacterium of claim 1, wherein the bacterium is an auxotroph
in a gene that is complemented when the bacterium is present in a
mammalian gut.
21. The bacterium of claim 20, wherein mammalian gut is a human
gut.
22. The bacterium of claim 20, wherein the bacterium is an
auxotroph in diaminopimelic acid or an enzyme in the thymine
biosynthetic pathway.
23. The bacterium of claim 1, wherein the bacterium is further
engineered to harbor an additional gene coding for a substance
toxic to the bacterium, wherein the additional gene is under the
control of a promoter that is directly or indirectly induced by an
environmental factor not naturally present in a mammalian gut.
24. The bacterium of claim 1, wherein the bacterium is further
engineered to harbor an additional gene coding for a substance
toxic to the bacterium, wherein the additional gene is under the
control of a promoter that is indirectly induced by the
transcription factor, and wherein the expression of the toxic
substance is delayed in time as compared to the expression of the
anti-inflammation molecule, the gut barrier enhancer molecule or
the gene cassette encoding the biosynthetic pathway.
25. A pharmaceutically acceptable composition comprising the
bacterium of claim 1; and a pharmaceutically acceptable
carrier.
26. The composition of claim 25 formulated for oral or rectal
administration.
27. A method of treating or preventing an autoimmune disorder,
comprising the step of administering to a patient in need thereof,
the composition of claim 25.
28. A method of treating a disease or condition associated with gut
inflammation and/or compromised gut barrier function, comprising
the step of administering to a patient in need thereof, the
composition of claim 25.
29. The method of claim 27, wherein the autoimmune disorder is
selected from the group consisting of acute disseminated
encephalomyelitis (ADEM), acute necrotizing hemorrhagic
leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia
areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM
nephritis, antiphospholipid syndrome (APS), autoimmune angioedema,
autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune
hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia,
autoimmune immunodeficiency, autoimmune inner ear disease (AIED),
autoimmune myocarditis, autoimmune oophoritis, autoimmune
pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic
purpura (ATP), autoimmune thyroid disease, autoimmune urticarial,
Axonal & neuronal neuropathies, Balo disease, Behcet's disease,
Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac
disease, Chagas disease, Chronic inflammatory demyelinating
polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis
(CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign
mucosal pemphigoid, Crohn's disease, Cogan syndrome, Cold
agglutinin disease, Congenital heart block, Coxsackie myocarditis,
CREST disease, Essential mixed cryoglobulinemia, Demyelinating
neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's
disease (neuromyelitis optica), Discoid lupus, Dressler's syndrome,
Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis,
Erythema nodosum, Experimental allergic encephalomyelitis, Evans
syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal
arteritis), Giant cell myocarditis, Glomerulonephritis,
Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA),
Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis,
Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein
purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic
thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related
sclerosing disease, Immunoregulatory lipoproteins, Inclusion body
myositis, Interstitial cystitis, Juvenile arthritis, Juvenile
idiopathic arthritis, Juvenile myositis, Kawasaki syndrome,
Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus,
Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease
(LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease,
Meniere's disease, Microscopic polyangiitis, Mixed connective
tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease,
Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy,
Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial
pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS
(Pediatric autoimmune Neuropsychiatric Disorders Associated with
Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal
nocturnal hemoglobinuria (PNH), Parry Romberg syndrome,
Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis),
Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis,
Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I,
II, & III autoimmune polyglandular syndromes, Polymyalgia
rheumatic, Polymyositis, Postmyocardial infarction syndrome,
Postpericardiotomy syndrome, Progesterone dermatitis, Primary
biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis,
Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma
gangrenosum, Pure red cell aplasia, Raynauds phenomenon, reactive
arthritis, reflex sympathetic dystrophy, Reiter's syndrome,
relapsing polychondritis, restless legs syndrome, retroperitoneal
fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis,
Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm
& testicular autoimmunity, stiff person syndrome, subacute
bacterial endocarditis (SBE), Susac's syndrome, sympathetic
ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell
arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome,
transverse myelitis, type 1 diabetes, asthma, ulcerative colitis,
undifferentiated connective tissue disease (UCTD), uveitis,
vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's
granulomatosis.
30. The method of claim 29, wherein the autoimmune disorder is
selected from the group consisting of type 1 diabetes, asthma,
multiple sclerosis, Crohn's disease, lupus, rheumatoid arthritis,
ulcerative colitis, juvenile arthritis, psoriasis, psoriatic
arthritis, celiac disease, and ankylosing spondylitis.
31. The method of claim 28, wherein the disease or disorder is
selected from an inflammatory bowel disease, and a diarrheal
disease.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 14/998,376, filed Dec. 22, 2015, which claims benefit of
priority to U.S. Provisional No. 62/095,415, filed Dec. 22, 2014;
U.S. Provisional No. 62/127,131, filed Mar. 2, 2015; U.S.
Provisional No. 62/248,825, filed Oct. 30, 2015; U.S. Provisional
No. 62/256,044, filed Nov. 16, 2015, which are incorporated herein
by reference in their entirety.
[0002] This disclosure relates to compositions and therapeutic
methods for inhibiting inflammatory mechanisms in the gut,
restoring and tightening gut mucosal barrier function, and/or
treating and preventing autoimmune disorders. In certain aspects,
the disclosure relates to genetically engineered bacteria that
reduce inflammation in the gut and/or enhance gut barrier function,
particularly in the presence of reactive nitrogen species. In some
embodiments, the genetically engineered bacteria reduce gut
inflammation and/or enhance gut barrier function, thereby
ameliorating or preventing an autoimmune disorder. In some aspects,
the compositions and methods disclosed herein may be used for
treating or preventing autoimmune disorders as well as diseases and
conditions associated with gut inflammation and/or compromised gut
barrier function, e.g., diarrheal diseases, inflammatory bowel
diseases, and related diseases.
[0003] Inflammatory bowel diseases (IBDs) are a group of diseases
characterized by significant local inflammation in the
gastrointestinal tract typically driven by T cells and activated
macrophages and by compromised function of the epithelial barrier
that separates the luminal contents of the gut from the host
circulatory system (Ghishan et al., 2014). IBD pathogenesis is
linked to both genetic and environmental factors and may be caused
by altered interactions between gut microbes and the intestinal
immune system. Current approaches to treat IBD are focused on
therapeutics that modulate the immune system and suppress
inflammation. These therapies include steroids, such as prednisone,
and tumor necrosis factor (TNF) inhibitors, such as Humira.RTM.
(Cohen et al., 2014). Drawbacks from this approach are associated
with systemic immunosuppression, which includes greater
susceptibility to infectious disease and cancer.
[0004] Other approaches have focused on treating compromised
barrier function by supplying the short-chain fatty acid butyrate
via enemas. Recently, several groups demonstrated the importance of
short-chain fatty acid production by commensal bacteria in
regulating the immune system in the gut (Smith et al., 2013). They
showed that butyrate plays a direct role in inducing the
differentiation of regulatory T cells and suppressing immune
responses associated with inflammation in IBD (Atarashi et al.,
2011; Furusawa et al., 2013). Butyrate is normally produced by
microbial fermentation of dietary fiber and plays a central role in
maintaining colonic epithelial cell homeostasis and barrier
function (Hamer et al., 2008). Studies with butyrate enemas have
shown some benefit to patients, but this treatment is not practical
for long term therapy. More recently, patients with IBD have been
treated with fecal transfer from healthy patients with some success
(Ianiro et al., 2014). This success illustrates the central role
that gut microbes play in disease pathology and suggests that
certain microbial functions are associated with ameliorating the
IBD disease process. However, this approach raises safety concerns
over the transmission of infectious disease from the donor to the
recipient. Moreover, the nature of this treatment has a negative
stigma and thus is unlikely to be widely accepted.
[0005] Compromised gut barrier function also plays "a central part
. . . in autoimmune diseases pathogenesis" (Lerner et al., 2015a;
Lerner et al., 2015b; Fasano et al., 2005; Fasano et al., 2012). A
single layer of epithelial cells separates the gut lumen from the
immune cells in the body. The epithelium is regulated by
intercellular tight junctions and "controls the equilibrium between
tolerance and immunity to nonself-antigens" (Fasano et al., 2005).
Disrupting the epithelial layer "can lead to pathological exposure
of the highly immunoreactive subepithelium to the vast number of
foreign antigens in the lumen" (Lerner et al., 2015a) and "both
intestinal and extraintestinal autoimmune disorders can occur"
(Fasano et al., 2005). Some foreign antigens "are postulated to
resemble self-antigens" and can induce "epitope-specific
cross-reactivity" that accelerates the progression of a
pre-existing autoimmune disease or initiates an autoimmune disease
(Fasano, 2012). Rheumatoid arthritis and celiac disease, for
example, are autoimmune disorders that are thought to involve
"increased intestinal permeability . . . as drivers of the
autoimmune cascade" (Lerner et al., 2015b). In individuals who are
genetically susceptible to autoimmune disorders, dysregulation of
intercellular tight junctions can lead to disease onset (Fasano,
2012). In fact, "the loss of protective function of mucosal
barriers that interact with the environment is necessary for
autoimmunity to develop" (Lerner et al., 2015a).
[0006] Changes in gut microbes can "alter the host immune response"
(Paun et al., 2015; Sanz et al., 2014; Sanz et al., 2015; Wen et
al., 2008). For example, in children with high genetic risk for
type 1 diabetes, there are "significant differences in the gut
microbiome between children who develop autoimmunity for the
disease and those who remain healthy" (Richardson et al., 2015).
Gut bacteria are "a potential therapeutic target in the prevention
of asthma" and exhibit "strong immunomodulatory capacity . . . in
lung inflammation" (Arrieta et al., 2015). Thus, enhancing barrier
function and reducing inflammation gastrointestinal tract are
potential therapeutic mechanisms for the treatment or prevention of
autoimmune disorders.
[0007] Recently there has been an effort to engineer microbes that
produce anti-inflammatory molecules, such as IL-10, and administer
them orally to a patient in order to deliver the therapeutic
directly to the site of inflammation in the gut. The advantage of
this approach is that it avoids systemic administration of
immunosuppressive drugs and delivers the therapeutic directly to
the gastrointestinal tract. While these engineered microbes have
shown efficacy in some pre-clinical models, efficacy in patients
has not been observed. One main reason why these engineered
microbes have not been successful in treating patients is that
their viability and stability are compromised, because they
constitutively produce large amounts of non-native proteins, e.g.,
human interleukin. Thus, there remains a great need for additional
therapies that reduce gut inflammation, enhance gut barrier
function, and/or treat autoimmune disorders, and that avoid
undesirable side effects.
[0008] Reactive nitrogen species (RNS) such as nitric oxide are
produced at sites of inflammation and are intimately associated
with the disease process. Certain bacterial transcription factors
have evolved to sense RNS and regulate the expression of a number
of proteins that protect the bacterial DNA from their damaging
effects.
[0009] The genetically engineered bacteria of the invention are
capable of producing therapeutic anti-inflammation and/or gut
barrier enhancer molecules, particularly in the presence of RNS.
The genetically engineered bacteria are functionally silent until
they reach an environment containing local RNS, wherein expression
of the therapeutic molecule is induced. In certain embodiments, the
genetically engineered bacteria are non-pathogenic and may be
introduced into the gut in order to reduce gut inflammation and/or
enhance gut barrier function and may thereby further ameliorate or
prevent an autoimmune disorder. In certain embodiments, the
anti-inflammation and/or gut barrier enhancer molecule is stably
produced by the genetically engineered bacteria, and/or the
genetically engineered bacteria are stably maintained in vivo
and/or in vitro. The invention also provides pharmaceutical
compositions comprising the genetically engineered bacteria, and
methods of treating diseases that benefit from reduced gut
inflammation and/or tightened gut mucosal barrier function, e.g.,
an inflammatory bowel disease or an autoimmune disorder.
[0010] The genetically engineered bacteria of the invention produce
a therapeutic molecule under the control of a RNS-responsive
regulatory region and a corresponding RNS-sensing transcription
factor. In some embodiments, the therapeutic molecule is butyrate;
in environment containing local RNS, the butyrate biosynthetic gene
cassette is activated, and butyrate is produced. Local production
of butyrate induces the differentiation of regulatory T cells in
the gut and/or promotes the barrier function of colonic epithelial
cells. The genetically engineered bacteria of the invention produce
their therapeutic effect only in environments environment
containing local ROS, thereby lowering the safety issues associated
with systemic exposure.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIGS. 1A and 1B depict the construction and gene
organization of two exemplary plasmids each comprising a gene
encoding NsrR, a regulatory sequence from norB, and a butyrate
operon. FIG. 1A shows the pLogic031-nsrR-norB-butyrate operon
construct, and FIG. 1B shows the pLogic046-nsrR-norB-butyrate
operon construct.
[0012] FIG. 2 depicts the gene organization of an exemplary
recombinant bacterium of the invention and its derepression in the
presence of nitric oxide (NO). In the upper panel, in the absence
of NO, the NsrR transcription factor (gray circle, "NsrR") binds to
and represses a corresponding regulatory region. Therefore, none of
the butyrate biosynthesis enzymes (bcd2, etfB3, etfA3, thiA1, hbd,
crt2, pbt, buk; black boxes) is expressed. In the lower panel, in
the presence of NO, the NsrR transcription factor interacts with
NO, and no longer binds to or represses the regulatory sequence.
This leads to expression of the butyrate biosynthesis enzymes
(indicated by gray arrows and black squiggles) and ultimately to
the production of butyrate.
[0013] FIG. 3 depicts the gene organization of another exemplary
recombinant bacterium of the invention and its derepression in the
presence of NO. In the upper panel, in the absence of NO, the NsrR
transcription factor (gray circle, "NsrR") binds to and represses a
corresponding regulatory region. Therefore, none of the butyrate
biosynthesis enzymes (ter, thiA1, hbd, crt2, pbt, buk; black boxes)
is expressed. In the lower panel, in the presence of NO, the NsrR
transcription factor interacts with NO, and no longer binds to or
represses the regulatory sequence. This leads to expression of the
butyrate biosynthesis enzymes (indicated by gray arrows and black
squiggles) and ultimately to the production of butyrate.
[0014] FIG. 4 depicts the nucleic acid sequence of an exemplary
RNS-regulated construct comprising a gene encoding nsrR, a
regulatory region of norB, and a butyrate operon
(pLogic031-nsrR-norB-butyrate operon construct; SEQ ID NO: 1). The
sequence encoding NsrR is underlined and bolded, and the NsrR
binding site, i.e., a regulatory region of norB is .
[0015] FIG. 5 depicts the nucleic acid sequence of an exemplary
RNS-regulated construct comprising a gene encoding nsrR, a
regulatory region of norB, and a butyrate operon
(pLogic046-nsrR-norB-butyrate operon construct; SEQ ID NO: 2). The
sequence encoding NsrR is underlined and bolded, and the NsrR
binding site, i.e., a regulatory region of norB is .
[0016] FIG. 6 depicts the nucleic acid sequence of an exemplary
tetracycline-regulated construct comprising a tet promoter and
butyrate operon (pLogic031-tet-butyrate operon construct; SEQ ID
NO: 12). The sequence encoding TetR is underlined, and the
overlapping tetR/tetA promoters are .
[0017] FIG. 7 depicts the nucleic acid sequence of an exemplary
tetracycline-regulated construct comprising a tet promoter and
butyrate operon (pLogic046-tet-butyrate operon construct; SEQ ID
NO: 13). The sequence encoding TetR is underlined, and the
overlapping tetR/tetA promoters are .
[0018] FIG. 8 depicts synthetic biology circuits comprising
parallel pathways, in vivo activation switches, auxotrophy and kill
switches, and peptide/protein export.
[0019] FIG. 9 depicts synthetic biology safety designs, e.g.,
auxotrophy and kill switches.
[0020] FIG. 10 depicts an exemplary schematic of the E. coli 1917
Nissle chromosome.
[0021] FIG. 11 depicts a schematic for inflammatory bowel disease
(IBD) therapies that target pro-inflammatory neutrophils and
macrophages and regulatory T cells (Treg), restore epithelial
barrier integrity, and maintain mucosal barrier function.
[0022] FIGS. 12 A, B, and C depict ATC or nitric oxide-inducible
reporter constructs. These constructs, when induced by their
cognate inducer, lead to expression of GFP. Nissle cells harboring
plasmids with either the control, ATC-inducible Ptet-GFP reporter
construct (FIGS. 12A and 12C) or the nitric oxide inducible
PnsrR-GFP reporter construct (FIGS. 12B and 12C) induced across a
range of concentrations. Promoter activity is expressed as relative
florescence units.
[0023] FIG. 13 the sequence of an exemplary nitric oxide-inducible
reporter construct. The bsrR sequence is bolded. The gfp sequence
is underlined. The PnsrR (NO regulated promoter and RBS) is
italicized. The constitutive promoter and RBS are .
[0024] FIG. 14 depicts a dot blot of bacteria harboring a plasmid
expressing NsrR under control of a constitutive promoter and the
reporter gene gfp (green fluorescent protein) under control of an
NsrR-inducible promoter. IBD is induced in mice by supplementing
drinking water with 2-3% dextran sodium sulfate (DSS).
Chemiluminescent is shown for NsrR-regulated promoters induced in
DSS-treated mice.
[0025] FIG. 15A depicts a schematic diagram of a wild-type clbA
construct (SEQ ID NO: 15). FIG. 15B depicts a schematic diagram of
a clbA knockout construct (SEQ ID NO: 16).
[0026] FIG. 16 depicts a map of exemplary integration sites within
the E. coli 1917 Nissle chromosome. These sites indicate regions
where circuit components may be inserted into the chromosome
without interfering with essential gene expression. Backslashes (/)
are used to show that the insertion will occur between divergently
or convergently expressed genes. Insertions within biosynthetic
genes, such as thyA, can be useful for creating nutrient
auxotrophies. In some embodiments, an individual circuit component
is inserted into more than one of the indicated sites.
DESCRIPTION OF EMBODIMENTS
[0027] The invention includes genetically engineered bacteria,
pharmaceutical compositions thereof, and methods of reducing gut
inflammation, enhancing gut barrier function, and/or and treating
or preventing autoimmune disorders. The genetically engineered
bacteria of the invention comprise a gene encoding a non-native
anti-inflammation and/or gut barrier function enhancer molecule, or
a gene cassette encoding a biosynthetic pathway for producing an
anti-inflammation and/or gut barrier function enhancer molecule.
The gene or gene cassette is further linked to a tunable regulatory
region that is directly or indirectly controlled by a transcription
factor that is capable of sensing at least one reactive nitrogen
species.
[0028] In order that the disclosure may be more readily understood,
certain terms are first defined. These definitions should be read
in light of the remainder of the disclosure and as understood by a
person of ordinary skill in the art. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood by a person of ordinary skill in the art.
Additional definitions are set forth throughout the detailed
description.
[0029] As used herein, "diseases and conditions associated with gut
inflammation and/or compromised gut barrier function" include, but
are not limited to, inflammatory bowel diseases, diarrheal
diseases, and related diseases. "Inflammatory bowel diseases" and
"IBD" are used interchangeably to refer to a group of diseases
associated with gut inflammation, which include, but are not
limited to, Crohn's disease, ulcerative colitis, collagenous
colitis, lymphocytic colitis, diversion colitis, Behcet's disease,
and indeterminate colitis. "Diarrheal diseases" include, but are
not limited to, acute watery diarrhea, e.g., cholera, acute bloody
diarrhea, e.g., dysentery, and persistent diarrhea. Related
diseases include, but are not limited to, short bowel syndrome,
ulcerative proctitis, proctosigmoiditis, left-sided colitis,
pancolitis, and fulminant colitis.
[0030] Symptoms associated with the aforementioned diseases and
conditions include, but are not limited to, one or more of
diarrhea, bloody stool, mouth sores, perianal disease, abdominal
pain, abdominal cramping, fever, fatigue, weight loss, iron
deficiency, anemia, appetite loss, weight loss, anorexia, delayed
growth, delayed pubertal development, inflammation of the skin,
inflammation of the eyes, inflammation of the joints, inflammation
of the liver, and inflammation of the bile ducts.
[0031] A disease or condition associated with gut inflammation
and/or compromised gut barrier function may be an autoimmune
disorder. A disease or condition associated with gut inflammation
and/or compromised gut barrier function may be co-morbid with an
autoimmune disorder. As used herein, "autoimmune disorders"
include, but are not limited to, acute disseminated
encephalomyelitis (ADEM), acute necrotizing hemorrhagic
leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia
areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM
nephritis, antiphospholipid syndrome (APS), autoimmune angioedema,
autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune
hemolytic anemia, autoimmune hepatitis, autoimmune hyperlipidemia,
autoimmune immunodeficiency, autoimmune inner ear disease (AIED),
autoimmune myocarditis, autoimmune oophoritis, autoimmune
pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic
purpura (ATP), autoimmune thyroid disease, autoimmune urticarial,
Axonal & neuronal neuropathies, Balo disease, Behcet's disease,
Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac
disease, Chagas disease, Chronic inflammatory demyelinating
polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis
(CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid/benign
mucosal pemphigoid, Crohn's disease, Cogan syndrome, Cold
agglutinin disease, Congenital heart block, Coxsackie myocarditis,
CREST disease, Essential mixed cryoglobulinemia, Demyelinating
neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's
disease (neuromyelitis optica), Discoid lupus, Dressler's syndrome,
Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis,
Erythema nodosum, Experimental allergic encephalomyelitis, Evans
syndrome, Fibrosing alveolitis, Giant cell arteritis (temporal
arteritis), Giant cell myocarditis, Glomerulonephritis,
Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA),
Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis,
Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein
purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic
thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related
sclerosing disease, Immunoregulatory lipoproteins, Inclusion body
myositis, Interstitial cystitis, Juvenile arthritis, Juvenile
idiopathic arthritis, Juvenile myositis, Kawasaki syndrome,
Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus,
Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease
(LAD), Lupus (Systemic Lupus Erythematosus), chronic Lyme disease,
Meniere's disease, Microscopic polyangiitis, Mixed connective
tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease,
Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy,
Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial
pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS
(Pediatric autoimmune Neuropsychiatric Disorders Associated with
Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal
nocturnal hemoglobinuria (PNH), Parry Romberg syndrome,
Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis),
Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis,
Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I,
II, & III autoimmune polyglandular syndromes, Polymyalgia
rheumatic, Polymyositis, Postmyocardial infarction syndrome,
Postpericardiotomy syndrome, Progesterone dermatitis, Primary
biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis,
Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma
gangrenosum, Pure red cell aplasia, Raynauds phenomenon, reactive
arthritis, reflex sympathetic dystrophy, Reiter's syndrome,
relapsing polychondritis, restless legs syndrome, retroperitoneal
fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis,
Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm
& testicular autoimmunity, stiff person syndrome, subacute
bacterial endocarditis (SBE), Susac's syndrome, sympathetic
ophthalmia, Takayasu's arteritis, temporal arteritis/giant cell
arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome,
transverse myelitis, type 1 diabetes, asthma, ulcerative colitis,
undifferentiated connective tissue disease (UCTD), uveitis,
vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's
granulomatosis.
[0032] "Anti-inflammation molecules" and/or "gut barrier function
enhancer molecules" include, but are not limited to, short-chain
fatty acids, butyrate, propionate, acetate, GLP-2, IL-10, IL-27,
TGF-.beta.1, TGF-.beta.2, N-acylphosphatidylethanolamines (NAPEs),
elafin (also called peptidase inhibitor 3 and SKALP), and trefoil
factor. Such molecules may also include compounds that inhibit
pro-inflammatory molecules, e.g., a single-chain variable fragment
(scFv), antisense RNA, siRNA, or shRNA that neutralizes
TNF-.alpha., IFN-.gamma., IL-1.beta., IL-6, IL-8, IL-17, and/or
chemokines, e.g., CXCL-8 and CCL2. A molecule may be primarily
anti-inflammatory, e.g., IL-10, or primarily gut barrier function
enhancing, e.g., GLP-2. A molecule may be both anti-inflammatory
and gut barrier function enhancing. An anti-inflammation and/or gut
barrier function enhancer molecule may be encoded by a single gene,
e.g., elafin is encoded by the PI3 gene. Alternatively, an
anti-inflammation and/or gut barrier function enhancer molecule may
be synthesized by a biosynthetic pathway requiring multiple genes,
e.g., butyrate. These molecules may also be referred to as
therapeutic molecules.
[0033] As used herein, a "gene cassette" or "operon" encoding a
biosynthetic pathway refers to the two or more genes that are
required to produce an anti-inflammation and/or gut barrier
function enhancer molecule, e.g., butyrate. In addition to encoding
a set of genes capable of producing said molecule, the gene
cassette or operon may also comprise additional transcription and
translation elements, e.g., a ribosome binding site.
[0034] A "butyrogenic gene cassette," "butyrate biosynthesis gene
cassette," and "butyrate operon" are used interchangeably to refer
to a set of genes capable of producing butyrate in a biosynthetic
pathway. Unmodified bacteria that are capable of producing butyrate
via an endogenous butyrate biosynthesis pathway include, but are
not limited to, Clostridium, Peptoclostridium, Fusobacterium,
Butyrivibrio, Eubacterium, and Treponema. The genetically
engineered bacteria of the invention may comprise butyrate
biosynthesis genes from a different species, strain, or substrain
of bacteria, or a combination of butyrate biosynthesis genes from
different species, strains, and/or substrains of bacteria. A
butyrogenic gene cassette may comprise, for example, the eight
genes of the butyrate production pathway from Peptoclostridium
difficile (also called Clostridium difficile): bcd2, etfB3, etfA3,
thiA1, hbd, crt2, pbt, and buk, which encode butyryl-CoA
dehydrogenase subunit, electron transfer flavoprotein subunit beta,
electron transfer flavoprotein subunit alpha, acetyl-CoA
C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase,
phosphate butyryltransferase, and butyrate kinase, respectively
(Aboulnaga et al., 2013). One or more of the butyrate biosynthesis
genes may be functionally replaced or modified. Peptoclostridium
difficile strain 630 and strain 1296 are both capable of producing
butyrate, but comprise different nucleic acid sequences for etfA3,
thiA1, hbd, crt2, pbt, and buk. A butyrogenic gene cassette may
comprise bcd2, etfB3, etfA3, and thiA1 from Peptoclostridium
difficile strain 630, and hbd, crt2, pbt, and buk from
Peptoclostridium difficile strain 1296. Alternatively, a single
gene from Treponema denticola (ter, encoding trans-2-enoynl-CoA
reductase) is capable of functionally replacing all three of the
bcd2, etfB3, and etfA3 genes from Peptoclostridium difficile. Thus,
a butyrogenic gene cassette may comprise thiA1, hbd, crt2, pbt, and
buk from Peptoclostridium difficile and ter from Treponema
denticola. The butyrogenic gene cassette may comprise genes for the
aerobic biosynthesis of butyrate and/or genes for the anaerobic or
microaerobic biosynthesis of butyrate.
[0035] Likewise, a "propionate gene cassette" or "propionate
operon" refers to a set of genes capable of producing propionate in
a biosynthetic pathway. Unmodified bacteria that are capable of
producing propionate via an endogenous propionate biosynthesis
pathway include, but are not limited to, Clostridium propionicum,
Megasphaera elsdenii, and Prevotella ruminicola. The genetically
engineered bacteria of the invention may comprise propionate
biosynthesis genes from a different species, strain, or substrain
of bacteria, or a combination of propionate biosynthesis genes from
different species, strains, and/or substrains of bacteria. In some
embodiments, the propionate gene cassette comprises acrylate
pathway propionate biosynthesis genes, e.g., pct, lcdA, lcdB, lcdC,
etfA, acrB, and acrC, which encode propionate CoA-transferase,
lactoyl-CoA dehydratase A, lactoyl-CoA dehydratase B, lactoyl-CoA
dehydratase C, electron transfer flavoprotein subunit A,
acryloyl-CoA reductase B, and acryloyl-CoA reductase C,
respectively (Hetzel et al., 2003, Selmer et al., 2002). In
alternate embodiments, the propionate gene cassette comprises
pyruvate pathway propionate biosynthesis genes (see, e.g., Tseng et
al., 2012), e.g., thrA.sup.fbr, thrB, thrC, ilvA.sup.fbr, aceE,
aceF, and Ipd, which encode homoserine dehydrogenase 1, homoserine
kinase, L-threonine synthase, L-threonine dehydratase, pyruvate
dehydrogenase, dihydrolipoamide acetyltransferase, and
dihydrolipoyl dehydrogenase, respectively. In some embodiments, the
propionate gene cassette further comprises tesB, which encodes
acyl-CoA thioesterase. The propionate gene cassette may comprise
genes for the aerobic biosynthesis of propionate and/or genes for
the anaerobic or microaerobic biosynthesis of propionate. One or
more of the butyrate biosynthesis genes may be functionally
replaced or modified, e.g., codon optimized.
[0036] An "acetate gene cassette" or "acetate operon" refers to a
set of genes capable of producing acetate in a biosynthetic
pathway. Bacteria "synthesize acetate from a number of carbon and
energy sources," including a variety of substrates such as
cellulose, lignin, and inorganic gases, and utilize different
biosynthetic mechanisms and genes, which are known in the art
(Ragsdale et al., 2008). The genetically engineered bacteria of the
invention may comprise acetate biosynthesis genes from a different
species, strain, or substrain of bacteria, or a combination of
acetate biosynthesis genes from different species, strains, and/or
substrains of bacteria. Escherichia coli are capable of consuming
glucose and oxygen to produce acetate and carbon dioxide during
aerobic growth (Kleman et al., 1994). Several bacteria, such as
Acetitomaculum, Acetoanaerobium, Acetohalobium, Acetonema, Balutia,
Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa, and
Thermoacetogenium, are acetogenic anaerobes that are capable of
converting CO or CO.sub.2+H.sub.2 into acetate, e.g., using the
Wood-Ljungdahl pathway (Schiel-Bengelsdorf et al, 2012). Genes in
the Wood-Ljungdahl pathway for various bacterial species are known
in the art. The acetate gene cassette may comprise genes for the
aerobic biosynthesis of acetate and/or genes for the anaerobic or
microaerobic biosynthesis of acetate. One or more of the acetate
biosynthesis genes may be functionally replaced or modified.
[0037] "Reactive nitrogen species" and "RNS" are used
interchangeably to refer to highly active molecules, ions, and/or
radicals derived from molecular nitrogen. RNS can cause deleterious
cellular effects such as nitrosative stress. RNS includes, but is
not limited to, nitric oxide (NO.cndot.), peroxynitrite or
peroxynitrite anion (ONOO.sup.-), nitrogen dioxide
(.cndot.NO.sub.2), dinitrogen trioxide (N.sub.2O.sub.3),
peroxynitrous acid (ONOOH), and nitroperoxycarbonate
(ONOOCO.sub.2.sup.-) (unpaired electrons denoted by .cndot.).
[0038] Bacteria have evolved transcription factors that are capable
of sensing RNS levels. Different RNS signaling pathways are
triggered by different RNS levels and occur with different
kinetics. "RNS-inducible regulatory region" refers to a nucleic
acid sequence to which one or more RNS-sensing transcription
factors is capable of binding, wherein the binding and/or
activation of the corresponding transcription factor activates
downstream gene expression; in the presence of RNS, the
transcription factor binds to and/or activates the regulatory
region. In some embodiments, the RNS-inducible regulatory region
comprises a promoter sequence. In some embodiments, the
transcription factor senses RNS and subsequently binds to the
RNS-inducible regulatory region, thereby activating downstream gene
expression. In alternate embodiments, the transcription factor is
bound to the RNS-inducible regulatory region in the absence of RNS;
in the presence of RNS, the transcription factor undergoes a
conformational change, thereby activating downstream gene
expression. The RNS-inducible regulatory region may be operatively
linked to a gene or gene cassette, e.g., a butyrogenic gene
cassette. For example, in the presence of RNS, a transcription
factor senses RNS and activates a corresponding RNS-inducible
regulatory region, thereby driving expression of an operatively
linked gene or gene cassette. Thus, RNS induces expression of the
gene or gene cassette.
[0039] "RNS-derepressible regulatory region" refers to a nucleic
acid sequence to which one or more RNS-sensing transcription
factors is capable of binding, wherein the binding of the
corresponding transcription factor represses downstream gene
expression; in the presence of RNS, the transcription factor does
not bind to and does not repress the regulatory region. In some
embodiments, the RNS-derepressible regulatory region comprises a
promoter sequence. The RNS-derepressible regulatory region may be
operatively linked to a gene or gene cassette, e.g., a butyrogenic
gene cassette. For example, in the presence of RNS, a transcription
factor senses RNS and no longer binds to and/or represses the
regulatory region, thereby derepressing an operatively linked gene
or gene cassette. Thus, RNS derepresses expression of the gene or
gene cassette.
[0040] "RNS-repressible regulatory region" refers to a nucleic acid
sequence to which one or more RNS-sensing transcription factors is
capable of binding, wherein the binding of the corresponding
transcription factor represses downstream gene expression; in the
presence of RNS, the transcription factor binds to and represses
the regulatory region. In some embodiments, the RNS-repressible
regulatory region comprises a promoter sequence. In some
embodiments, the transcription factor that senses RNS is capable of
binding to a regulatory region that overlaps with part of the
promoter sequence. In alternate embodiments, the transcription
factor that senses RNS is capable of binding to a regulatory region
that is upstream or downstream of the promoter sequence. The
RNS-repressible regulatory region may be operatively linked to a
gene or gene cassette. For example, in the presence of RNS, a
transcription factor senses RNS and binds to a corresponding
RNS-repressible regulatory region, thereby blocking expression of
an operatively linked gene or gene cassette. Thus, RNS represses
expression of the gene or gene cassette.
[0041] A "RNS-responsive regulatory region" refers to a
RNS-inducible regulatory region, a RNS-repressible regulatory
region, and/or a RNS-derepressible regulatory region. In some
embodiments, the RNS-responsive regulatory region comprises a
promoter sequence. Each regulatory region is capable of binding at
least one corresponding RNS-sensing transcription factor. Examples
of transcription factors that sense RNS and their corresponding
RNS-responsive genes, promoters, and/or regulatory regions include,
but are not limited to, those shown in Table 1.
[0042] A "tunable regulatory region" refers to a nucleic acid
sequence under direct or indirect control of a transcription factor
and which is capable of activating, repressing, derepressing, or
otherwise controlling gene expression relative to levels of an
inducer. In some embodiments, the tunable regulatory region
comprises a promoter sequence. The inducer may be RNS, and the
tunable regulatory region may be a RNS-responsive regulatory
region. The tunable regulatory region may be operatively linked to
a gene or gene cassette, e.g., a butyrogenic gene cassette. For
example, the tunable regulatory region is a RNS-derepressible
regulatory region, and when RNS is present, a RNS-sensing
transcription factor no longer binds to and/or represses the
regulatory region, thereby permitting expression of the operatively
linked gene or gene cassette. In this instance, the tunable
regulatory region derepresses gene or gene cassette expression
relative to RNS levels.
[0043] A gene or gene cassette for producing a therapeutic molecule
may be operatively linked to a tunable regulatory region that is
directly or indirectly controlled by a transcription factor that is
capable of sensing at least one RNS. "Directly controlled" refers
to a RNS-inducible or RNS-derepressible regulatory region, in which
the regulatory region is operatively linked to said gene or gene
cassette; in the presence of RNS, the therapeutic molecule is
expressed. "Indirectly controlled" refers to a RNS-repressible
regulatory region, wherein a RNS-sensing repressor inhibits
transcription of a second repressor, which inhibits the
transcription of the gene or gene cassette for producing a
therapeutic molecule; in the presence of RNS, the second repressor
does not inhibit transcription of said gene or gene cassette, and
the therapeutic molecule is expressed. "Operatively linked" refers
a nucleic acid sequence, e.g., a gene or gene cassette for an
anti-inflammation and/or gut barrier enhancer molecule, that is
joined to a regulatory region sequence in a manner which allows
expression of the nucleic acid sequence, e.g., acts in cis.
TABLE-US-00001 TABLE 1 Examples of RNS-sensing transcription
factors and RNS-responsive genes Primarily RNS-sensing capable of
Examples of responsive genes, transcription factor: sensing:
promoters, and/or regulatory regions: NsrR NO norB, aniA, nsrR,
hmpA, ytfE, ygbA, hcp, hcr, nrfA, aox NorR NO norVW, norR DNR NO
norCB, nir, nor, nos
[0044] As used herein, a "non-native" nucleic acid sequence refers
to a nucleic acid sequence not normally present in a bacterium,
e.g., an extra copy of an endogenous sequence, or a heterologous
sequence, e.g., a sequence from a different species, strain, or
substrain of bacteria, or a sequence that is modified and/or
mutated as compared to the unmodified sequence from bacteria of the
same subtype. In some embodiments, the non-native nucleic acid
sequence is a synthetic, non-naturally occurring sequence (see,
e.g., Purcell et al., 2013). The non-native nucleic acid sequence
may be a regulatory region, a promoter, a gene, and/or one or more
genes in gene cassette. The non-native nucleic acid sequence may be
present on a plasmid or chromosome.
[0045] "Gut" refers to the organs, glands, tracts, and systems that
are responsible for the transfer and digestion of food, absorption
of nutrients, and excretion of waste. In humans, the gut comprises
the gastrointestinal (GI) tract, which starts at the mouth and ends
at the anus, and additionally comprises the esophagus, stomach,
small intestine, and large intestine. The gut also comprises
accessory organs and glands, such as the spleen, liver,
gallbladder, and pancreas. The upper gastrointestinal tract
comprises the esophagus, stomach, and duodenum of the small
intestine. The lower gastrointestinal tract comprises the remainder
of the small intestine, i.e., the jejunum and ileum, and all of the
large intestine, i.e., the cecum, colon, rectum, and anal canal.
Bacteria can be found throughout the gut, e.g., in the
gastrointestinal tract, and particularly in the intestines.
[0046] "Non-pathogenic bacteria" refer to bacteria that are not
capable of causing disease or harmful responses in a host. Examples
of non-pathogenic bacteria include, but are not limited to
Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium,
Enterococcus, Escherichia, Lactobacillus, Lactococcus,
Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans,
Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis,
Bacteroides thetaiotaomicron, Bifidobacterium bifidum,
Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium
longum, Clostridium butyricum, Enterococcus faecium, Escherichia
coli, Lactobacillus acidophilus, Lactobacillus bulgaricus,
Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus
paracasei, Lactobacillus plantarum, Lactobacillus reuteri,
Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces
boulardii (Sonnenborn et al., 2009; Dinleyici et al., 2014; U.S.
Pat. No. 6,835,376; U.S. Pat. No. 6,203,797; U.S. Pat. No.
5,589,168; U.S. Pat. No. 7,731,976).
[0047] "Probiotic" is used to refer to live, non-pathogenic
microorganisms, e.g., bacteria, which can confer health benefits to
a host organism that contains an appropriate amount of the
microorganism. In some embodiments, the host organism is a mammal.
In some embodiments, the host organism is a human. Some species,
strains, and/or subtypes of non-pathogenic bacteria are currently
recognized as probiotic. Examples of probiotic bacteria include,
but are not limited to, Bifidobacteria, Escherichia, Lactobacillus,
and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus
faecium, Escherichia coli, Escherichia coli strain Nissle,
Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus
paracasei, Lactobacillus plantarum, and Saccharomyces boulardii
(Dinleyici et al., 2014; U.S. Pat. No. 5,589,168; U.S. Pat. No.
6,203,797; U.S. Pat. No. 6,835,376). The probiotic may be a variant
or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos
et al., 2010; Olier et al., 2012; Nougayrede et al., 2006).
Non-pathogenic bacteria may be genetically engineered to enhance or
improve desired biological properties, e.g., survivability.
Non-pathogenic bacteria may be genetically engineered to provide
probiotic properties. Probiotic bacteria may be genetically
engineered to enhance or improve probiotic properties.
[0048] As used herein, "stably maintained" or "stable" bacterium is
used to refer to a bacterial host cell carrying non-native genetic
material, e.g., a butyrogenic gene cassette, that is incorporated
into the host genome or propagated on a self-replicating
extra-chromosomal plasmid, such that the non-native genetic
material is retained, expressed, and propagated. The stable
bacterium is capable of survival and/or growth in vitro, e.g., in
medium, and/or in vivo, e.g., in the gut. For example, the stable
bacterium may be a genetically modified bacterium comprising a
butyrogenic gene cassette, in which the plasmid or chromosome
carrying the butyrogenic gene cassette is stably maintained in the
host cell, such that butyrate can be expressed in the host cell,
and the host cell is capable of survival and/or growth in vitro
and/or in vivo.
[0049] As used herein, the term "treat" and its cognates refer to
an amelioration of a disease or disorder, or at least one
discernible symptom thereof. In another embodiment, "treat" refers
to an amelioration of at least one measurable physical parameter,
not necessarily discernible by the patient. In another embodiment,
"treat" refers to inhibiting the progression of a disease or
disorder, either physically (e.g., stabilization of a discernible
symptom), physiologically (e.g., stabilization of a physical
parameter), or both. In another embodiment, "treat" refers to
slowing the progression or reversing the progression of a disease
or disorder. As used herein, "prevent" and its cognates refer to
delaying the onset or reducing the risk of acquiring a given
disease or disorder.
[0050] Those in need of treatment may include individuals already
having a particular medical disorder, as well as those at risk of
having, or who may ultimately acquire the disorder. The need for
treatment is assessed, for example, by the presence of one or more
risk factors associated with the development of a disorder, the
presence or progression of a disorder, or likely receptiveness to
treatment of a subject having the disorder. Treating autoimmune
disorders and/or diseases and conditions associated with gut
inflammation and/or compromised gut barrier function may encompass
reducing or eliminating excess inflammation and/or associated
symptoms, and does not necessarily encompass the elimination of the
underlying disease or disorder. In some instances, the "initial
colonization of the newborn intestine is particularly relevant to
the proper development of the host's immune and metabolic functions
and to determine disease risk in early and later life" (Sanz et
al., 2015). In some embodiments, early intervention (e.g.,
prenatal, perinatal, neonatal) using the genetically engineered
bacteria of the invention may be sufficient to prevent or delay the
onset of the disease or disorder.
[0051] As used herein a "pharmaceutical composition" refers to a
preparation of genetically engineered bacteria of the invention
with other components such as a physiologically suitable carrier
and/or excipient.
[0052] The phrases "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier" which may be used
interchangeably refer to a carrier or a diluent that does not cause
significant irritation to an organism and does not abrogate the
biological activity and properties of the administered bacterial
compound. An adjuvant is included under these phrases.
[0053] The term "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of
an active ingredient. Examples include, but are not limited to,
calcium bicarbonate, calcium phosphate, various sugars and types of
starch, cellulose derivatives, gelatin, vegetable oils,
polyethylene glycols, and surfactants, including, for example,
polysorbate 20.
[0054] The terms "therapeutically effective dose" and
"therapeutically effective amount" are used to refer to an amount
of a compound that results in prevention, delay of onset of
symptoms, or amelioration of symptoms of a condition, e.g.,
inflammation, diarrhea. A therapeutically effective amount may, for
example, be sufficient to treat, prevent, reduce the severity,
delay the onset, and/or reduce the risk of occurrence of one or
more symptoms of an autoimmune disorder and/or a disease or
condition associated with gut inflammation and/or compromised gut
barrier function. A therapeutically effective amount, as well as a
therapeutically effective frequency of administration, can be
determined by methods known in the art and discussed below.
[0055] The articles "a" and "an," as used herein, should be
understood to mean "at least one," unless clearly indicated to the
contrary.
[0056] The phrase "and/or," when used between elements in a list,
is intended to mean either (1) that only a single listed element is
present, or (2) that more than one element of the list is present.
For example, "A, B, and/or C" indicates that the selection may be A
alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C.
The phrase "and/or" may be used interchangeably with "at least one
of" or "one or more of" the elements in a list.
[0057] Bacteria
[0058] The genetically engineered bacteria of the invention
comprise a gene encoding a non-native anti-inflammation and/or gut
barrier function enhancer molecule, or a gene cassette encoding a
non-native biosynthetic pathway capable of producing an
anti-inflammation and/or gut barrier function enhancer molecule,
wherein the gene or gene cassette is operatively linked to a
tunable regulatory region that is directly or indirectly controlled
by a transcription factor that is capable of sensing at least one
reactive nitrogen species. In some embodiments, the gene or gene
cassette is an additional copy of a native gene or gene cassette.
In some embodiments, the gene or gene cassette is from a different
species. In some embodiments, the gene or gene cassette is operably
linked to a directly or indirectly inducible promoter. In some
embodiments, the inducible promoter is not associated with the gene
or gene cassette in nature.
[0059] In some embodiments, the genetically engineered bacteria are
naturally non-pathogenic bacteria. In some embodiments, the
genetically engineered bacteria are naturally pathogenic bacteria
that are modified or mutated to reduce or eliminate pathogenicity.
In some embodiments, the genetically engineered bacteria are
commensal bacteria. In some embodiments, the genetically engineered
bacteria are probiotic bacteria. In certain embodiments, the
genetically engineered bacteria are selected from the group
consisting of Bacteroides fragilis, Bacteroides thetaiotaomicron,
Bacteroides subtilis, Bifidobacterium bifidum, Bifidobacterium
infantis, Bifidobacterium lactis, Clostridium butyricum,
Escherichia coli Nissle, Lactobacillus acidophilus, Lactobacillus
plantarum, Lactobacillus reuteri, and Lactococcus lactis.
[0060] In some embodiments, the genetically engineered bacteria are
Escherichia coli strain Nissle 1917 (E. coli Nissle), a
Gram-positive bacterium of the Enterobacteriaceae family that "has
evolved into one of the best characterized probiotics" (Ukena et
al., 2007). The strain is characterized by its "complete
harmlessness" (Schultz, 2008), and "has GRAS (generally recognized
as safe) status" (Reister et al., 2014, emphasis added). Genomic
sequencing confirmed that E. coli Nissle "lacks prominent virulence
factors (e.g., E. coli .alpha.-hemolysin, P-fimbrial adhesins)"
(Schultz, 2008), and E. coli Nissle "does not carry pathogenic
adhesion factors and does not produce any enterotoxins or
cytotoxins, it is not invasive, not uropathogenic" (Sonnenborn et
al., 2009). As early as in 1917, E. coli Nissle was packaged into
medicinal capsules, called Mutaflor, for therapeutic use. E. coli
Nissle has since been used to treat ulcerative colitis in humans in
vivo (Rembacken et al., 1999), to treat inflammatory bowel disease,
Crohn's disease, and pouchitis in humans in vivo (Schultz, 2008),
and to inhibit enteroinvasive Salmonella, Legionella, Yersinia, and
Shigella in vitro (Altenhoefer et al., 2004). It is commonly
accepted that E. coli Nissle's "therapeutic efficacy and safety
have convincingly been proven" (Ukena et al., 2007).
[0061] One of ordinary skill in the art would appreciate that the
genetic modifications disclosed herein may be adapted for other
species, strains, and subtypes of bacteria. It is known, for
example, that "the clostridial butyrogenic pathway [genes] . . .
are widespread in the genome-sequenced clostridia and related
species" (Aboulnaga et al., 2013). Furthermore, genes from one or
more different species of bacteria can be introduced into one
another, e.g., the butyrogenic genes from Peptoclostridium
difficile have been expressed in Escherichia coli (Aboulnaga et
al., 2013).
[0062] Reducing Gut Inflammation, Tightening Gut Mucosal Barrier,
and/or Treating or Preventing Autoimmune Disorders
[0063] The genetically engineered bacteria of the invention
comprise a gene encoding a non-native anti-inflammation and/or gut
barrier function enhancer molecule, or a gene cassette encoding a
biosynthetic pathway capable of producing an anti-inflammation
and/or gut barrier function enhancer molecule. In some embodiments,
the molecule is selected from the group consisting of a short-chain
fatty acid, butyrate, propionate, acetate, GLP-2, IL-10, IL-27,
TGF-.beta.1, TGF-.beta.2, elafin (also known as peptidase inhibitor
3 or SKALP), and trefoil factor. A molecule may be primarily
anti-inflammatory, e.g., IL-10, or primarily gut barrier function
enhancing, e.g., GLP-2. Alternatively, a molecule may be both
anti-inflammatory and gut barrier function enhancing.
[0064] In some embodiments, the genetically engineered bacteria of
the invention express an anti-inflammation and/or gut barrier
function enhancer molecule that is encoded by a single gene, e.g.,
the molecule is elafin and encoded by the PI3 gene, or the molecule
is interleukin-10 and encoded by the IL10 gene. In alternate
embodiments, the genetically engineered bacteria of the invention
encode an anti-inflammation and/or gut barrier function enhancer
molecule, e.g., butyrate, that is synthesized by a biosynthetic
pathway requiring multiple genes.
[0065] In some embodiments, the genetically engineered bacteria of
the invention comprise a butyrogenic gene cassette and produce
butyrate in the presence of RNS. Unmodified bacteria comprising
butyrate biosynthesis genes are known and include, but are not
limited to, Peptoclostridium, Clostridium, Fusobacterium,
Butyrivibrio, Eubacterium, and Treponema. The genetically
engineered bacteria may include any suitable set of butyrogenic
genes. In some embodiments, the genetically engineered bacteria of
the invention comprise butyrate biosynthesis genes from a different
species, strain, or substrain of bacteria. In some embodiments, the
genetically engineered bacteria comprise the eight genes of the
butyrate biosynthesis pathway from Peptoclostridium difficile,
e.g., Peptoclostridium difficile strain 630: bcd2, etfB3, etfA3,
thiA1, hbd, crt2, pbt, and buk (Aboulnaga et al., 2013), and
produce butyrate in the presence of RNS. Peptoclostridium difficile
strain 630 and strain 1296 are both capable of producing butyrate,
but comprise different nucleic acid sequences for etfA3, thiA1,
hbd, crt2, pbt, and buk. In some embodiments, the genetically
engineered bacteria comprise a combination of butyrogenic genes
from different species, strains, and/or substrains of bacteria, and
produce butyrate in the presence of RNS. For example, in some
embodiments, the genetically engineered bacteria comprise bcd2,
etfB3, etfA3, and thiA1 from Peptoclostridium difficile strain 630,
and hbd, crt2, pbt, and buk from Peptoclostridium difficile strain
1296.
[0066] The gene products of the bcd2, etfA3, and etfB3 genes in
Clostridium difficile form a complex that converts crotonyl-CoA to
butyryl-CoA, which may function as an oxygen-dependent co-oxidant.
Because the genetically engineered bacteria of the invention are
designed to produce butyrate in a microaerobic or oxygen-limited
environment, e.g., the mammalian gut, oxygen-dependence could have
a negative effect on butyrate production in the gut. It has been
shown that a single gene from Treponema denticola (ter, encoding
trans-2-enoynl-CoA reductase) can functionally replace this
three-gene complex in an oxygen-independent manner. In some
embodiments, the genetically engineered bacteria comprise a ter
gene, e.g., from Treponema denticola, which can functionally
replace all three of the bcd2, etfB3, and etfA3 genes, e.g., from
Peptoclostridium difficile. In this embodiment, the genetically
engineered bacteria comprise thiA1, hbd, crt2, pbt, and buk, e.g.,
from Peptoclostridium difficile, and ter, e.g., from Treponema
denticola, and produce butyrate in the presence of RNS (see, e.g.,
FIG. 5). In some embodiments, the genetically engineered bacteria
comprise genes for aerobic butyrate biosynthesis and/or genes for
anaerobic or microaerobic butyrate biosynthesis. In some
embodiments, the genetically engineered bacteria of the invention
comprise thiA1, hbd, crt2, pbt, and buk, e.g., from
Peptoclostridium difficile; ter, e.g., from Treponema denticola;
one or more of bcd2, etfB3, and etfA3, e.g., from Peptoclostridium
difficile; and produce butyrate in the presence of RNS. In some
embodiments, one or more of the butyrate biosynthesis genes is
functionally replaced, modified, and/or mutated in order to enhance
stability and/or increase butyrate production in the presence of
RNS. In some embodiments, the local production of butyrate induces
the differentiation of regulatory T cells in the gut and/or
promotes the barrier function of colonic epithelial cells.
[0067] In some embodiments, the genetically engineered bacteria of
the invention comprise a propionate gene cassette and produce
propionate in the presence of RNS. Unmodified bacteria that are
capable of producing propionate via an endogenous propionate
biosynthesis pathway include, but are not limited to, Clostridium
propionicum, Megasphaera elsdenii, and Prevotella ruminicola. The
genetically engineered bacteria may include any suitable set of
propionate biosynthesis genes. In some embodiments, the genetically
engineered bacteria of the invention comprise propionate
biosynthesis genes from a different species, strain, or substrain
of bacteria. In some embodiments, the genetically engineered
bacteria comprise the genes pct, lcd, and acr from Clostridium
propionicum. In some embodiments, the genetically engineered
bacteria comprise acrylate pathway propionate biosynthesis genes,
e.g., pct, lcdA, lcdB, lcdC, etfA, acrB, and acrC. In alternate
embodiments, the genetically engineered bacteria comprise pyruvate
pathway propionate biosynthesis genes, e.g., thrA.sup.fbr, thrB,
thrC, ilvA.sup.fbr, aceE, aceF, and lpd, and optionally further
comprise tesB.
[0068] In some embodiments, one or more of the propionate
biosynthesis genes is a synthetic propionate biosynthesis gene. In
some embodiments, one or more of the propionate biosynthesis genes
is an E. coli propionate biosynthesis gene. In some embodiments,
one or more of the propionate biosynthesis genes is a C. glutamicum
propionate biosynthesis gene. In some embodiments, one or more of
the propionate biosynthesis genes is a C. propionicum propionate
biosynthesis gene. In some embodiments, one or more of the
propionate biosynthesis genes is a synthetic propionate
biosynthesis gene. The propionate gene cassette may comprise genes
for the aerobic biosynthesis of propionate and/or genes for the
anaerobic or microaerobic biosynthesis of propionate. One or more
of the butyrate biosynthesis genes may be functionally replaced or
modified, e.g., codon optimized. In some embodiments, the
genetically engineered bacteria comprise a combination of
propionate biosynthesis genes from different species, strains,
and/or substrains of bacteria, and produce propionate in low-oxygen
conditions. In some embodiments, one or more of the propionate
biosynthesis genes is functionally replaced, modified, and/or
mutated in order to enhance stability and/or increase propionate
production in low-oxygen conditions.
[0069] In some embodiments, the genetically engineered bacteria of
the invention comprise an acetate gene cassette and produce acetate
in the presence of RNS. Unmodified bacteria comprising acetate
biosynthesis genes are known in the art and are capable of
consuming various substrates to produce acetate under aerobic
and/or anaerobic conditions (see, e.g., Ragsdale et al., 2008). The
genetically engineered bacteria may include any suitable set of
acetate biosynthesis genes. In some embodiments, the genetically
engineered bacteria of the invention comprise acetate biosynthesis
genes from a different species, strain, or substrain of bacteria.
In some embodiments, the native acetate biosynthesis genes in the
genetically engineered bacteria are enhanced. In some embodiments,
the genetically engineered bacteria comprise aerobic acetate
biosynthesis genes, e.g., from Escherichia coli. In some
embodiments, the genetically engineered bacteria comprise anaerobic
acetate biosynthesis genes, e.g., from Acetitomaculum,
Acetoanaerobium, Acetohalobium, Acetonema, Balutia,
Butyribacterium, Clostridium, Moorella, Oxobacter, Sporomusa,
and/or Thermoacetogenium. The genetically engineered bacteria may
comprise genes for aerobic acetate biosynthesis or genes for
anaerobic or microaerobic acetate biosynthesis. In some
embodiments, the genetically engineered bacteria comprise both
aerobic and anaerobic or microaerobic acetate biosynthesis genes.
In some embodiments, the genetically engineered bacteria comprise a
combination of acetate biosynthesis genes from different species,
strains, and/or substrains of bacteria, and produce acetate in the
presence of RNS. In some embodiments, one or more of the acetate
biosynthesis genes is functionally replaced, modified, and/or
mutated in order to enhance stability and/or acetate production in
the presence of RNS.
[0070] In some embodiments, the genetically engineered bacteria of
the invention express IL-10 in the presence of RNS. Interleukin-10
(IL-10) is a class 2 cytokine, a category which includes cytokines,
interferons, and interferon-like molecules, such as IL-19, IL-20,
IL-22, IL-24, IL-26, IL-28A, IL-28B, IL-29, IFN-.alpha.,
IFN-.beta., IFN-.delta., IFN-.epsilon., IFN-.kappa., IFN-.tau.,
IFN-.omega., and limitin. IL-10 is an anti-inflammatory cytokine
that signals through two receptors, IL-10R1 and IL-10R2.
Deficiencies in IL-10 and/or its receptors are associated with IBD
and intestinal sensitivity (Nielsen 2014). Bacteria expressing
IL-10 or protease inhibitors may ameliorate conditions such as
Crohn's disease and ulcerative colitis (Simpson et al., 2014). The
genetically engineered bacteria may comprise any suitable gene
encoding IL-10, e.g., human IL-10. In some embodiments, the gene
encoding IL-10 is modified and/or mutated, e.g., to enhance
stability, increase IL-10 production, and/or increase
anti-inflammatory potency in the presence of RNS.
[0071] In some embodiments, the genetically engineered bacteria of
the invention express GLP-2 or proglucagon in the presence of RNS.
Glucagon-like peptide 2 (GLP-2) is produced by intestinal endocrine
cells and stimulates intestinal growth and enhances gut barrier
function. Post-translational proteolytic cleavage of proglucagon
produces GLP-2 and GLP-1. GLP-2 administration has therapeutic
potential in treating IBD, short bowel syndrome, and small bowel
enteritis (Yazbeck et al., 2009). The genetically engineered
bacteria may comprise any suitable gene encoding GLP-2 or
proglucagon, e.g., human GLP-2 or proglucagon. In some embodiments,
a protease inhibitor, e.g., an inhibitor of dipeptidyl peptidase,
is also administered to decrease GLP-2 degradation. In some
embodiments, the genetically engineered bacteria express a
degradation resistant GLP-2 analog, e.g., Teduglutide (Yazbeck et
al., 2009). In some embodiments, the gene encoding GLP-2 or
proglucagon is modified and/or mutated, e.g., to enhance stability,
increase GLP-2 production, and/or increase gut barrier enhancing
potency in the presence of RNS.
[0072] In some embodiments, the genetically engineered bacteria of
the invention express a molecule that is capable of inhibiting a
pro-inflammatory molecule. The genetically engineered bacteria may
express any suitable inhibitory molecule, e.g., a single-chain
variable fragment (scFv), antisense RNA, siRNA, or shRNA, that is
capable of neutralizing one or more pro-inflammatory molecules,
e.g., TNF, IFN-.gamma., IL-1.beta., IL-6, IL-8, IL-17, or
chemokines (Keates et al., 2008; Ahmad et al., 2012). The
genetically engineered bacteria may inhibit one or more
pro-inflammatory molecules, e.g., TNF, IL-17.
[0073] RNA interference (RNAi) is a post-transcriptional gene
silencing mechanism in plants and animals. RNAi is activated when
microRNA (miRNA), double-stranded RNA (dsRNA), or short hairpin RNA
(shRNA) is processed into short interfering RNA (siRNA) duplexes
(Keates et al., 2008). RNAi can be "activated in vitro and in vivo
by non-pathogenic bacteria engineered to manufacture and deliver
shRNA to target cells" such as mammalian cells (Keates et al.,
2008). In some embodiments, the genetically engineered bacteria of
the invention induce RNAi-mediated gene silencing of one or more
pro-inflammatory molecules in the presence of RNS. In some
embodiments, the genetically engineered bacteria produce siRNA
targeting TNF in the presence of RNS.
[0074] Single-chain variable fragments (scFv) are "widely used
antibody fragments . . . produced in prokaryotes" (Frenzel et al.,
2013). scFv lacks the constant domain of a traditional antibody and
expresses the antigen-binding domain as a single peptide. Bacteria
such as Escherichia coli are capable of producing scFv that target
pro-inflammatory cytokines, e.g., TNF (Hristodorov et al., 2014).
In some embodiments, the genetically engineered bacteria of the
invention express a binding protein for neutralizing one or more
pro-inflammatory molecules in the presence of RNS. In some
embodiments, the genetically engineered bacteria produce scFv
targeting TNF in the presence of RNS. In some embodiments, the
genetically engineered bacteria produce both scFv and siRNA
targeting one or more pro-inflammatory molecules in the presence of
RNS (see, e.g., Xiao et al., 2014).
[0075] One of skill in the art would appreciate that additional
genes and gene cassettes capable of producing anti-inflammation
and/or gut barrier function enhancer molecules are known in the art
and may be expressed by the genetically engineered bacteria of the
invention. In some embodiments, the gene or gene cassette for
producing a therapeutic molecule also comprises additional
transcription and translation elements, e.g., a ribosome binding
site, to enhance expression of the therapeutic molecule.
[0076] In some embodiments, the genetically engineered bacteria
produce two or more anti-inflammation and/or gut barrier function
enhancer molecules. In certain embodiments, the two or more
molecules behave synergistically to reduce gut inflammation and/or
enhance gut barrier function. In some embodiments, the genetically
engineered bacteria express at least one anti-inflammation molecule
and at least one gut barrier function enhancer molecule. In certain
embodiments, the genetically engineered bacteria express IL-10 and
GLP-2. In alternate embodiments, the genetically engineered
bacteria express IL-10 and butyrate.
[0077] RNS Tunable Regulatory Region
[0078] The genetically engineered bacteria of the invention
comprise a tunable regulatory region that is directly or indirectly
controlled by a transcription factor that is capable of sensing at
least one reactive nitrogen species. The tunable regulatory region
is operatively linked to a gene or gene cassette capable of
directly or indirectly driving the expression of an
anti-inflammation and/or gut barrier function enhancer molecule,
thus controlling expression of the molecule relative to RNS levels.
For example, the tunable regulatory region is a RNS-inducible
regulatory region, and the molecule is butyrate; when RNS is
present, e.g., in an inflamed tissue, a RNS-sensing transcription
factor binds to and/or activates the regulatory region and drives
expression of the butyrate operon, thereby producing butyrate,
which exerts anti-inflammatory and/or gut barrier enhancing
effects. Subsequently, when inflammation is ameliorated, RNS levels
are reduced, and butyrate production is decreased or
eliminated.
[0079] In some embodiments, the tunable regulatory region is a
RNS-inducible regulatory region; in the presence of RNS, a
transcription factor senses RNS and activates the RNS-inducible
regulatory region, thereby driving expression of an operatively
linked gene or gene cassette. In some embodiments, the
transcription factor senses RNS and subsequently binds to the
RNS-inducible regulatory region, thereby activating downstream gene
expression. In alternate embodiments, the transcription factor is
bound to the RNS-inducible regulatory region in the absence of RNS;
when the transcription factor senses RNS, it undergoes a
conformational change, thereby inducing downstream gene
expression.
[0080] In some embodiments, the tunable regulatory region is a
RNS-inducible regulatory region, and the transcription factor that
senses RNS is NorR. NorR "is an NO-responsive transcriptional
activator that regulates expression of the norVW genes encoding
flavorubredoxin and an associated flavoprotein, which reduce NO to
nitrous oxide" (Spiro 2006). The genetically engineered bacteria of
the invention may comprise any suitable RNS-responsive regulatory
region from a gene that is activated by NorR. Genes that are
capable of being activated by NorR are known in the art (see, e.g.,
Spiro 2006; Vine et al., 2011; Karlinsey et al., 2012; Table 1). In
certain embodiments, the genetically engineered bacteria of the
invention comprise a RNS-inducible regulatory region from norVW
that is operatively linked to a gene or gene cassette, e.g., a
butyrogenic gene cassette. In the presence of RNS, a NorR
transcription factor senses RNS and activates to the norVW
regulatory region, thereby driving expression of the operatively
linked butyrogenic gene cassette and producing butyrate.
[0081] In some embodiments, the tunable regulatory region is a
RNS-inducible regulatory region, and the transcription factor that
senses RNS is DNR. DNR (dissimilatory nitrate respiration
regulator) "promotes the expression of the nir, the nor and the nos
genes" in the presence of nitric oxide (Castiglione et al., 2009).
The genetically engineered bacteria of the invention may comprise
any suitable RNS-responsive regulatory region from a gene that is
activated by DNR. Genes that are capable of being activated by DNR
are known in the art (see, e.g., Castiglione et al., 2009; Giardina
et al., 2008; Table 1). In certain embodiments, the genetically
engineered bacteria of the invention comprise a RNS-inducible
regulatory region from norCB that is operatively linked to a gene
or gene cassette, e.g., a butyrogenic gene cassette. In the
presence of RNS, a DNR transcription factor senses RNS and
activates to the norCB regulatory region, thereby driving
expression of the operatively linked butyrogenic gene cassette and
producing butyrate. In some embodiments, the DNR is Pseudomonas
aeruginosa DNR.
[0082] In some embodiments, the tunable regulatory region is a
RNS-derepressible regulatory region, and binding of a corresponding
transcription factor represses downstream gene expression; in the
presence of RNS, the transcription factor no longer binds to the
regulatory region, thereby derepressing the operatively linked gene
or gene cassette.
[0083] In some embodiments, the tunable regulatory region is a
RNS-derepressible regulatory region, and the transcription factor
that senses RNS is NsrR. NsrR is "an Rrf2-type transcriptional
repressor [that] can sense NO and control the expression of genes
responsible for NO metabolism" (Isabella et al., 2009). The
genetically engineered bacteria of the invention may comprise any
suitable RNS-responsive regulatory region from a gene that is
repressed by NsrR. In some embodiments, the NsrR is Neisseria
gonorrhoeae NsrR. Genes that are capable of being repressed by NsrR
are known in the art (see, e.g., Isabella et al., 2009; Dunn et
al., 2010; Table 1). In certain embodiments, the genetically
engineered bacteria of the invention comprise a RNS-derepressible
regulatory region from norB that is operatively linked to a gene or
gene cassette, e.g., a butyrogenic gene cassette. In the presence
of RNS, an NsrR transcription factor senses RNS and no longer binds
to the norB regulatory region, thereby derepressing the operatively
linked butyrogenic gene cassette and producing butyrate.
[0084] In some embodiments, it is advantageous for the genetically
engineered bacteria to express a RNS-sensing transcription factor
that does not regulate the expression of a significant number of
native genes in the bacteria. In some embodiments, the genetically
engineered bacterium of the invention expresses a RNS-sensing
transcription factor from a different species, strain, or substrain
of bacteria, wherein the transcription factor does not bind to
regulatory sequences in the genetically engineered bacterium of the
invention. In some embodiments, the genetically engineered
bacterium of the invention is Escherichia coli, and the RNS-sensing
transcription factor is NsrR, e.g., from is Neisseria gonorrhoeae,
wherein the Escherichia coli does not comprise binding sites for
said NsrR. In some embodiments, the heterologous transcription
factor minimizes or eliminates off-target effects on endogenous
regulatory regions and genes in the genetically engineered
bacteria.
[0085] In some embodiments, the tunable regulatory region is a
RNS-repressible regulatory region, and binding of a corresponding
transcription factor represses downstream gene expression; in the
presence of RNS, the transcription factor senses RNS and binds to
the RNS-repressible regulatory region, thereby repressing
expression of the operatively linked gene or gene cassette. In some
embodiments, the RNS-sensing transcription factor is capable of
binding to a regulatory region that overlaps with part of the
promoter sequence. In alternate embodiments, the RNS-sensing
transcription factor is capable of binding to a regulatory region
that is upstream or downstream of the promoter sequence.
[0086] In these embodiments, the genetically engineered bacteria
may comprise a two repressor activation regulatory circuit, which
is used to express an anti-inflammation and/or gut barrier function
enhancer molecule. The two repressor activation regulatory circuit
comprises a first RNS-sensing repressor and a second repressor,
which is operatively linked to a gene or gene cassette, e.g., a
butyrogenic gene cassette. In one aspect of these embodiments, the
RNS-sensing repressor inhibits transcription of the second
repressor, which inhibits the transcription of the gene or gene
cassette. Examples of second repressors useful in these embodiments
include, but are not limited to, TetR, C1, and LexA. In the absence
of binding by the first repressor (which occurs in the absence of
RNS), the second repressor is transcribed, which represses
expression of the gene or gene cassette, e.g., a butyrogenic gene
cassette. In the presence of binding by the first repressor (which
occurs in the presence of RNS), expression of the second repressor
is repressed, and the gene or gene cassette, e.g., a butyrogenic
gene cassette, is expressed.
[0087] A RNS-responsive transcription factor may induce, derepress,
or repress gene expression depending upon the regulatory region
sequence used in the genetically engineered bacteria. One or more
types of RNS-sensing transcription factors and corresponding
regulatory region sequences may be present in genetically
engineered bacteria. In some embodiments, the genetically
engineered bacteria comprise one type of RNS-sensing transcription
factor, e.g., NsrR, and one corresponding regulatory region
sequence, e.g., from norB. In some embodiments, the genetically
engineered bacteria comprise one type of RNS-sensing transcription
factor, e.g., NsrR, and two or more different corresponding
regulatory region sequences, e.g., from norB and aniA. In some
embodiments, the genetically engineered bacteria comprise two or
more types of RNS-sensing transcription factors, e.g., NsrR and
NorR, and two or more corresponding regulatory region sequences,
e.g., from norB and norR, respectively. One RNS-responsive
regulatory region may be capable of binding more than one
transcription factor. In some embodiments, the genetically
engineered bacteria comprise two or more types of RNS-sensing
transcription factors and one corresponding regulatory region
sequence. Nucleic acid sequences of several RNS-regulated
regulatory regions are known in the art (see, e.g., Spiro 2006;
Isabella et al., 2009; Dunn et al., 2010; Vine et al., 2011;
Karlinsey et al., 2012).
[0088] In some embodiments, the genetically engineered bacteria of
the invention comprise a gene encoding a RNS-sensing transcription
factor, e.g., the nsrR gene, that is controlled by its native
promoter, an inducible promoter, a promoter that is stronger than
the native promoter, e.g., the GlnRS promoter or the P(Bla)
promoter, or a constitutive promoter. In some instances, it may be
advantageous to express the RNS-sensing transcription factor under
the control of an inducible promoter in order to enhance expression
stability. In some embodiments, expression of the RNS-sensing
transcription factor is controlled by a different promoter than the
promoter that controls expression of the therapeutic molecule. In
some embodiments, expression of the RNS-sensing transcription
factor is controlled by the same promoter that controls expression
of the therapeutic molecule. In some embodiments, the RNS-sensing
transcription factor and therapeutic molecule are divergently
transcribed from a promoter region.
[0089] In some embodiments, the genetically engineered bacteria of
the invention comprise a gene for a RNS-sensing transcription
factor from a different species, strain, or substrain of bacteria.
In some embodiments, the genetically engineered bacteria comprise a
RNS-responsive regulatory region from a different species, strain,
or substrain of bacteria. In some embodiments, the genetically
engineered bacteria comprise a RNS-sensing transcription factor and
corresponding RNS-responsive regulatory region from a different
species, strain, or substrain of bacteria. The heterologous
RNS-sensing transcription factor and regulatory region may increase
the transcription of genes operatively linked to said regulatory
region in the presence of RNS, as compared to the native
transcription factor and regulatory region from bacteria of the
same subtype under the same conditions.
[0090] In some embodiments, the genetically engineered bacteria
comprise a RNS-sensing transcription factor, NsrR, and
corresponding regulatory region, nsrR, from Neisseria gonorrhoeae.
In some embodiments, the native RNS-sensing transcription factor,
e.g., NsrR, is left intact and retains wild-type activity. In
alternate embodiments, the native RNS-sensing transcription factor,
e.g., NsrR, is deleted or mutated to reduce or eliminate wild-type
activity.
[0091] In some embodiments, the genetically engineered bacteria of
the invention comprise multiple copies of the endogenous gene
encoding the RNS-sensing transcription factor, e.g., the nsrR gene.
In some embodiments, the gene encoding the RNS-sensing
transcription factor is present on a plasmid. In some embodiments,
the gene encoding the RNS-sensing transcription factor and the gene
or gene cassette for producing the therapeutic molecule are present
on different plasmids. In some embodiments, the gene encoding the
RNS-sensing transcription factor and the gene or gene cassette for
producing the therapeutic molecule are present on the same plasmid.
In some embodiments, the gene encoding the RNS-sensing
transcription factor is present on a chromosome. In some
embodiments, the gene encoding the RNS-sensing transcription factor
and the gene or gene cassette for producing the therapeutic
molecule are present on different chromosomes. In some embodiments,
the gene encoding the RNS-sensing transcription factor and the gene
or gene cassette for producing the therapeutic molecule are present
on the same chromosome.
[0092] In some embodiments, the genetically engineered bacteria
comprise a wild-type gene encoding a RNS-sensing transcription
factor, e.g., the NsrR gene, and a corresponding regulatory region,
e.g., a norB regulatory region, that is mutated relative to the
wild-type regulatory region from bacteria of the same subtype. The
mutated regulatory region increases the expression of the
anti-inflammatory and/or gut barrier enhancer molecule in the
presence of RNS, as compared to the wild-type regulatory region
under the same conditions. In some embodiments, the genetically
engineered bacteria comprise a wild-type RNS-responsive regulatory
region, e.g., the norB regulatory region, and a corresponding
transcription factor, e.g., NsrR, that is mutated relative to the
wild-type transcription factor from bacteria of the same subtype.
The mutant transcription factor increases the expression of the
anti-inflammatory and/or gut barrier enhancer molecule in the
presence of RNS, as compared to the wild-type transcription factor
under the same conditions. In some embodiments, both the
RNS-sensing transcription factor and corresponding regulatory
region are mutated relative to the wild-type sequences from
bacteria of the same subtype in order to increase expression of the
anti-inflammatory and/or gut barrier enhancer molecule in the
presence of RNS. Nucleic acid sequences of exemplary RNS-regulated
constructs comprising a gene encoding NsrR and a norB promoter are
shown in FIGS. 4 and 5.
[0093] The genetically engineered bacteria comprise a stably
maintained plasmid or chromosome carrying the gene(s) or gene
cassette(s) capable of producing an anti-inflammation and/or gut
barrier function enhancer molecule, such that said gene(s) or gene
cassette(s) can be expressed in the host cell, and the host cell is
capable of survival and/or growth in vitro, e.g., in medium, and/or
in vivo, e.g., in the gut.
[0094] In some embodiments, the genetically engineered bacteria may
comprise multiple copies of the gene(s) or gene cassette(s) capable
of producing an anti-inflammation and/or gut barrier function
enhancer molecule. In some embodiments, the gene(s) or gene
cassette(s) capable of producing an anti-inflammation and/or gut
barrier function enhancer molecule is present on a plasmid and
operatively linked to a RNS-responsive regulatory region. In some
embodiments, the gene(s) or gene cassette(s) capable of producing
an anti-inflammation and/or gut barrier function enhancer molecule
is present in a chromosome and operatively linked to a
RNS-responsive regulatory region.
[0095] In some embodiments, any of the gene(s) or gene cassette(s)
of the present disclosure may be integrated into the bacterial
chromosome at one or more integration sites. For example, one or
more copies of the butryogenic gene cassette may be integrated into
the bacterial chromosome. Having multiple copies of the butryogenic
gene cassette integrated into the chromosome allows for greater
production of the butyrate and also permits fine-tuning of the
level of expression. Alternatively, different circuits described
herein, such as any of the kill-switch circuits, in addition to the
therapeutic gene(s) or gene cassette(s) could be integrated into
the bacterial chromosome at one or more different integration sites
to perform multiple different functions.
[0096] In some embodiments, the genetically engineered bacteria of
the invention produce at least one anti-inflammation and/or gut
barrier enhancer molecule in the presence of RNS to reduce local
gut inflammation by at least about 1.5-fold, at least about 2-fold,
at least about 10-fold, at least about 15-fold, at least about
20-fold, at least about 30-fold, at least about 50-fold, at least
about 100-fold, at least about 200-fold, at least about 300-fold,
at least about 400-fold, at least about 500-fold, at least about
600-fold, at least about 700-fold, at least about 800-fold, at
least about 900-fold, at least about 1,000-fold, or at least about
1,500-fold as compared to unmodified bacteria of the same subtype
under the same conditions. Inflammation may be measured by methods
known in the art, e.g., counting disease lesions using endoscopy;
detecting T regulatory cell differentiation in peripheral blood,
e.g., by fluorescence activated sorting; measuring T regulatory
cell levels; measuring cytokine levels; measuring areas of mucosal
damage; assaying inflammatory biomarkers, e.g., by qPCR; PCR
arrays; transcription factor phosphorylation assays; immunoassays;
and/or cytokine assay kits (Mesoscale, Cayman Chemical,
Qiagen).
[0097] In some embodiments, the genetically engineered bacteria
produce at least about 1.5-fold, at least about 2-fold, at least
about 10-fold, at least about 15-fold, at least about 20-fold, at
least about 30-fold, at least about 50-fold, at least about
100-fold, at least about 200-fold, at least about 300-fold, at
least about 400-fold, at least about 500-fold, at least about
600-fold, at least about 700-fold, at least about 800-fold, at
least about 900-fold, at least about 1,000-fold, or at least about
1,500-fold more of an anti-inflammation and/or gut barrier enhancer
molecule in the presence of RNS than unmodified bacteria of the
same subtype under the same conditions. Certain unmodified bacteria
will not have detectable levels of the anti-inflammation and/or gut
barrier enhancer molecule. In embodiments using genetically
modified forms of these bacteria, the anti-inflammation and/or gut
barrier enhancer molecule will be detectable in the presence of
RNS.
[0098] In certain embodiments, the anti-inflammation and/or gut
barrier enhancer molecule is butyrate. Methods of measuring
butyrate levels, e.g., by mass spectrometry, gas chromatography,
high-performance liquid chromatography (HPLC), are known in the art
(see, e.g., Aboulnaga et al., 2013). In some embodiments, butyrate
is measured as butyrate level/bacteria optical density (OD). In
some embodiments, measuring the activity and/or expression of one
or more gene products in the butyrogenic gene cassette serves as a
proxy measurement for butyrate production. In some embodiments, the
bacterial cells of the invention are harvested and lysed to measure
butyrate production. In alternate embodiments, butyrate production
is measured in the bacterial cell medium. In some embodiments, the
genetically engineered bacteria produce at least about 1 nM/OD, at
least about 10 nM/OD, at least about 100 nM/OD, at least about 500
nM/OD, at least about 1 .mu.M/OD, at least about 10 .mu.M/OD, at
least about 100 .mu.M/OD, at least about 500 .mu.M/OD, at least
about 1 mM/OD, at least about 2 mM/OD, at least about 3 mM/OD, at
least about 5 mM/OD, at least about 10 mM/OD, at least about 20
mM/OD, at least about 30 mM/OD, or at least about 50 mM/OD of
butyrate in the presence of RNS.
[0099] Secretion
[0100] In some embodiments, the genetically engineered bacteria
further comprise a non-native secretion mechanism that is capable
of secreting the anti-inflammation and/or gut barrier enhancer
molecule from the bacterial cytoplasm. Many bacteria have evolved
sophisticated secretion systems to transport substrates across the
bacterial cell envelope. Substrates, such as small molecules,
proteins, and DNA, may be released into the extracellular space or
periplasm, injected into a target cell, or associated with the
bacterial membrane.
[0101] In Gram-negative bacteria, secretion machineries may span
one or both of the inner and outer membranes. In some embodiments,
the genetically engineered bacteria further comprise a non-native
double membrane-spanning secretion system. Double membrane-spanning
secretion systems include, but are not limited to, the type I
secretion system (T1SS), the type II secretion system (T2SS), the
type III secretion system (T3SS), the type IV secretion system
(T4SS), the type VI secretion system (T6SS), and the
resistance-nodulation-division (RND) family of multi-drug efflux
pumps (Pugsley 1993; Gerlach et al., 2007; Collinson et al., 2015;
Costa et al., 2015; Reeves et al., 2015; WO2014138324A1,
incorporated herein by reference). Mycobacteria, which have a
Gram-negative-like cell envelope, may also encode a type VII
secretion system (T7SS) (Stanley et al., 2003). With the exception
of the T2SS, double membrane-spanning secretions generally
transport substrates from the bacterial cytoplasm directly into the
extracellular space or into the target cell. In contrast, the T2SS
and secretion systems that span only the outer membrane may use a
two-step mechanism, wherein substrates are first translocated to
the periplasm by inner membrane-spanning transporters, and then
transferred to the outer membrane or secreted into the
extracellular space. Outer membrane-spanning secretion systems
include, but are not limited to, the type V secretion or
autotransporter system (T5SS), the curli secretion system, and the
chaperone-usher pathway for pili assembly (Saier, 2006; Costa et
al., 2015).
[0102] In some embodiments, the genetically engineered bacteria of
the invention further comprise a type III or a type Ill-like
secretion system (T3SS) from Shigella, Salmonella, E. coli, Bivrio,
Burkholderia, Yersinia, Chlamydia, or Pseudomonas. The T3SS is
capable of transporting a protein from the bacterial cytoplasm to
the host cytoplasm through a needle complex. The T3SS may be
modified to secrete the molecule from the bacterial cytoplasm, but
not inject the molecule into the host cytoplasm. In some
embodiments, the genetically engineered bacteria comprise said
modified T3SS and are capable of secreting the anti-inflammation
and/or gut barrier enhancer molecule from the bacterial
cytoplasm.
[0103] In alternate embodiments, the genetically engineered
bacteria further comprise a non-native single membrane-spanning
secretion system. Single membrane-spanning transporters may act as
a component of a secretion system, or may export substrates
independently. Such transporters include, but are not limited to,
ATP-binding cassette translocases, flagellum/virulence-related
translocases, conjugation-related translocases, the general
secretory system (e.g., the SecYEG complex in E. coli), the
accessory secretory system in mycobacteria and several types of
Gram-positive bacteria (e.g., Bacillus anthracis, Lactobacillus
johnsonii, Corynebacterium glutamicum, Streptococcus gordonii,
Staphylococcus aureus), and the twin-arginine translocation (TAT)
system (Saier, 2006; Rigel and Braunstein, 2008; Albiniak et al.,
2013). It is known that the general secretory and TAT systems can
both export substrates with cleavable N-terminal signal peptides
into the periplasm, and have been explored in the context of
biopharmaceutical production. The TAT system may offer particular
advantages, however, in that it is able to transport folded
substrates, thus eliminating the potential for premature or
incorrect folding. In certain embodiments, the genetically
engineered bacteria comprise a TAT or a TAT-like system and are
capable of secreting the anti-inflammation and/or gut barrier
enhancer molecule from the bacterial cytoplasm.
[0104] One of ordinary skill in the art would appreciate that the
secretion systems disclosed herein may be modified to act in
different species, strains, and subtypes of bacteria, and/or
adapted to deliver different payloads.
[0105] Treatment In Vivo
[0106] The genetically engineered bacteria of the invention may be
evaluated in vivo, e.g., in an animal model. Any suitable animal
model of a disease or condition associated with gut inflammation,
compromised gut barrier function, and/or an autoimmune disorder may
be used (see, e.g., Mizoguchi 2012). The animal model may be a
mouse model of IBD, and IBD may be induced by treatment with
dextran sodium sulfate. The animal model may be a mouse model of
type 1 diabetes (T1D), and T1D may be induced by treatment with
streptozotocin. In some embodiments, the genetically engineered
bacteria of the invention is administered to the animal, e.g., by
oral gavage, and treatment efficacy is determined, e.g., by
endoscopy, colon translucency, fibrin attachment, mucosal and
vascular pathology, and/or stool characteristics. In some
embodiments, the animal is sacrificed, and tissue samples are
collected and analyzed, e.g., colonic sections are fixed and scored
for inflammation and ulceration, and/or homogenized and analyzed
for myeloperoxidase activity and cytokine levels (e.g., IL-1.beta.,
TNF-.alpha., IL-6, IFN-.gamma. and IL-10).
[0107] Essential Genes and Auxotrophs
[0108] As used herein, the term "essential gene" refers to a gene
which is necessary to for cell growth and/or survival. Bacterial
essential genes are well known to one of ordinary skill in the art,
and can be identified by directed deletion of genes and/or random
mutagenesis and screening (see, for example, Zhang and Lin, 2009,
DEG 5.0, a database of essential genes in both prokaryotes and
eukaryotes, Nucl. Acids Res., 37:D455-D458 and Gerdes et al.,
Essential genes on metabolic maps, Curr. Opin. Biotechnol.,
17(5):448-456, the entire contents of each of which are expressly
incorporated herein by reference).
[0109] An "essential gene" may be dependent on the circumstances
and environment in which an organism lives. For example, a mutation
of, modification of, or excision of an essential gene may result in
the recombinant bacteria of the disclosure becoming an auxotroph.
An auxotrophic modification is intended to cause bacteria to die in
the absence of an exogenously added nutrient essential for survival
or growth because they lack the gene(s) necessary to produce that
essential nutrient.
[0110] An auxotrophic modification is intended to cause bacteria to
die in the absence of an exogenously added nutrient essential for
survival or growth because they lack the gene(s) necessary to
produce that essential nutrient. In some embodiments, any of the
genetically engineered bacteria described herein also comprise a
deletion or mutation in a gene required for cell survival and/or
growth. In one embodiment, the essential gene is a DNA synthesis
gene, for example, thyA. In another embodiment, the essential gene
is a cell wall synthesis gene, for example, dapA. In yet another
embodiment, the essential gene is an amino acid gene, for example,
serA or MetA. Any gene required for cell survival and/or growth may
be targeted, including but not limited to, cysE, glnA, ilvD, leuB,
lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA,
thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB,
and thi1, as long as the corresponding wild-type gene product is
not produced in the bacteria. For example, thymine is a nucleic
acid that is required for bacterial cell growth; in its absence,
bacteria undergo cell death. The thyA gene encodes thimidylate
synthetase, an enzyme that catalyzes the first step in thymine
synthesis by converting dUMP to dTMP (Sat et al., 2003). In some
embodiments, the bacterial cell of the disclosure is a thyA
auxotroph in which the thyA gene is deleted and/or replaced with an
unrelated gene. A thyA auxotroph can grow only when sufficient
amounts of thymine are present, e.g., by adding thymine to growth
media in vitro, or in the presence of high thymine levels found
naturally in the human gut in vivo. In some embodiments, the
bacterial cell of the disclosure is auxotrophic in a gene that is
complemented when the bacterium is present in the mammalian gut.
Without sufficient amounts of thymine, the thyA auxotroph dies. In
some embodiments, the auxotrophic modification is used to ensure
that the bacterial cell does not survive in the absence of the
auxotrophic gene product (e.g., outside of the gut).
[0111] Diaminopimelic acid (DAP) is an amino acid synthetized
within the lysine biosynthetic pathway and is required for
bacterial cell wall growth (Meadow et al., 1959; Clarkson et al.,
1971). In some embodiments, any of the genetically engineered
bacteria described herein is a dapD auxotroph in which dapD is
deleted and/or replaced with an unrelated gene. A dapD auxotroph
can grow only when sufficient amounts of DAP are present, e.g., by
adding DAP to growth media in vitro. Without sufficient amounts of
DAP, the dapD auxotroph dies. In some embodiments, the auxotrophic
modification is used to ensure that the bacterial cell does not
survive in the absence of the auxotrophic gene product (e.g.,
outside of the gut).
[0112] In other embodiments, the genetically engineered bacterium
of the present disclosure is a uraA auxotroph in which uraA is
deleted and/or replaced with an unrelated gene. The uraA gene codes
for UraA, a membrane-bound transporter that facilitates the uptake
and subsequent metabolism of the pyrimidine uracil (Andersen et
al., 1995). A uraA auxotroph can grow only when sufficient amounts
of uracil are present, e.g., by adding uracil to growth media in
vitro. Without sufficient amounts of uracil, the uraA auxotroph
dies. In some embodiments, auxotrophic modifications are used to
ensure that the bacteria do not survive in the absence of the
auxotrophic gene product (e.g., outside of the gut).
[0113] In complex communities, it is possible for bacteria to share
DNA. In very rare circumstances, an auxotrophic bacterial strain
may receive DNA from a non-auxotrophic strain, which repairs the
genomic deletion and permanently rescues the auxotroph. Therefore,
engineering a bacterial strain with more than one auxotroph may
greatly decrease the probability that DNA transfer will occur
enough times to rescue the auxotrophy. In some embodiments, the
genetically engineered bacteria of the invention comprise a
deletion or mutation in two or more genes required for cell
survival and/or growth.
[0114] Other examples of essential genes include, but are not
limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs,
ispA, dnaX, adk, hemH, lpxH, cysS, fold, rplT, infC, thrS, nadE,
gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA,
nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS,
ispG, suhB, tadA, acpS, era, rnc, ftsB, eno, pyrG, chpR, Igt, fbaA,
pgk, yqgD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF,
yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rplJ, rplL, rpoB,
rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE,
rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, IspA, ispH, dapB,
folA, imp, yabQ ftsL, ftsl, murE, murF, mraY, murD, ftsW, murG,
murC, ftsQ ftsA, ftsZ, lpxC, secM, secA, can, folK, hemL, yadR,
dapD, map, rpsB, infB, nusA, ftsH, obgE, rpmA, rplU, ispB, murA,
yrbB, yrbK, yhbN, rpsl, rplM, degS, mreD, mreC, mreB, accB, accC,
yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA,
nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB,
csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS,
yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA,
coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA,
yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG,
secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN,
rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, lpxD,
fabZ, lpxA, lpxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA,
rlpB, leuS, Int, ginS, fldA, cydA, infA, cydC, ftsK, lolA, serS,
rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, rne,
yceQ fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK,
minE, mind, pth, rsA, ispE, IolB, hemA, prfA, prmC, kdsA, topA,
ribA, fabI, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ
bcsB, glyQ yibJ, and gpsA. Other essential genes are known to those
of ordinary skill in the art.
[0115] In some embodiments, the genetically engineered bacterium of
the present disclosure is a synthetic ligand-dependent essential
gene (SLiDE) bacterial cell. SLiDE bacterial cells are synthetic
auxotrophs with a mutation in one or more essential genes that only
grow in the presence of a particular ligand (see Lopez and Anderson
"Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a
BL21 (DE3 Biosafety Strain," ACS Synthetic Biology (2015) DOI:
10.1021/acssynbio.5b00085, the entire contents of which are
expressly incorporated herein by reference).
[0116] In some embodiments, the SLiDE bacterial cell comprises a
mutation in an essential gene. In some embodiments, the essential
gene is selected from the group consisting of pheS, dnaN, tyrS,
metG and adk. In some embodiments, the essential gene is dnaN
comprising one or more of the following mutations: H191N, R240C,
I317S, F319V, L340T, V347I, and S345C. In some embodiments, the
essential gene is dnaN comprising the mutations H191N, R240C,
I317S, F319V, L340T, V347I, and S345C. In some embodiments, the
essential gene is pheS comprising one or more of the following
mutations: F125G, P183T, P184A, R186A, and I188L. In some
embodiments, the essential gene is pheS comprising the mutations
F125G, P183T, P184A, R186A, and I188L. In some embodiments, the
essential gene is tyrS comprising one or more of the following
mutations: L36V, C38A and F40G. In some embodiments, the essential
gene is tyrS comprising the mutations L36V, C38A and F40G. In some
embodiments, the essential gene is metG comprising one or more of
the following mutations: E45Q, N47R, I49G, and A51C. In some
embodiments, the essential gene is metG comprising the mutations
E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene
is adk comprising one or more of the following mutations: I4L, L5I
and L6G. In some embodiments, the essential gene is adk comprising
the mutations I4L, L5I and L6G.
[0117] In some embodiments, the genetically engineered bacterium is
complemented by a ligand. In some embodiments, the ligand is
selected from the group consisting of benzothiazole, indole,
2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid,
and L-histidine methyl ester. For example, bacterial cells
comprising mutations in metG (E45Q, N47R, I49G, and A51C) are
complemented by benzothiazole, indole, 2-aminobenzothiazole,
indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl
ester. Bacterial cells comprising mutations in dnaN (H191N, R240C,
I317S, F319V, L340T, V347I, and S345C) are complemented by
benzothiazole, indole or 2-aminobenzothiazole. Bacterial cells
comprising mutations in pheS (F125G, P183T, P184A, R186A, and
I188L) are complemented by benzothiazole or 2-aminobenzothiazole.
Bacterial cells comprising mutations in tyrS (L36V, C38A, and F40G)
are complemented by benzothiazole or 2-aminobenzothiazole.
Bacterial cells comprising mutations in adk (I4L, L5I and L6G) are
complemented by benzothiazole or indole.
[0118] In some embodiments, the genetically engineered bacterium
comprises more than one mutant essential gene that renders it
auxotrophic to a ligand. In some embodiments, the bacterial cell
comprises mutations in two essential genes. For example, in some
embodiments, the bacterial cell comprises mutations in tyrS (L36V,
C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C). In other
embodiments, the bacterial cell comprises mutations in three
essential genes. For example, in some embodiments, the bacterial
cell comprises mutations in tyrS (L36V, C38A, and F40G), metG
(E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A,
and I188L). In some embodiments, the genetically engineered
bacterium is a conditional auxotroph whose essential gene(s) is
replaced using the arabinose system.
[0119] In some embodiments, the genetically engineered bacterium of
the disclosure is an auxotroph and also comprises kill-switch
circuitry, such as any of the kill-switch components and systems
described herein. For example, the recombinant bacteria may
comprise a deletion or mutation in an essential gene required for
cell survival and/or growth, for example, in a DNA synthesis gene,
for example, thyA, cell wall synthesis gene, for example, dapA
and/or an amino acid gene, for example, serA or MetA and may also
comprise a toxin gene that is regulated by one or more
transcriptional activators that are expressed in response to an
environmental condition(s) and/or signal(s) (such as the described
arabinose system) or regulated by one or more recombinases that are
expressed upon sensing an exogenous environmental condition(s)
and/or signal(s). Other embodiments are described in Wright et al.,
"GeneGuard: A Modular Plasmid System Designed for Biosafety," ACS
Synthetic Biology (2015) 4: 307-16, the entire contents of which
are expressly incorporated herein by reference). In some
embodiments, the genetically engineered bacterium of the disclosure
is an auxotroph and also comprises kill-switch circuitry, such as
any of the kill-switch components and systems described herein, as
well as another biosecurity system, such a conditional origin of
replication (see Wright et al., supra).
[0120] Kill Switch
[0121] In some embodiments, the genetically engineered bacteria of
the invention also comprise a kill switch (see, e.g., U.S.
Provisional Application Nos. 62/183,935 and 62/263,329 incorporated
herein by reference in their entireties). The kill switch is
intended to actively kill engineered microbes in response to
external stimuli. As opposed to an auxotrophic mutation where
bacteria die because they lack an essential nutrient for survival,
the kill switch is triggered by a particular factor in the
environment that induces the production of toxic molecules within
the microbe that cause cell death.
[0122] Bacteria engineered with kill switches have been engineered
for in vitro research purposes, e.g., to limit the spread of a
biofuel-producing microorganism outside of a laboratory
environment. Bacteria engineered for in vivo administration to
treat a disease or disorder may also be programmed to die at a
specific time after the expression and delivery of a heterologous
gene or genes, for example, a therapeutic gene(s) or after the
subject has experienced the therapeutic effect. In some
embodiments, the kill switch is activated to kill the bacteria
after a period of time following RNS-mediated expression the
anti-inflammation and/or gut barrier function enhancer molecule. In
some embodiments, the kill switch is activated in a delayed fashion
following RNS-mediated expression the anti-inflammation and/or gut
barrier function enhancer molecule. Alternatively, the bacteria may
be engineered to die after the bacteria have spread outside of a
disease site. Specifically, it may be useful to prevent long-term
colonization of subjects by the microorganism, spread of the
microorganism outside the area of interest (for example, outside
the gut) within the subject, or spread of the microorganism outside
of the subject into the environment (for example, spread to the
environment through the stool of the subject). Examples of such
toxins that can be used in kill-switches include, but are not
limited to, bacteriocins, lysins, and other molecules that cause
cell death by lysing cell membranes, degrading cellular DNA, or
other mechanisms. Such toxins can be used individually or in
combination. The switches that control their production can be
based on, for example, transcriptional activation (toggle switches;
see, e.g., Gardner et al., 2000), translation (riboregulators), or
DNA recombination (recombinase-based switches), and can sense
environmental stimuli such as anaerobiosis or reactive oxygen
species. These switches can be activated by a single environmental
factor or may require several activators in AND, OR, NAND and NOR
logic configurations to induce cell death. For example, an AND
riboregulator switch is activated by tetracycline, isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG), and arabinose to induce
the expression of lysins, which permeabilize the cell membrane and
kill the cell. IPTG induces the expression of the endolysin and
holin mRNAs, which are then derepressed by the addition of
arabinose and tetracycline. All three inducers must be present to
cause cell death. Examples of kill switches are known in the art
(Callura et al., 2010).
[0123] Kill-switches can be designed such that a toxin is produced
in response to an environmental condition or external signal (e.g.,
the bacteria is killed in response to an external cue) or,
alternatively designed such that a toxin is produced once an
environmental condition no longer exists or an external signal is
ceased.
[0124] Thus, in some embodiments, the genetically engineered
bacteria of the disclosure are further programmed to die after
sensing an exogenous environmental signal, for example, in a low
oxygen environment. In some embodiments, the genetically engineered
bacteria of the present disclosure comprise one or more genes
encoding one or more recombinase(s), whose expression is induced in
response to an environmental condition or signal and causes one or
more recombination events that ultimately lead to the expression of
a toxin which kills the cell. In some embodiments, the at least one
recombination event is the flipping of an inverted heterologous
gene encoding a bacterial toxin which is then constitutively
expressed after it is flipped by the first recombinase. In one
embodiment, constitutive expression of the bacterial toxin kills
the genetically engineered bacterium. In these types of kill-switch
systems once the engineered bacterial cell senses the exogenous
environmental condition and expresses the heterologous gene of
interest, the recombinant bacterial cell is no longer viable.
[0125] In another embodiment in which the genetically engineered
bacteria of the present disclosure express one or more
recombinase(s) in response to an environmental condition or signal
causing at least one recombination event, the genetically
engineered bacterium further expresses a heterologous gene encoding
an anti-toxin in response to an exogenous environmental condition
or signal. In one embodiment, the at least one recombination event
is flipping of an inverted heterologous gene encoding a bacterial
toxin by a first recombinase. In one embodiment, the inverted
heterologous gene encoding the bacterial toxin is located between a
first forward recombinase recognition sequence and a first reverse
recombinase recognition sequence. In one embodiment, the
heterologous gene encoding the bacterial toxin is constitutively
expressed after it is flipped by the first recombinase. In one
embodiment, the anti-toxin inhibits the activity of the toxin,
thereby delaying death of the genetically engineered bacterium. In
one embodiment, the genetically engineered bacterium is killed by
the bacterial toxin when the heterologous gene encoding the
anti-toxin is no longer expressed when the exogenous environmental
condition is no longer present.
[0126] In another embodiment, the at least one recombination event
is flipping of an inverted heterologous gene encoding a second
recombinase by a first recombinase, followed by the flipping of an
inverted heterologous gene encoding a bacterial toxin by the second
recombinase. In one embodiment, the inverted heterologous gene
encoding the second recombinase is located between a first forward
recombinase recognition sequence and a first reverse recombinase
recognition sequence. In one embodiment, the inverted heterologous
gene encoding the bacterial toxin is located between a second
forward recombinase recognition sequence and a second reverse
recombinase recognition sequence. In one embodiment, the
heterologous gene encoding the second recombinase is constitutively
expressed after it is flipped by the first recombinase. In one
embodiment, the heterologous gene encoding the bacterial toxin is
constitutively expressed after it is flipped by the second
recombinase. In one embodiment, the genetically engineered
bacterium is killed by the bacterial toxin. In one embodiment, the
genetically engineered bacterium further expresses a heterologous
gene encoding an anti-toxin in response to the exogenous
environmental condition. In one embodiment, the anti-toxin inhibits
the activity of the toxin when the exogenous environmental
condition is present, thereby delaying death of the genetically
engineered bacterium. In one embodiment, the genetically engineered
bacterium is killed by the bacterial toxin when the heterologous
gene encoding the anti-toxin is no longer expressed when the
exogenous environmental condition is no longer present.
[0127] In one embodiment, the at least one recombination event is
flipping of an inverted heterologous gene encoding a second
recombinase by a first recombinase, followed by flipping of an
inverted heterologous gene encoding a third recombinase by the
second recombinase, followed by flipping of an inverted
heterologous gene encoding a bacterial toxin by the third
recombinase.
[0128] In one embodiment, the at least one recombination event is
flipping of an inverted heterologous gene encoding a first excision
enzyme by a first recombinase. In one embodiment, the inverted
heterologous gene encoding the first excision enzyme is located
between a first forward recombinase recognition sequence and a
first reverse recombinase recognition sequence. In one embodiment,
the heterologous gene encoding the first excision enzyme is
constitutively expressed after it is flipped by the first
recombinase. In one embodiment, the first excision enzyme excises a
first essential gene. In one embodiment, the programmed recombinant
bacterial cell is not viable after the first essential gene is
excised.
[0129] In one embodiment, the first recombinase further flips an
inverted heterologous gene encoding a second excision enzyme. In
one embodiment, the wherein the inverted heterologous gene encoding
the second excision enzyme is located between a second forward
recombinase recognition sequence and a second reverse recombinase
recognition sequence. In one embodiment, the heterologous gene
encoding the second excision enzyme is constitutively expressed
after it is flipped by the first recombinase. In one embodiment,
the genetically engineered bacterium dies or is no longer viable
when the first essential gene and the second essential gene are
both excised. In one embodiment, the genetically engineered
bacterium dies or is no longer viable when either the first
essential gene is excised or the second essential gene is excised
by the first recombinase.
[0130] In one embodiment, the genetically engineered bacterium dies
after the at least one recombination event occurs. In another
embodiment, the genetically engineered bacterium is no longer
viable after the at least one recombination event occurs.
[0131] In any of these embodiment, the recombinase can be a
recombinase selected from the group consisting of: Bxbl, PhiC31,
TP901, Bxbl, PhiC31, TP901, HK022, HP1, R4, Int1, Int2, Int3, Int4,
Int5, Int6, Int7, Int8, Int9, Int10, Int11, Int12, Int13, Int14,
Int15, Int16, Int17, Int18, Int19, Int20, Int21, Int22, Int23,
Int24, Int25, Int26, Int27, Int28, Int29, Int30, Int31, Int32,
Int33, and Int34, or a biologically active fragment thereof. In the
above-described kill-switch circuits, a toxin is produced in the
presence of an environmental factor or signal. In another aspect of
kill-switch circuitry, a toxin may be repressed in the presence of
an environmental factor (not produced) and then produced once the
environmental condition or external signal is no longer
present.
[0132] The disclosure provides recombinant bacterial cells which
express one or more heterologous gene(s) upon sensing arabinose or
other sugar in the exogenous environment. In this aspect, the
recombinant bacterial cells contain the araC gene, which encodes
the AraC transcription factor, as well as one or more genes under
the control of the araBAD promoter. In the absence of arabinose,
the AraC transcription factor adopts a conformation that represses
transcription of genes under the control of the araBAD promoter. In
the presence of arabinose, the AraC transcription factor undergoes
a conformational change that allows it to bind to and activate the
AraBAD promoter, which induces expression of the desired gene.
[0133] Thus, in some embodiments in which one or more heterologous
gene(s) are expressed upon sensing arabinose in the exogenous
environment, the one or more heterologous genes are directly or
indirectly under the control of the araBAD promoter. In some
embodiments, the expressed heterologous gene is selected from one
or more of the following: a heterologous therapeutic gene, a
heterologous gene encoding an antitoxin, a heterologous gene
encoding a repressor protein or polypeptide, for example, a TetR
repressor, a heterologous gene encoding an essential protein not
found in the bacterial cell, and/or a heterologous encoding a
regulatory protein or polypeptide.
[0134] Arabinose inducible promoters are known in the art,
including P.sub.ara, P.sub.araB, P.sub.araC, and P.sub.araBAD. In
one embodiment, the arabinose inducible promoter is from E. coli.
In some embodiments, the P.sub.araC promoter and the P.sub.araBAD
promoter operate as a bidirectional promoter, with the P.sub.araBAD
promoter controlling expression of a heterologous gene(s) in one
direction, and the P.sub.araC (in close proximity to, and on the
opposite strand from the P.sub.araBAD promoter), controlling
expression of a heterologous gene(s) in the other direction. In the
presence of arabinose, transcription of both heterologous genes
from both promoters is induced. However, in the absence of
arabinose, transcription of both heterologous genes from both
promoters is not induced.
[0135] In one exemplary embodiment of the disclosure, the
engineered bacteria of the present disclosure contains a
kill-switch having at least the following sequences: a P.sub.araBAD
promoter operably linked to a heterologous gene encoding a
Tetracycline Repressor Protein (TetR), a P.sub.araC promoter
operably linked to a heterologous gene encoding AraC transcription
factor, and a heterologous gene encoding a bacterial toxin operably
linked to a promoter which is repressed by the Tetracycline
Repressor Protein (P.sub.TetR). In the presence of arabinose, the
AraC transcription factor activates the P.sub.araBAD promoter,
which activates transcription of the TetR protein which, in turn,
represses transcription of the toxin. In the absence of arabinose,
however, AraC suppresses transcription from the P.sub.araBAD
promoter and no TetR protein is expressed. In this case, expression
of the heterologous toxin gene is activated, and the toxin is
expressed. The toxin builds up in the recombinant bacterial cell,
and the recombinant bacterial cell is killed. In one embodiment,
the AraC gene encoding the AraC transcription factor is under the
control of a constitutive promoter and is therefore constitutively
expressed.
[0136] In one embodiment of the disclosure, the recombinant
bacterial cell further comprises an antitoxin under the control of
a constitutive promoter. In this situation, in the presence of
arabinose, the toxin is not expressed due to repression by TetR
protein, and the antitoxin protein builds-up in the cell. However,
in the absence of arabinose, TetR protein is not expressed, and
expression of the toxin is induced. The toxin begins to build-up
within the recombinant bacterial cell. The recombinant bacterial
cell is no longer viable once the toxin protein is present at
either equal or greater amounts than that of the anti-toxin protein
in the cell, and the recombinant bacterial cell will be killed by
the toxin.
[0137] In another embodiment of the disclosure, the recombinant
bacterial cell further comprises an antitoxin under the control of
the P.sub.araBAD promoter. In this situation, in the presence of
arabinose, TetR and the anti-toxin are expressed, the anti-toxin
builds up in the cell, and the toxin is not expressed due to
repression by TetR protein. However, in the absence of arabinose,
both the TetR protein and the anti-toxin are not expressed, and
expression of the toxin is induced. The toxin begins to build-up
within the recombinant bacterial cell. The recombinant bacterial
cell is no longer viable once the toxin protein is expressed, and
the recombinant bacterial cell will be killed by the toxin.
[0138] In another exemplary embodiment of the disclosure, the
engineered bacteria of the present disclosure contains a
kill-switch having at least the following sequences: a P.sub.araBAD
promoter operably linked to a heterologous gene encoding an
essential polypeptide not found in the recombinant bacterial cell
(and required for survival), and a P.sub.araC promoter operably
linked to a heterologous gene encoding AraC transcription factor.
In the presence of arabinose, the AraC transcription factor
activates the P.sub.araBAD promoter, which activates transcription
of the heterologous gene encoding the essential polypeptide,
allowing the recombinant bacterial cell to survive. In the absence
of arabinose, however, AraC suppresses transcription from the
P.sub.araBAD promoter and the essential protein required for
survival is not expressed. In this case, the recombinant bacterial
cell dies in the absence of arabinose. In some embodiments, the
sequence of P.sub.araBAD promoter operably linked to a heterologous
gene encoding an essential polypeptide not found in the recombinant
bacterial cell can be present in the bacterial cell in conjunction
with the TetR/toxin kill-switch system described directly above. In
some embodiments, the sequence of P.sub.araBAD promoter operably
linked to a heterologous gene encoding an essential polypeptide not
found in the recombinant bacterial cell can be present in the
bacterial cell in conjunction with the TetR/toxin/anto-toxin
kill-switch system described directly above.
[0139] In any of the above-described embodiments, the bacterial
toxin is selected from the group consisting of a lysin, Hok, Fst,
TisB, LdrD, Kid, SymE, MazF, FlmA, lbs, XCV2162, dinJ, CcdB, MazF,
ParE, YafO, Zeta, hicB, relB, yhaV, yoeB, chpBK, hipA, microcin B,
microcin B17, microcin C, microcin C7-C51, microcin J25, microcin
ColV, microcin 24, microcin L, microcin D93, microcin L, microcin
E492, microcin H47, microcin I47, microcin M, colicin A, colicin
E1, colicin K, colicin N, colicin U, colicin B, colicin Ia, colicin
Ib, colicin 5, colicin10, colicin S4, colicin Y, colicin E2,
colicin E7, colicin E8, colicin E9, colicin E3, colicin E4, colicin
E6; colicin E5, colicin D, colicin M, and cloacin DF13, or a
biologically active fragment thereof.
[0140] In any of the above-described embodiments, the anti-toxin is
selected from the group consisting of an anti-lysin, Sok, RNAII,
lstR, RdlD, Kis, SymR, MazE, FlmB, Sib, ptaRNA1, yafQ, CcdA, MazE,
ParD, yafN, Epsilon, HicA, relE, prlF, yefM, chpBI, hipB, MccE,
MccE.sup.CTD, MccF, Cai, ImmE1, Cki, Cni, Cui, Cbi, Iia, Imm, Cfi,
Im10, Csi, Cyi, Im2, Im7, Im8, Im9, Im3, Im4, ImmE6, cloacin
immunity protein (Cim), ImmE5, ImmD, and Cmi, or a biologically
active fragment thereof.
[0141] In one embodiment, the bacterial toxin is bactericidal to
the genetically engineered bacterium. In one embodiment, the
bacterial toxin is bacteriostatic to the genetically engineered
bacterium.
[0142] Mutagenesis
[0143] In some embodiments, a RNS-responsive regulatory region is
operatively linked to a detectable product, e.g., GFP, and can be
used to screen for mutants. In some embodiments, the RNS-responsive
regulatory region is mutagenized, and mutants are selected based
upon the level of detectable product, e.g., by flow cytometry,
fluorescence-activated cell sorting (FACS) when the detectable
product fluoresces. In some embodiments, one or more transcription
factor binding sites (see, e.g., sequences in FIGS. 4 and 5) are
mutagenized to increase or decrease binding. In alternate
embodiments, the wild-type binding sites are left in tact and the
remainder of the regulatory region is subjected to mutagenesis. In
some embodiments, the mutant regulatory region is inserted into the
genetically engineered bacteria of the invention to increase
expression of the anti-inflammation and/or gut barrier enhancer
molecule in the presence of RNS, as compared to unmutated bacteria
of the same subtype under the same conditions. In some embodiments,
the RNS-sensing transcription factor and/or the RNS-responsive
regulatory region is a synthetic, non-naturally occurring
sequence.
[0144] In some embodiments, the gene encoding an anti-inflammation
and/or gut barrier enhancer molecule is mutated to increase
expression and/or stability of said molecule in the presence of
RNS, as compared to unmutated bacteria of the same subtype under
the same conditions. In some embodiments, one or more of the genes
in a gene cassette for producing an anti-inflammation and/or gut
barrier enhancer molecule is mutated to increase expression of said
molecule in the presence of RNS, as compared to unmutated bacteria
of the same subtype under the same conditions.
[0145] Methods of Reporting Disease
[0146] In addition to producing therapeutic molecules, the
genetically engineered bacteria of the invention may also be used
to report disease pathology, progression, and/or resolution. Kotula
et al. (2014) reported a binary toggle switch engineered into
Escherichia coli to be induced in the gut of a mouse and remain
stable in this configuration for at least seven days. These
bacteria were able to sense a molecular event that flipped a toggle
switch, which reported .beta.-galactosidase activity seven days
later in stool samples. In some embodiments, the genetically
engineered bacteria of the invention comprise a molecular switch,
e.g., a toggle switch, a cis/trans riboregulator, or an adjustable
threshold switch (Gardner et al., 2000; Callura et al., 2010;
Kobayashi et al., 2004), for detecting a disease marker associated
with gut inflammation and/or compromised gut barrier function,
e.g., RNS, inflammatory cytokines, wherein the molecular switch is
capable of producing a reporter, e.g., GFP, that is detected
externally, e.g., in a stool sample, by endoscopy, in peripheral
blood cells. In some embodiments, the reporter is expressed under
the control of a RNS-responsive regulatory region. This molecular
switch and reporter system may provide direct detection of
molecular events and changes in disease pathology. In some
embodiments, the molecular switch and reporter system is
incorporated into the same bacteria that are delivering the
anti-inflammation and/or gut barrier enhancer molecule. In
alternate embodiments, the molecular switch and reporter system is
incorporated into different bacteria than those delivering the
anti-inflammation and/or gut barrier enhancer molecule. In some
embodiments, methods using the molecular switch and reporter system
may be used to develop therapies, screen for disease, stratify
disease states, monitor disease progression, assess responsiveness
to treatment, and/or to tailor and direct therapies.
[0147] Pharmaceutical Compositions and Formulations
[0148] Pharmaceutical compositions comprising the genetically
engineered bacteria of the invention may be used to treat, manage,
ameliorate, and/or prevent a disorder associated with
hyperammonemia or symptom(s) associated with hyperammonemia.
Pharmaceutical compositions of the invention comprising one or more
genetically engineered bacteria, alone or in combination with
prophylactic agents, therapeutic agents, and/or pharmaceutically
acceptable carriers are provided.
[0149] In certain embodiments, the pharmaceutical composition
comprises one species, strain, or subtype of bacteria that are
engineered to comprise the genetic modifications described herein.
In alternate embodiments, the pharmaceutical composition comprises
two or more species, strains, and/or subtypes of bacteria that are
each engineered to comprise the genetic modifications described
herein.
[0150] The pharmaceutical compositions of the invention may be
formulated in a conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the active ingredients
into compositions for pharmaceutical use. Methods of formulating
pharmaceutical compositions are known in the art (see, e.g.,
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton,
Pa.). In some embodiments, the pharmaceutical compositions are
subjected to tabletting, lyophilizing, direct compression,
conventional mixing, dissolving, granulating, levigating,
emulsifying, encapsulating, entrapping, or spray drying to form
tablets, granulates, nanoparticles, nanocapsules, microcapsules,
microtablets, pellets, or powders, which may be enterically coated
or uncoated. Appropriate formulation depends on the route of
administration.
[0151] The genetically engineered bacteria of the invention may be
formulated into pharmaceutical compositions in any suitable dosage
form (e.g., liquids, capsules, sachet, hard capsules, soft
capsules, tablets, enteric coated tablets, suspension powders,
granules, or matrix sustained release formations for oral
administration) and for any suitable type of administration (e.g.,
oral, topical, immediate-release, pulsatile-release,
delayed-release, or sustained release). Suitable dosage amounts for
the genetically engineered bacteria may range from about 10.sup.5
to 10.sup.12 bacteria. The composition may be administered once or
more daily, weekly, or monthly. The genetically engineered bacteria
may be formulated into pharmaceutical compositions comprising one
or more pharmaceutically acceptable carriers, thickeners, diluents,
buffers, surface active agents, neutral or cationic lipids, lipid
complexes, liposomes, penetration enhancers, carrier compounds, and
other pharmaceutically acceptable carriers or agents.
[0152] The genetically engineered bacteria of the invention may be
administered topically and formulated in the form of an ointment,
cream, transdermal patch, lotion, gel, shampoo, spray, aerosol,
solution, emulsion, or other form well-known to one of skill in the
art. See, e.g., "Remington's Pharmaceutical Sciences," Mack
Publishing Co., Easton, Pa. In an embodiment, for non-sprayable
topical dosage forms, viscous to semi-solid or solid forms
comprising a carrier or one or more excipients compatible with
topical application and having a dynamic viscosity greater than
water are employed. Suitable formulations include, but are not
limited to, solutions, suspensions, emulsions, creams, ointments,
powders, liniments, salves, etc., which may be sterilized or mixed
with auxiliary agents (e.g., preservatives, stabilizers, wetting
agents, buffers, or salts) for influencing various properties,
e.g., osmotic pressure. Other suitable topical dosage forms include
sprayable aerosol preparations wherein the active ingredient in
combination with a solid or liquid inert carrier, is packaged in a
mixture with a pressurized volatile (e.g., a gaseous propellant,
such as freon) or in a squeeze bottle. Moisturizers or humectants
can also be added to pharmaceutical compositions and dosage forms.
Examples of such additional ingredients are well known in the
art.
[0153] The genetically engineered bacteria of the invention may be
administered orally and formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions, etc.
Pharmacological compositions for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients include, but are not limited to, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose
compositions such as maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP) or polyethylene glycol (PEG). Disintegrating agents may also
be added, such as cross-linked polyvinylpyrrolidone, agar, alginic
acid or a salt thereof such as sodium alginate.
[0154] Tablets or capsules can be prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone,
hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene
glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and
tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or
calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum,
zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate,
starch, sodium benzoate, L-leucine, magnesium stearate, talc, or
silica); disintegrants (e.g., starch, potato starch, sodium starch
glycolate, sugars, cellulose derivatives, silica powders); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. A coating shell may be
present, and common membranes include, but are not limited to,
polylactide, polyglycolic acid, polyanhydride, other biodegradable
polymers, alginate-polylysine-alginate (APA),
alginate-polymethylene-co-guanidine-alginate (A-PMCG-A),
hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered
HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC),
acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene
glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane
(PEG/PD5/PDMS), poly N,N-dimethyl acrylamide (PDMAAm), siliceous
encapsulates, cellulose sulphate/sodium
alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate
phthalate, calcium alginate, k-carrageenan-locust bean gum gel
beads, gellan-xanthan beads, poly(lactide-co-glycolides),
carrageenan, starch poly-anhydrides, starch polymethacrylates,
polyamino acids, and enteric coating polymers.
[0155] In some embodiments, the genetically engineered bacteria are
enterically coated for release into the gut or a particular region
of the gut, for example, the large intestine. The typical pH
profile from the stomach to the colon is about 1-4 (stomach), 5.5-6
(duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon). In some diseases,
the pH profile may be modified. In some embodiments, the coating is
degraded in specific pH environments in order to specify the site
of release. In some embodiments, at least two coatings are used. In
some embodiments, the outside coating and the inside coating are
degraded at different pH levels.
[0156] Liquid preparations for oral administration may take the
form of solutions, syrups, suspensions, or a dry product for
constitution with water or other suitable vehicle before use. Such
liquid preparations may be prepared by conventional means with
pharmaceutically acceptable agents such as suspending agents (e.g.,
sorbitol syrup, cellulose derivatives, or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol, or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring, and sweetening
agents as appropriate. Preparations for oral administration may be
suitably formulated for slow release, controlled release, or
sustained release of the genetically engineered bacteria of the
invention.
[0157] In certain embodiments, the genetically engineered bacteria
of the invention may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The compound may
also be enclosed in a hard or soft shell gelatin capsule,
compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the compounds
may be incorporated with excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. To administer a compound
of the invention by other than parenteral administration, it may be
necessary to coat the compound with, or co-administer the compound
with, a material to prevent its inactivation.
[0158] In some embodiments, the composition is formulated for
intraintestinal administration, intrajejunal administration,
intraduodenal administration, intraileal administration, gastric
shunt administration, or intracolic administration, via
nanoparticles, nanocapsules, microcapsules, or microtablets, which
are enterically coated or uncoated. The pharmaceutical compositions
of the present invention may also be formulated in rectal
compositions such as suppositories or retention enemas, using,
e.g., conventional suppository bases such as cocoa butter or other
glycerides. The compositions may be suspensions, solutions, or
emulsions in oily or aqueous vehicles, and may contain suspending,
stabilizing and/or dispersing agents.
[0159] The genetically engineered bacteria of the invention may be
administered intranasally, formulated in an aerosol form, spray,
mist, or in the form of drops, and conveniently delivered in the
form of an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
Pressurized aerosol dosage units may be determined by providing a
valve to deliver a metered amount. Capsules and cartridges (e.g.,
of gelatin) for use in an inhaler or insufflator may be formulated
containing a powder mix of the compound and a suitable powder base
such as lactose or starch.
[0160] The genetically engineered bacteria of the invention may be
administered and formulated as depot preparations. Such long acting
formulations may be administered by implantation or by injection.
For example, the compositions may be formulated with suitable
polymeric or hydrophobic materials (e.g., as an emulsion in an
acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives (e.g., as a sparingly soluble salt).
[0161] In some embodiments, the invention provides pharmaceutically
acceptable compositions in single dosage forms. Single dosage forms
may be in a liquid or a solid form. Single dosage forms may be
administered directly to a patient without modification or may be
diluted or reconstituted prior to administration. In certain
embodiments, a single dosage form may be administered in bolus
form, e.g., single injection, single oral dose, including an oral
dose that comprises multiple tablets, capsule, pills, etc. In
alternate embodiments, a single dosage form may be administered
over a period of time, e.g., by infusion.
[0162] Single dosage forms of the pharmaceutical composition of the
invention may be prepared by portioning the pharmaceutical
composition into smaller aliquots, single dose containers, single
dose liquid forms, or single dose solid forms, such as tablets,
granulates, nanoparticles, nanocapsules, microcapsules,
microtablets, pellets, or powders, which may be enterically coated
or uncoated. A single dose in a solid form may be reconstituted by
adding liquid, typically sterile water or saline solution, prior to
administration to a patient.
[0163] Dosage regimens may be adjusted to provide a therapeutic
response. For example, a single bolus may be administered at one
time, several divided doses may be administered over a
predetermined period of time, or the dose may be reduced or
increased as indicated by the therapeutic situation. The
specification for the dosage is dictated by the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved. Dosage values may vary with the
type and severity of the condition to be alleviated. For any
particular subject, specific dosage regimens may be adjusted over
time according to the individual need and the professional judgment
of the treating clinician.
[0164] In another embodiment, the composition can be delivered in a
controlled release or sustained release system. In one embodiment,
a pump may be used to achieve controlled or sustained release. In
another embodiment, polymeric materials can be used to achieve
controlled or sustained release of the therapies of the present
disclosure (see e.g., U.S. Pat. No. 5,989,463). Examples of
polymers used in sustained release formulations include, but are
not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl
methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The
polymer used in a sustained release formulation may be inert, free
of leachable impurities, stable on storage, sterile, and
biodegradable. In some embodiments, a controlled or sustained
release system can be placed in proximity of the prophylactic or
therapeutic target, thus requiring only a fraction of the systemic
dose. Any suitable technique known to one of skill in the art may
be used.
[0165] The genetically engineered bacteria of the invention may be
administered and formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions
such as those derived from hydrochloric, phosphoric, acetic,
oxalic, tartaric acids, etc., and those formed with cations such as
those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,
histidine, procaine, etc.
[0166] The ingredients are supplied either separately or mixed
together in unit dosage form, for example, as a dry lyophilized
powder or water-free concentrate in a hermetically sealed container
such as an ampoule or sachet indicating the quantity of active
agent. If the mode of administration is by injection, an ampoule of
sterile water for injection or saline can be provided so that the
ingredients may be mixed prior to administration.
[0167] The pharmaceutical compositions of the invention may be
packaged in a hermetically sealed container such as an ampoule or
sachet indicating the quantity of the agent. In one embodiment, one
or more of the pharmaceutical compositions of the invention is
supplied as a dry sterilized lyophilized powder or water-free
concentrate in a hermetically sealed container and can be
reconstituted (e.g., with water or saline) to the appropriate
concentration for administration to a subject. In an embodiment,
one or more of the prophylactic or therapeutic agents or
pharmaceutical compositions of the invention is supplied as a dry
sterile lyophilized powder in a hermetically sealed container
stored between 2.degree. C. and 8.degree. C. and administered
within 1 hour, within 3 hours, within 5 hours, within 6 hours,
within 12 hours, within 24 hours, within 48 hours, within 72 hours,
or within one week after being reconstituted. Cryoprotectants can
be included for a lyophilized dosage form, principally 0-10%
sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants
include trehalose and lactose. Other suitable bulking agents
include glycine and arginine, either of which can be included at a
concentration of 0-0.05%, and polysorbate-80 (optimally included at
a concentration of 0.005-0.01%). Additional surfactants include but
are not limited to polysorbate 20 and BRIJ surfactants. The
pharmaceutical composition may be prepared as an injectable
solution and can further comprise an agent useful as an adjuvant,
such as those used to increase absorption or dispersion, e.g.,
hyaluronidase.
[0168] Dosing can depend on several factors, including severity and
responsiveness of the disease, route of administration, time course
of treatment (days to months to years), and time to amelioration of
the disease. Toxicity and therapeutic efficacy of compounds
provided herein can be determined by standard pharmaceutical
procedures in cell culture or animal models. For example,
LD.sub.50, ED.sub.50, EC.sub.50, and IC.sub.50 may be determined,
and the dose ratio between toxic and therapeutic effects
(LD.sub.50/ED.sub.50) may be calculated as the therapeutic index.
Compositions that exhibit toxic side effects may be used, with
careful modifications to minimize potential damage to reduce side
effects. Dosing may be estimated initially from cell culture assays
and animal models. The data obtained from in vitro and in vivo
assays and animal studies can be used in formulating a range of
dosage for use in humans.
[0169] Methods of Treatment
[0170] Another aspect of the invention provides methods of treating
autoimmune disorders, diarrheal diseases, IBD, related diseases,
and other diseases that benefit from reduced gut inflammation
and/or enhanced gut barrier function. In some embodiments, the
disease or condition is selected from the group consisting of
Crohn's disease, ulcerative colitis, collagenous colitis,
lymphocytic colitis, diversion colitis, Behcet's disease,
intermediate colitis, short bowel syndrome, ulcerative proctitis,
proctosigmoiditis, left-sided colitis, pancolitis, and fulminant
colitis. In some embodiments, the disease or condition is an
autoimmune disorder selected from the group consisting of acute
disseminated encephalomyelitis (ADEM), acute necrotizing
hemorrhagic leukoencephalitis, Addison's disease,
agammaglobulinemia, alopecia areata, amyloidosis, ankylosing
spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome
(APS), autoimmune angioedema, autoimmune aplastic anemia,
autoimmune dysautonomia, autoimmune hemolytic anemia, autoimmune
hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency,
autoimmune inner ear disease (AIED), autoimmune myocarditis,
autoimmune oophoritis, autoimmune pancreatitis, autoimmune
retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune
thyroid disease, autoimmune urticarial, Axonal & neuronal
neuropathies, Balo disease, Behcet's disease, Bullous pemphigoid,
Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease,
Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic
recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome,
Cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease,
Cogan syndrome, Cold agglutinin disease, Congenital heart block,
Coxsackie myocarditis, CREST disease, Essential mixed
cryoglobulinemia, Demyelinating neuropathies, Dermatitis
herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis
optica), Discoid lupus, Dressler's syndrome, Endometriosis,
Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum,
Experimental allergic encephalomyelitis, Evans syndrome, Fibrosing
alveolitis, Giant cell arteritis (temporal arteritis), Giant cell
myocarditis, Glomerulonephritis, Goodpasture's syndrome,
Granulomatosis with Polyangiitis (GPA), Graves' disease,
Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's
thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes
gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic
purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease,
Immunoregulatory lipoproteins, Inclusion body myositis,
Interstitial cystitis, Juvenile arthritis, Juvenile idiopathic
arthritis, Juvenile myositis, Kawasaki syndrome, Lambert-Eaton
syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen
sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus
(Systemic Lupus Erythematosus), chronic Lyme disease, Meniere's
disease, Microscopic polyangiitis, Mixed connective tissue disease
(MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple
sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis
optica (Devic's), Neutropenia, Ocular cicatricial pemphigoid, Optic
neuritis, Palindromic rheumatism, PANDAS (Pediatric autoimmune
Neuropsychiatric Disorders Associated with Streptococcus),
Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal
hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner
syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral
neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS
syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune
polyglandular syndromes, Polymyalgia rheumatic, Polymyositis,
Postmyocardial infarction syndrome, Postpericardiotomy syndrome,
Progesterone dermatitis, Primary biliary cirrhosis, Primary
sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic
pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia,
Raynauds phenomenon, reactive arthritis, reflex sympathetic
dystrophy, Reiter's syndrome, relapsing polychondritis, restless
legs syndrome, retroperitoneal fibrosis, rheumatic fever,
rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis,
scleroderma, Sjogren's syndrome, sperm & testicular
autoimmunity, stiff person syndrome, subacute bacterial
endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia,
Takayasu's arteritis, temporal arteritis/giant cell arteritis,
thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse
myelitis, type 1 diabetes, asthma, ulcerative colitis,
undifferentiated connective tissue disease (UCTD), uveitis,
vasculitis, vesiculobullous dermatosis, vitiligo, and Wegener's
granulomatosis. In some embodiments, the invention provides methods
for reducing, ameliorating, or eliminating one or more symptom(s)
associated with these diseases, including but not limited to
diarrhea, bloody stool, mouth sores, perianal disease, abdominal
pain, abdominal cramping, fever, fatigue, weight loss, iron
deficiency, anemia, appetite loss, weight loss, anorexia, delayed
growth, delayed pubertal development, and inflammation of the skin,
eyes, joints, liver, and bile ducts. In some embodiments, the
invention provides methods for reducing gut inflammation and/or
enhancing gut barrier function, thereby ameliorating or preventing
a systemic autoimmune disorder, e.g., asthma (Arrieta et al.,
2015).
[0171] The method may comprise preparing a pharmaceutical
composition with at least one genetically engineered species,
strain, or subtype of bacteria described herein, and administering
the pharmaceutical composition to a subject in a therapeutically
effective amount. In some embodiments, the genetically engineered
bacteria of the invention are administered orally in a liquid
suspension. In some embodiments, the genetically engineered
bacteria of the invention are lyophilized in a gel cap and
administered orally. In some embodiments, the genetically
engineered bacteria of the invention are administered via a feeding
tube. In some embodiments, the genetically engineered bacteria of
the invention are administered rectally, e.g., by enema.
[0172] In certain embodiments, the pharmaceutical composition
described herein is administered to reduce gut inflammation,
enhance gut barrier function, and/or treat or prevent an autoimmune
disorder in a subject. In some embodiments, the methods of the
present disclosure may reduce gut inflammation in a subject by at
least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,
90%, 95%, or more as compared to levels in an untreated or control
subject. In some embodiments, the methods of the present disclosure
may enhance gut barrier function in a subject by at least about
10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or
more as compared to levels in an untreated or control subject. In
some embodiments, changes in inflammation and/or gut barrier
function are measured by comparing a subject before and after
administration of the pharmaceutical composition. In some
embodiments, the method of treating or ameliorating the autoimmune
disorder and/or the disease or condition associated with gut
inflammation and/or compromised gut barrier function allows one or
more symptoms of the disease or condition to improve by at least
about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or
more.
[0173] Before, during, and after the administration of the
pharmaceutical composition, gut inflammation and/or barrier
function in the subject may be measured in a biological sample,
such as blood, serum, plasma, urine, fecal matter, peritoneal
fluid, intestinal mucosal scrapings, a sample collected from a
tissue, and/or a sample collected from the contents of one or more
of the following: the stomach, duodenum, jejunum, ileum, cecum,
colon, rectum, and anal canal. In some embodiments, the methods may
include administration of the compositions of the invention to
enhance gut barrier function and/or to reduce gut inflammation to
baseline levels, e.g., levels comparable to those of a healthy
control, in a subject. In some embodiments, the methods may include
administration of the compositions of the invention to reduce gut
inflammation to undetectable levels in a subject, or to less than
about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or
80% of the subject's levels prior to treatment. In some
embodiments, the methods may include administration of the
compositions of the invention to enhance gut barrier function in a
subject by about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%,
70%, 75%, 80%, 90%, 100% or more of the subject's levels prior to
treatment.
[0174] In certain embodiments, the genetically engineered bacteria
are E. coli Nissle. The genetically engineered bacteria may be
destroyed, e.g., by defense factors in the gut or blood serum
(Sonnenborn et al., 2009) or by activation of a kill switch,
several hours or days after administration. Thus, the
pharmaceutical composition comprising the genetically engineered
bacteria may be re-administered at a therapeutically effective dose
and frequency. In alternate embodiments, the genetically engineered
bacteria are not destroyed within hours or days after
administration and may propagate and colonize the gut.
[0175] The pharmaceutical composition may be administered alone or
in combination with one or more additional therapeutic agents,
e.g., corticosteroids, aminosalicylates, anti-inflammatory agents.
An important consideration in the selection of the one or more
additional therapeutic agents is that the agent(s) should be
compatible with the genetically engineered bacteria of the
invention, e.g., the agent(s) must not kill the bacteria. The
dosage of the pharmaceutical composition and the frequency of
administration may be selected based on the severity of the
symptoms and the progression of the disorder. The appropriate
therapeutically effective dose and/or frequency of administration
can be selected by a treating clinician.
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EXAMPLES
[0243] The following examples provide illustrative embodiments of
the disclosure. One of ordinary skill in the art will recognize the
numerous modifications and variations that may be performed without
altering the spirit or scope of the disclosure. Such modifications
and variations are encompassed within the scope of the disclosure.
The Examples do not in any way limit the disclosure.
Example 1. Construction of Vectors for Overproducing Butyrate
[0244] To facilitate inducible production of butyrate in
Escherichia coli Nissle, the eight genes of the butyrate production
pathway from Peptoclostridium difficile 630 (bcd2, etfB3, etfA3,
thiA1, hbd, crt2, pbt, and buk; NCBI; SEQ ID NO: 3-10), as well as
transcriptional and translational elements, were synthesized (Gen9,
Cambridge, Mass.) and cloned into vector pBR322 to create
pLogic031. The butyrate gene cassette was placed under control of a
tetracycline-inducible promoter, with the tet repressor (TetR)
expressed constitutively on another portion of the plasmid. For
efficient translation of butyrate genes, each synthetic gene in the
operon was separated by a 15 base pair ribosome binding site
derived from the T7 promoter/translational start site.
[0245] The gene products of the bcd2-etfA3-etfB3 genes form a
complex that converts crotonyl-CoA to butyryl-CoA, and may show
some dependence on oxygen as a co-oxidant. Because the recombinant
bacteria of the invention are designed to produce butyrate in an
oxygen-limited environment (e.g. the mammalian gut), that
dependence on oxygen could have a negative effect of butyrate
production in the gut. It has been shown that a single gene from
Treponema denticola, trans-2-enoynl-CoA reductase (ter), can
functionally replace this three gene complex in an
oxygen-independent manner. Therefore, we created a second plasmid
capable of butyrate production in E. coli. Inverse PCR was used to
amplify the entire sequence of pLogic031 outside of the
bcd-etfA3-etfB3 region. The ter gene was codon optimized for E.
coli codon usage using Integrated DNA Technologies online codon
optimization tool (https://www.idtdna.com/CodonOpt), synthesized
(Genewiz, Cambridge, Mass.), and cloned into this inverse PCR
fragment using Gibson assembly to create pLogic046.
Example 2. Transforming E. coli with pLogic031 or pLogic046
[0246] The plasmid pLogic031 or pLogic046 was transformed into E.
coli Nissle or E. coli DH5a. All tubes, solutions, and cuvettes are
pre-chilled to 4.degree. C. An overnight culture of E. coli Nissle
or E. coli DH5a was diluted 1:100 in 5 mL of lysogeny broth (LB)
containing ampicillin and grown until it reached an OD.sub.600 of
0.4-0.6. The E. coli cells were then centrifuged at 2,000 rpm for 5
min. at 4.degree. C., the supernatant was removed, and the cells
were resuspended in 1 mL of 4.degree. C. water. The E. coli were
again centrifuged at 2,000 rpm for 5 min. at 4.degree. C., the
supernatant was removed, and the cells were resuspended in 0.5 mL
of 4.degree. C. water. The E. coli were again centrifuged at 2,000
rpm for 5 min. at 4.degree. C., the supernatant was removed, and
the cells were finally resuspended in 0.1 mL of 4.degree. C. water.
The electroporator was set to 2.5 kV. 0.5 .mu.g of one of the two
pLogic plasmids was added to the cells, mixed by pipetting, and
pipetted into a sterile, chilled cuvette. The dry cuvette was
placed into the sample chamber, and the electric pulse was applied.
One mL of room-temperature SOC media was immediately added, and the
mixture is transferred to a culture tube and incubated at
37.degree. C. for 1 hr. The cells were spread out on an LB plate
containing ampicillin and incubated overnight.
Example 3. Production of Butyrate in Recombinant E. coli
[0247] All incubations are performed at 37.degree. C. Cultures of
E. coli strains DH5a and Nissle transformed with either pLogic031
or pLogic046 are grown overnight in LB and then diluted 1:50 into 4
mL of M9 minimal medium containing 0.5% glucose. The cells are
grown with shaking (200 rpm) for 2 h, at which time anhydrous
tetracycline (ATC) is added to cultures at a concentration of 100
ng/mL to induce expression the butyrate operon from the plasmids.
Samples are collected at 2 h after addition of inducer for analysis
of butyrate concentration by LC-MS.
[0248] Production of butyrate is assessed in E. coli Nissle strains
containing pLogic031 and pLogic046 under microaerobic conditions in
order to determine the effect of oxygen on butyrate production from
these two plasmid variants. Overnight cultures are diluted 1:50 in
M9 media containing 0.5% glucose and grown shaking (200 rpm) for 2
hours, at which point ATC is added to cultures (100 ng/mL). One mL
culture aliquots are prepared in 1.5 mL capped tubes and incubated
in a stationary incubator to limit culture aeration. One tube is
removed at each time point (0, 1, 2, 4, and 20 hours) and analyzed
for butyrate concentration by LC-MS to confirm that butyrate
production in these recombinant strains can be achieved in a
low-oxygen environment.
Example 4. Construction of Vectors Encoding Butyrate Biosynthesis
Cassette Under Control of norB Regulatory Region
[0249] To create plasmids capable of nitric oxide-mediated
induction of the butyrate operon, inverse PCR, using appropriate
primers, is used to amplify the entire region of pLogic031 and
pLogic046 outside of the tetR gene and tet promoter region. The
nucleic acid sequence of pLogic031, comprising a tet promoter and
butyrate operon (SEQ ID NO: 12), is shown in FIG. 6. The sequence
encoding TetR is underlined, and the overlapping tetR/tetA
promoters are . The nucleic acid sequence of pLogic046, comprising
a tet promoter and butyrate operon (SEQ ID NO: 13), is shown in
FIG. 7. The sequence encoding TetR is underlined, and the
overlapping tetR/tetA promoters are . The nsrR gene and norB
regulatory region from Neisseria gonorrhoeae are PCR amplified with
overhangs homologous to the ends of these PCR fragments so that it
may be inserted/cloned by Gibson assembly into the pLogic031 or
pLogic046 derived PCR products. These constructs have tetracycline
control region (tetR and tet promoter) replaced by the nsrR gene
and norB regulatory region, which is induced by NsrR in the
presence of NO. These newly assembled constructs,
pLogic031-nsrR-norB and pLogic046-nsrR-norB, are used to transform
E. coli Nissle through electroporation as described above, and
transformants selected on ampicillin at 100 .mu.g/mL. Butyrate
production from pLogic031-nsrR-norB and pLogic046-nsrR-norB in
vitro is achieved by the addition of nitric oxide to cultures at
100 .mu.M. Butyrate levels in in vitro cultures are quantitated in
culture supernatants by LC-MS as described above for the parent
plasmids, pLogic031 and pLogic046.
Example 5. Construction of Vectors Encoding NsrR
[0250] Alternatively, nsrR may be expressed in a different plasmid
than the one comprising the norB regulatory region and operatively
linked gene(s) or gene cassette(s). The nsrR gene from Neisseria
gonorrhoeae, which encodes the nitric oxide-sensitive protein
responsible for driving expression from the norB regulatory region,
is overexpressed from a medium copy plasmid in order to provide
NsrR protein necessary for expression of the butyrate cassettes
from pLogic031-norB or pLogic046-norB. The nsrR gene is cloned into
the medium copy number plasmid under control of a constitutive
promoter, such as Plac, to create a plasmid that produces NsrR in a
bacterium transformed therewith. This plasmid, which also bears an
antibiotic resistance gene, such as kanamycin resistance, is used
to transform E. coli Nissle already harboring pLogic031-norB or
pLogic046-norB through electroporation. Transformants are selected
in the presence of the antibiotic whose resistance is encoded in
the plasmid (e.g., kanamycin at 50 .mu.g/mL) to select for those
carrying the NsrR plasmid and ampicillin at 100 .mu.g/mL to
maintain the plasmid already present (pLogic031-norB or
pLogic046-norB). The resulting strains carry two plasmids, one
containing the butyrate cassette, and the other containing the NsrR
to control said cassette's induction. Butyrate production from
these strains in vitro is achieved by the addition of nitric oxide
to cultures at 100 .mu.M. Butyrate levels in culture supernatants
are measured by LC-MS.
Example 6. Efficacy of Butyrate-Expressing Bacteria in a Mouse
Model of IBD
[0251] Bacteria harboring both a plasmid expressing NsrR under
control of a constitutive promoter and either pLogic031-nsrR-norB
or pLogic046-nsrR-norB are grown overnight in LB supplemented with
ampicillin. Bacteria are then diluted 1:100 into LB containing
ampicillin and grown to an optical density of 0.4-0.5 and then
pelleted by centrifugation. Bacteria are resuspended in phosphate
buffered saline and 100 microliters is administered by oral gavage
to mice. IBD is induced in mice by supplementing drinking water
with 3% dextran sodium sulfate for 7 days prior to bacterial
gavage. Mice are treated daily for 1 week and bacteria in stool
samples are detected by plating stool homogenate on agar plates
supplemented with ampicillin. After 5 days of bacterial treatment,
colitis is scored in live mice using endoscopy. Endoscopic damage
score is determined by assessing colon translucency, fibrin
attachment, mucosal and vascular pathology, and/or stool
characteristics. Mice are sacrificed and colonic tissues are
isolated. Distal colonic sections are fixed and scored for
inflammation and ulceration. Colonic tissue is homogenized and
measurements are made for myeloperoxidase activity using an
enzymatic assay kit and for cytokine levels (IL-1.beta.,
TNF-.alpha., IL-6, IFN-.gamma. and IL-10).
Example 7. Nitric Oxide-Inducible Reporter Constructs
[0252] ATC and nitric oxide-inducible reporter constructs were
synthesized (Genewiz, Cambridge, Mass.). When induced by their
cognate inducers, these constructs express GFP, which is detected
by monitoring fluorescence in a plate reader at an
excitation/emission of 395/509 nm, respectively. Nissle cells
harboring plasmids with either the control, ATC-inducible Ptet-GFP
reporter construct, or the nitric oxide inducible PnsrR-GFP
reporter construct were first grown to early log phase (OD600 of
about 0.4-0.6), at which point they were transferred to 96-well
microtiter plates containing LB and two-fold decreased inducer (ATC
or the long half-life NO donor, DETA-NO (Sigma)). Both ATC and NO
were able to induce the expression of GFP in their respective
constructs across a range of concentrations (FIGS. 12A-C); promoter
activity is expressed as relative florescence units. An exemplary
sequence of a nitric oxide-inducible reporter construct is shown in
FIG. 13. The bsrR sequence is bolded. The gfp sequence is
underlined. The PnsrR (NO regulated promoter and RBS) is
italicized. The constitutive promoter and RBS are .
Example 8. Nitric Oxide-Inducible Reporter Constructs in Mouse
Model of IBD
[0253] Bacteria harboring a plasmid expressing NsrR under control
of a constitutive promoter and the reporter gene gfp (green
fluorescent protein) under control of an NsrR-inducible promoter
were grown overnight in LB supplemented with kanamycin. Bacteria
are then diluted 1:100 into LB containing kanamycin and grown to an
optical density of about 0.4-0.5 and then pelleted by
centrifugation. Bacteria are resuspended in phosphate buffered
saline and 100 microliters were administered by oral gavage to
mice. IBD is induced in mice by supplementing drinking water with
2-3% dextran sodium sulfate for 7 days prior to bacterial gavage.
At 4 hours post-gavage, mice were sacrificed and bacteria were
recovered from colonic samples. Colonic contents were boiled in
SDS, and the soluble fractions were used to perform a dot blot for
GFP detection (induction of NsrR-regulated promoters) (FIG. 14).
Detection of GFP was performed by binding of anti-GFP antibody
conjugated to to HRP (horse radish peroxidase). Detection was
visualized using Pierce chemiluminescent detection kit. FIG. 14
shows NsrR-regulated promoters are induced in DSS-treated mice, but
not in untreated mice.
Sequence CWU 1
1
1618575DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 1ttattatcgc accgcaatcg ggattttcga
ttcataaagc aggtcgtagg tcggcttgtt 60gagcaggtct tgcagcgtga aaccgtccag
atacgtgaaa aacgacttca ttgcaccgcc 120gagtatgccc gtcagccggc
aggacggcgt aatcaggcat tcgttgttcg ggcccataca 180ctcgaccagc
tgcatcggtt cgaggtggcg gacgaccgcg ccgatattga tgcgttcggg
240cggcgcggcc agcctcagcc cgccgccttt cccgcgtacg ctgtgcaaga
acccgccttt 300gaccagcgcg gtaaccactt tcatcaaatg gcttttggaa
atgccgtagg tcgaggcgat 360ggtggcgata ttgaccagcg cgtcgtcgtt
gacggcggtg tagatgagga cgcgcagccc 420gtagtcggta tgttgggtca
gatacataca acctccttag tacatgcaaa attatttcta 480gagcaacata
cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagttgagtt
540gaggaattat aacaggaaga aatattcctc atacgcttgt aattcctcta
tggttgttga 600caattaatca tcggctcgta taatgtataa cattcatatt
ttgtgaattt taaactctag 660aaataatttt gtttaacttt aagaaggaga
tatacatatg gatttaaatt ctaaaaaata 720tcagatgctt aaagagctat
atgtaagctt cgctgaaaat gaagttaaac ctttagcaac 780agaacttgat
gaagaagaaa gatttcctta tgaaacagtg gaaaaaatgg caaaagcagg
840aatgatgggt ataccatatc caaaagaata tggtggagaa ggtggagaca
ctgtaggata 900tataatggca gttgaagaat tgtctagagt ttgtggtact
acaggagtta tattatcagc 960tcatacatct cttggctcat ggcctatata
tcaatatggt aatgaagaac aaaaacaaaa 1020attcttaaga ccactagcaa
gtggagaaaa attaggagca tttggtctta ctgagcctaa 1080tgctggtaca
gatgcgtctg gccaacaaac aactgctgtt ttagacgggg atgaatacat
1140acttaatggc tcaaaaatat ttataacaaa cgcaatagct ggtgacatat
atgtagtaat 1200ggcaatgact gataaatcta aggggaacaa aggaatatca
gcatttatag ttgaaaaagg 1260aactcctggg tttagctttg gagttaaaga
aaagaaaatg ggtataagag gttcagctac 1320gagtgaatta atatttgagg
attgcagaat acctaaagaa aatttacttg gaaaagaagg 1380tcaaggattt
aagatagcaa tgtctactct tgatggtggt agaattggta tagctgcaca
1440agctttaggt ttagcacaag gtgctcttga tgaaactgtt aaatatgtaa
aagaaagagt 1500acaatttggt agaccattat caaaattcca aaatacacaa
ttccaattag ctgatatgga 1560agttaaggta caagcggcta gacaccttgt
atatcaagca gctataaata aagacttagg 1620aaaaccttat ggagtagaag
cagcaatggc aaaattattt gcagctgaaa cagctatgga 1680agttactaca
aaagctgtac aacttcatgg aggatatgga tacactcgtg actatccagt
1740agaaagaatg atgagagatg ctaagataac tgaaatatat gaaggaacta
gtgaagttca 1800aagaatggtt atttcaggaa aactattaaa atagtaagaa
ggagatatac atatggagga 1860aggatttatg aatatagtcg tttgtataaa
acaagttcca gatacaacag aagttaaact 1920agatcctaat acaggtactt
taattagaga tggagtacca agtataataa accctgatga 1980taaagcaggt
ttagaagaag ctataaaatt aaaagaagaa atgggtgctc atgtaactgt
2040tataacaatg ggacctcctc aagcagatat ggctttaaaa gaagctttag
caatgggtgc 2100agatagaggt atattattaa cagatagagc atttgcgggt
gctgatactt gggcaacttc 2160atcagcatta gcaggagcat taaaaaatat
agattttgat attataatag ctggaagaca 2220ggcgatagat ggagatactg
cacaagttgg acctcaaata gctgaacatt taaatcttcc 2280atcaataaca
tatgctgaag aaataaaaac tgaaggtgaa tatgtattag taaaaagaca
2340atttgaagat tgttgccatg acttaaaagt taaaatgcca tgccttataa
caactcttaa 2400agatatgaac acaccaagat acatgaaagt tggaagaata
tatgatgctt tcgaaaatga 2460tgtagtagaa acatggactg taaaagatat
agaagttgac ccttctaatt taggtcttaa 2520aggttctcca actagtgtat
ttaaatcatt tacaaaatca gttaaaccag ctggtacaat 2580atacaatgaa
gatgcgaaaa catcagctgg aattatcata gataaattaa aagagaagta
2640tatcatataa taagaaggag atatacatat gggtaacgtt ttagtagtaa
tagaacaaag 2700agaaaatgta attcaaactg tttctttaga attactagga
aaggctacag aaatagcaaa 2760agattatgat acaaaagttt ctgcattact
tttaggtagt aaggtagaag gtttaataga 2820tacattagca cactatggtg
cagatgaggt aatagtagta gatgatgaag ctttagcagt 2880gtatacaact
gaaccatata caaaagcagc ttatgaagca ataaaagcag ctgaccctat
2940agttgtatta tttggtgcaa cttcaatagg tagagattta gcgcctagag
tttctgctag 3000aatacataca ggtcttactg ctgactgtac aggtcttgca
gtagctgaag atacaaaatt 3060attattaatg acaagacctg cctttggtgg
aaatataatg gcaacaatag tttgtaaaga 3120tttcagacct caaatgtcta
cagttagacc aggggttatg aagaaaaatg aacctgatga 3180aactaaagaa
gctgtaatta accgtttcaa ggtagaattt aatgatgctg ataaattagt
3240tcaagttgta caagtaataa aagaagctaa aaaacaagtt aaaatagaag
atgctaagat 3300attagtttct gctggacgtg gaatgggtgg aaaagaaaac
ttagacatac tttatgaatt 3360agctgaaatt ataggtggag aagtttctgg
ttctcgtgcc actatagatg caggttggtt 3420agataaagca agacaagttg
gtcaaactgg taaaactgta agaccagacc tttatatagc 3480atgtggtata
tctggagcaa tacaacatat agctggtatg gaagatgctg agtttatagt
3540tgctataaat aaaaatccag aagctccaat atttaaatat gctgatgttg
gtatagttgg 3600agatgttcat aaagtgcttc cagaacttat cagtcagtta
agtgttgcaa aagaaaaagg 3660tgaagtttta gctaactaat aagaaggaga
tatacatatg agagaagtag taattgccag 3720tgcagctaga acagcagtag
gaagttttgg aggagcattt aaatcagttt cagcggtaga 3780gttaggggta
acagcagcta aagaagctat aaaaagagct aacataactc cagatatgat
3840agatgaatct cttttagggg gagtacttac agcaggtctt ggacaaaata
tagcaagaca 3900aatagcatta ggagcaggaa taccagtaga aaaaccagct
atgactataa atatagtttg 3960tggttctgga ttaagatctg tttcaatggc
atctcaactt atagcattag gtgatgctga 4020tataatgtta gttggtggag
ctgaaaacat gagtatgtct ccttatttag taccaagtgc 4080gagatatggt
gcaagaatgg gtgatgctgc ttttgttgat tcaatgataa aagatggatt
4140atcagacata tttaataact atcacatggg tattactgct gaaaacatag
cagagcaatg 4200gaatataact agagaagaac aagatgaatt agctcttgca
agtcaaaata aagctgaaaa 4260agctcaagct gaaggaaaat ttgatgaaga
aatagttcct gttgttataa aaggaagaaa 4320aggtgacact gtagtagata
aagatgaata tattaagcct ggcactacaa tggagaaact 4380tgctaagtta
agacctgcat ttaaaaaaga tggaacagtt actgctggta atgcatcagg
4440aataaatgat ggtgctgcta tgttagtagt aatggctaaa gaaaaagctg
aagaactagg 4500aatagagcct cttgcaacta tagtttctta tggaacagct
ggtgttgacc ctaaaataat 4560gggatatgga ccagttccag caactaaaaa
agctttagaa gctgctaata tgactattga 4620agatatagat ttagttgaag
ctaatgaggc atttgctgcc caatctgtag ctgtaataag 4680agacttaaat
atagatatga ataaagttaa tgttaatggt ggagcaatag ctataggaca
4740tccaatagga tgctcaggag caagaatact tactacactt ttatatgaaa
tgaagagaag 4800agatgctaaa actggtcttg ctacactttg tataggcggt
ggaatgggaa ctactttaat 4860agttaagaga tagtaagaag gagatataca
tatgaaatta gctgtaatag gtagtggaac 4920tatgggaagt ggtattgtac
aaacttttgc aagttgtgga catgatgtat gtttaaagag 4980tagaactcaa
ggtgctatag ataaatgttt agctttatta gataaaaatt taactaagtt
5040agttactaag ggaaaaatgg atgaagctac aaaagcagaa atattaagtc
atgttagttc 5100aactactaat tatgaagatt taaaagatat ggatttaata
atagaagcat ctgtagaaga 5160catgaatata aagaaagatg ttttcaagtt
actagatgaa ttatgtaaag aagatactat 5220cttggcaaca aatacttcat
cattatctat aacagaaata gcttcttcta ctaagcgccc 5280agataaagtt
ataggaatgc atttctttaa tccagttcct atgatgaaat tagttgaagt
5340tataagtggt cagttaacat caaaagttac ttttgataca gtatttgaat
tatctaagag 5400tatcaataaa gtaccagtag atgtatctga atctcctgga
tttgtagtaa atagaatact 5460tatacctatg ataaatgaag ctgttggtat
atatgcagat ggtgttgcaa gtaaagaaga 5520aatagatgaa gctatgaaat
taggagcaaa ccatccaatg ggaccactag cattaggtga 5580tttaatcgga
ttagatgttg ttttagctat aatgaacgtt ttatatactg aatttggaga
5640tactaaatat agacctcatc cacttttagc taaaatggtt agagctaatc
aattaggaag 5700aaaaactaag ataggattct atgattataa taaataataa
gaaggagata tacatatgag 5760tacaagtgat gttaaagttt atgagaatgt
agctgttgaa gtagatggaa atatatgtac 5820agtgaaaatg aatagaccta
aagcccttaa tgcaataaat tcaaagactt tagaagaact 5880ttatgaagta
tttgtagata ttaataatga tgaaactatt gatgttgtaa tattgacagg
5940ggaaggaaag gcatttgtag ctggagcaga tattgcatac atgaaagatt
tagatgctgt 6000agctgctaaa gattttagta tcttaggagc aaaagctttt
ggagaaatag aaaatagtaa 6060aaaagtagtg atagctgctg taaacggatt
tgctttaggt ggaggatgtg aacttgcaat 6120ggcatgtgat ataagaattg
catctgctaa agctaaattt ggtcagccag aagtaactct 6180tggaataact
ccaggatatg gaggaactca aaggcttaca agattggttg gaatggcaaa
6240agcaaaagaa ttaatcttta caggtcaagt tataaaagct gatgaagctg
aaaaaatagg 6300gctagtaaat agagtcgttg agccagacat tttaatagaa
gaagttgaga aattagctaa 6360gataatagct aaaaatgctc agcttgcagt
tagatactct aaagaagcaa tacaacttgg 6420tgctcaaact gatataaata
ctggaataga tatagaatct aatttatttg gtctttgttt 6480ttcaactaaa
gaccaaaaag aaggaatgtc agctttcgtt gaaaagagag aagctaactt
6540tataaaaggg taataagaag gagatataca tatgagaagt tttgaagaag
taattaagtt 6600tgcaaaagaa agaggaccta aaactatatc agtagcatgt
tgccaagata aagaagtttt 6660aatggcagtt gaaatggcta gaaaagaaaa
aatagcaaat gccattttag taggagatat 6720agaaaagact aaagaaattg
caaaaagcat agacatggat atcgaaaatt atgaactgat 6780agatataaaa
gatttagcag aagcatctct aaaatctgtt gaattagttt cacaaggaaa
6840agccgacatg gtaatgaaag gcttagtaga cacatcaata atactaaaag
cagttttaaa 6900taaagaagta ggtcttagaa ctggaaatgt attaagtcac
gtagcagtat ttgatgtaga 6960gggatatgat agattatttt tcgtaactga
cgcagctatg aacttagctc ctgatacaaa 7020tactaaaaag caaatcatag
aaaatgcttg cacagtagca cattcattag atataagtga 7080accaaaagtt
gctgcaatat gcgcaaaaga aaaagtaaat ccaaaaatga aagatacagt
7140tgaagctaaa gaactagaag aaatgtatga aagaggagaa atcaaaggtt
gtatggttgg 7200tgggcctttt gcaattgata atgcagtatc tttagaagca
gctaaacata aaggtataaa 7260tcatcctgta gcaggacgag ctgatatatt
attagcccca gatattgaag gtggtaacat 7320attatataaa gctttggtat
tcttctcaaa atcaaaaaat gcaggagtta tagttggggc 7380taaagcacca
ataatattaa cttctagagc agacagtgaa gaaactaaac taaactcaat
7440agctttaggt gttttaatgg cagcaaaggc ataataagaa ggagatatac
atatgagcaa 7500aatatttaaa atcttaacaa taaatcctgg ttcgacatca
actaaaatag ctgtatttga 7560taatgaggat ttagtatttg aaaaaacttt
aagacattct tcagaagaaa taggaaaata 7620tgagaaggtg tctgaccaat
ttgaatttcg taaacaagta atagaagaag ctctaaaaga 7680aggtggagta
aaaacatctg aattagatgc tgtagtaggt agaggaggac ttcttaaacc
7740tataaaaggt ggtacttatt cagtaagtgc tgctatgatt gaagatttaa
aagtgggagt 7800tttaggagaa cacgcttcaa acctaggtgg aataatagca
aaacaaatag gtgaagaagt 7860aaatgttcct tcatacatag tagaccctgt
tgttgtagat gaattagaag atgttgctag 7920aatttctggt atgcctgaaa
taagtagagc aagtgtagta catgctttaa atcaaaaggc 7980aatagcaaga
agatatgcta gagaaataaa caagaaatat gaagatataa atcttatagt
8040tgcacacatg ggtggaggag tttctgttgg agctcataaa aatggtaaaa
tagtagatgt 8100tgcaaacgca ttagatggag aaggaccttt ctctccagaa
agaagtggtg gactaccagt 8160aggtgcatta gtaaaaatgt gctttagtgg
aaaatatact caagatgaaa ttaaaaagaa 8220aataaaaggt aatggcggac
tagttgcata cttaaacact aatgatgcta gagaagttga 8280agaaagaatt
gaagctggtg atgaaaaagc taaattagta tatgaagcta tggcatatca
8340aatctctaaa gaaataggag ctagtgctgc agttcttaag ggagatgtaa
aagcaatatt 8400attaactggt ggaatcgcat attcaaaaat gtttacagaa
atgattgcag atagagttaa 8460atttatagca gatgtaaaag tttatccagg
tgaagatgaa atgattgcat tagctcaagg 8520tggacttaga gttttaactg
gtgaagaaga ggctcaagtt tatgataact aataa 857526787DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
2ttattatcgc accgcaatcg ggattttcga ttcataaagc aggtcgtagg tcggcttgtt
60gagcaggtct tgcagcgtga aaccgtccag atacgtgaaa aacgacttca ttgcaccgcc
120gagtatgccc gtcagccggc aggacggcgt aatcaggcat tcgttgttcg
ggcccataca 180ctcgaccagc tgcatcggtt cgaggtggcg gacgaccgcg
ccgatattga tgcgttcggg 240cggcgcggcc agcctcagcc cgccgccttt
cccgcgtacg ctgtgcaaga acccgccttt 300gaccagcgcg gtaaccactt
tcatcaaatg gcttttggaa atgccgtagg tcgaggcgat 360ggtggcgata
ttgaccagcg cgtcgtcgtt gacggcggtg tagatgagga cgcgcagccc
420gtagtcggta tgttgggtca gatacataca acctccttag tacatgcaaa
attatttcta 480gagcaacata cgagccggaa gcataaagtg taaagcctgg
ggtgcctaat gagttgagtt 540gaggaattat aacaggaaga aatattcctc
atacgcttgt aattcctcta tggttgttga 600caattaatca tcggctcgta
taatgtataa cattcatatt ttgtgaattt taaactctag 660aaataatttt
gtttaacttt aagaaggaga tatacatatg atcgtaaaac ctatggtacg
720caacaatatc tgcctgaacg cccatcctca gggctgcaag aagggagtgg
aagatcagat 780tgaatatacc aagaaacgca ttaccgcaga agtcaaagct
ggcgcaaaag ctccaaaaaa 840cgttctggtg cttggctgct caaatggtta
cggcctggcg agccgcatta ctgctgcgtt 900cggatacggg gctgcgacca
tcggcgtgtc ctttgaaaaa gcgggttcag aaaccaaata 960tggtacaccg
ggatggtaca ataatttggc atttgatgaa gcggcaaaac gcgagggtct
1020ttatagcgtg acgatcgacg gcgatgcgtt ttcagacgag atcaaggccc
aggtaattga 1080ggaagccaaa aaaaaaggta tcaaatttga tctgatcgta
tacagcttgg ccagcccagt 1140acgtactgat cctgatacag gtatcatgca
caaaagcgtt ttgaaaccct ttggaaaaac 1200gttcacaggc aaaacagtag
atccgtttac tggcgagctg aaggaaatct ccgcggaacc 1260agcaaatgac
gaggaagcag ccgccactgt taaagttatg gggggtgaag attgggaacg
1320ttggattaag cagctgtcga aggaaggcct cttagaagaa ggctgtatta
ccttggccta 1380tagttatatt ggccctgaag ctacccaagc tttgtaccgt
aaaggcacaa tcggcaaggc 1440caaagaacac ctggaggcca cagcacaccg
tctcaacaaa gagaacccgt caatccgtgc 1500cttcgtgagc gtgaataaag
gcctggtaac ccgcgcaagc gccgtaatcc cggtaatccc 1560tctgtatctc
gccagcttgt tcaaagtaat gaaagagaag ggcaatcatg aaggttgtat
1620tgaacagatc acgcgtctgt acgccgagcg cctgtaccgt aaagatggta
caattccagt 1680tgatgaggaa aatcgcattc gcattgatga ttgggagtta
gaagaagacg tccagaaagc 1740ggtatccgcg ttgatggaga aagtcacggg
tgaaaacgca gaatctctca ctgacttagc 1800ggggtaccgc catgatttct
tagctagtaa cggctttgat gtagaaggta ttaattatga 1860agcggaagtt
gaacgcttcg accgtatctg ataagaagga gatatacata tgagagaagt
1920agtaattgcc agtgcagcta gaacagcagt aggaagtttt ggaggagcat
ttaaatcagt 1980ttcagcggta gagttagggg taacagcagc taaagaagct
ataaaaagag ctaacataac 2040tccagatatg atagatgaat ctcttttagg
gggagtactt acagcaggtc ttggacaaaa 2100tatagcaaga caaatagcat
taggagcagg aataccagta gaaaaaccag ctatgactat 2160aaatatagtt
tgtggttctg gattaagatc tgtttcaatg gcatctcaac ttatagcatt
2220aggtgatgct gatataatgt tagttggtgg agctgaaaac atgagtatgt
ctccttattt 2280agtaccaagt gcgagatatg gtgcaagaat gggtgatgct
gcttttgttg attcaatgat 2340aaaagatgga ttatcagaca tatttaataa
ctatcacatg ggtattactg ctgaaaacat 2400agcagagcaa tggaatataa
ctagagaaga acaagatgaa ttagctcttg caagtcaaaa 2460taaagctgaa
aaagctcaag ctgaaggaaa atttgatgaa gaaatagttc ctgttgttat
2520aaaaggaaga aaaggtgaca ctgtagtaga taaagatgaa tatattaagc
ctggcactac 2580aatggagaaa cttgctaagt taagacctgc atttaaaaaa
gatggaacag ttactgctgg 2640taatgcatca ggaataaatg atggtgctgc
tatgttagta gtaatggcta aagaaaaagc 2700tgaagaacta ggaatagagc
ctcttgcaac tatagtttct tatggaacag ctggtgttga 2760ccctaaaata
atgggatatg gaccagttcc agcaactaaa aaagctttag aagctgctaa
2820tatgactatt gaagatatag atttagttga agctaatgag gcatttgctg
cccaatctgt 2880agctgtaata agagacttaa atatagatat gaataaagtt
aatgttaatg gtggagcaat 2940agctatagga catccaatag gatgctcagg
agcaagaata cttactacac ttttatatga 3000aatgaagaga agagatgcta
aaactggtct tgctacactt tgtataggcg gtggaatggg 3060aactacttta
atagttaaga gatagtaaga aggagatata catatgaaat tagctgtaat
3120aggtagtgga actatgggaa gtggtattgt acaaactttt gcaagttgtg
gacatgatgt 3180atgtttaaag agtagaactc aaggtgctat agataaatgt
ttagctttat tagataaaaa 3240tttaactaag ttagttacta agggaaaaat
ggatgaagct acaaaagcag aaatattaag 3300tcatgttagt tcaactacta
attatgaaga tttaaaagat atggatttaa taatagaagc 3360atctgtagaa
gacatgaata taaagaaaga tgttttcaag ttactagatg aattatgtaa
3420agaagatact atcttggcaa caaatacttc atcattatct ataacagaaa
tagcttcttc 3480tactaagcgc ccagataaag ttataggaat gcatttcttt
aatccagttc ctatgatgaa 3540attagttgaa gttataagtg gtcagttaac
atcaaaagtt acttttgata cagtatttga 3600attatctaag agtatcaata
aagtaccagt agatgtatct gaatctcctg gatttgtagt 3660aaatagaata
cttataccta tgataaatga agctgttggt atatatgcag atggtgttgc
3720aagtaaagaa gaaatagatg aagctatgaa attaggagca aaccatccaa
tgggaccact 3780agcattaggt gatttaatcg gattagatgt tgttttagct
ataatgaacg ttttatatac 3840tgaatttgga gatactaaat atagacctca
tccactttta gctaaaatgg ttagagctaa 3900tcaattagga agaaaaacta
agataggatt ctatgattat aataaataat aagaaggaga 3960tatacatatg
agtacaagtg atgttaaagt ttatgagaat gtagctgttg aagtagatgg
4020aaatatatgt acagtgaaaa tgaatagacc taaagccctt aatgcaataa
attcaaagac 4080tttagaagaa ctttatgaag tatttgtaga tattaataat
gatgaaacta ttgatgttgt 4140aatattgaca ggggaaggaa aggcatttgt
agctggagca gatattgcat acatgaaaga 4200tttagatgct gtagctgcta
aagattttag tatcttagga gcaaaagctt ttggagaaat 4260agaaaatagt
aaaaaagtag tgatagctgc tgtaaacgga tttgctttag gtggaggatg
4320tgaacttgca atggcatgtg atataagaat tgcatctgct aaagctaaat
ttggtcagcc 4380agaagtaact cttggaataa ctccaggata tggaggaact
caaaggctta caagattggt 4440tggaatggca aaagcaaaag aattaatctt
tacaggtcaa gttataaaag ctgatgaagc 4500tgaaaaaata gggctagtaa
atagagtcgt tgagccagac attttaatag aagaagttga 4560gaaattagct
aagataatag ctaaaaatgc tcagcttgca gttagatact ctaaagaagc
4620aatacaactt ggtgctcaaa ctgatataaa tactggaata gatatagaat
ctaatttatt 4680tggtctttgt ttttcaacta aagaccaaaa agaaggaatg
tcagctttcg ttgaaaagag 4740agaagctaac tttataaaag ggtaataaga
aggagatata catatgagaa gttttgaaga 4800agtaattaag tttgcaaaag
aaagaggacc taaaactata tcagtagcat gttgccaaga 4860taaagaagtt
ttaatggcag ttgaaatggc tagaaaagaa aaaatagcaa atgccatttt
4920agtaggagat atagaaaaga ctaaagaaat tgcaaaaagc atagacatgg
atatcgaaaa 4980ttatgaactg atagatataa aagatttagc agaagcatct
ctaaaatctg ttgaattagt 5040ttcacaagga aaagccgaca tggtaatgaa
aggcttagta gacacatcaa taatactaaa 5100agcagtttta aataaagaag
taggtcttag aactggaaat gtattaagtc acgtagcagt 5160atttgatgta
gagggatatg atagattatt tttcgtaact gacgcagcta tgaacttagc
5220tcctgataca aatactaaaa agcaaatcat agaaaatgct tgcacagtag
cacattcatt 5280agatataagt gaaccaaaag ttgctgcaat atgcgcaaaa
gaaaaagtaa atccaaaaat 5340gaaagataca gttgaagcta aagaactaga
agaaatgtat gaaagaggag aaatcaaagg 5400ttgtatggtt ggtgggcctt
ttgcaattga taatgcagta tctttagaag cagctaaaca 5460taaaggtata
aatcatcctg tagcaggacg agctgatata ttattagccc cagatattga
5520aggtggtaac atattatata aagctttggt attcttctca aaatcaaaaa
atgcaggagt 5580tatagttggg gctaaagcac caataatatt aacttctaga
gcagacagtg aagaaactaa 5640actaaactca atagctttag gtgttttaat
ggcagcaaag gcataataag aaggagatat 5700acatatgagc aaaatattta
aaatcttaac aataaatcct ggttcgacat caactaaaat 5760agctgtattt
gataatgagg atttagtatt tgaaaaaact ttaagacatt cttcagaaga
5820aataggaaaa tatgagaagg tgtctgacca atttgaattt cgtaaacaag
taatagaaga 5880agctctaaaa gaaggtggag taaaaacatc tgaattagat
gctgtagtag gtagaggagg 5940acttcttaaa cctataaaag gtggtactta
ttcagtaagt gctgctatga ttgaagattt 6000aaaagtggga gttttaggag
aacacgcttc aaacctaggt ggaataatag caaaacaaat 6060aggtgaagaa
gtaaatgttc cttcatacat agtagaccct gttgttgtag atgaattaga
6120agatgttgct agaatttctg gtatgcctga aataagtaga gcaagtgtag
tacatgcttt 6180aaatcaaaag gcaatagcaa gaagatatgc tagagaaata
aacaagaaat atgaagatat 6240aaatcttata gttgcacaca tgggtggagg
agtttctgtt ggagctcata aaaatggtaa 6300aatagtagat gttgcaaacg
cattagatgg agaaggacct ttctctccag aaagaagtgg 6360tggactacca
gtaggtgcat tagtaaaaat gtgctttagt ggaaaatata ctcaagatga
6420aattaaaaag aaaataaaag gtaatggcgg actagttgca tacttaaaca
ctaatgatgc 6480tagagaagtt gaagaaagaa ttgaagctgg tgatgaaaaa
gctaaattag tatatgaagc 6540tatggcatat caaatctcta aagaaatagg
agctagtgct gcagttctta agggagatgt 6600aaaagcaata ttattaactg
gtggaatcgc atattcaaaa atgtttacag aaatgattgc 6660agatagagtt
aaatttatag cagatgtaaa agtttatcca ggtgaagatg aaatgattgc
6720attagctcaa ggtggactta gagttttaac tggtgaagaa gaggctcaag
tttatgataa 6780ctaataa 678731137DNAPeptoclostridium difficile
3atggatttaa attctaaaaa atatcagatg cttaaagagc tatatgtaag cttcgctgaa
60aatgaagtta aacctttagc aacagaactt gatgaagaag aaagatttcc ttatgaaaca
120gtggaaaaaa tggcaaaagc aggaatgatg ggtataccat atccaaaaga
atatggtgga 180gaaggtggag acactgtagg atatataatg gcagttgaag
aattgtctag agtttgtggt 240actacaggag ttatattatc agctcataca
tctcttggct catggcctat atatcaatat 300ggtaatgaag aacaaaaaca
aaaattctta agaccactag caagtggaga aaaattagga 360gcatttggtc
ttactgagcc taatgctggt acagatgcgt ctggccaaca aacaactgct
420gttttagacg gggatgaata catacttaat ggctcaaaaa tatttataac
aaacgcaata 480gctggtgaca tatatgtagt aatggcaatg actgataaat
ctaaggggaa caaaggaata 540tcagcattta tagttgaaaa aggaactcct
gggtttagct ttggagttaa agaaaagaaa 600atgggtataa gaggttcagc
tacgagtgaa ttaatatttg aggattgcag aatacctaaa 660gaaaatttac
ttggaaaaga aggtcaagga tttaagatag caatgtctac tcttgatggt
720ggtagaattg gtatagctgc acaagcttta ggtttagcac aaggtgctct
tgatgaaact 780gttaaatatg taaaagaaag agtacaattt ggtagaccat
tatcaaaatt ccaaaataca 840caattccaat tagctgatat ggaagttaag
gtacaagcgg ctagacacct tgtatatcaa 900gcagctataa ataaagactt
aggaaaacct tatggagtag aagcagcaat ggcaaaatta 960tttgcagctg
aaacagctat ggaagttact acaaaagctg tacaacttca tggaggatat
1020ggatacactc gtgactatcc agtagaaaga atgatgagag atgctaagat
aactgaaata 1080tatgaaggaa ctagtgaagt tcaaagaatg gttatttcag
gaaaactatt aaaatag 11374783DNAPeptoclostridium difficile
4atgaatatag tcgtttgtat aaaacaagtt ccagatacaa cagaagttaa actagatcct
60aatacaggta ctttaattag agatggagta ccaagtataa taaaccctga tgataaagca
120ggtttagaag aagctataaa attaaaagaa gaaatgggtg ctcatgtaac
tgttataaca 180atgggacctc ctcaagcaga tatggcttta aaagaagctt
tagcaatggg tgcagataga 240ggtatattat taacagatag agcatttgcg
ggtgctgata cttgggcaac ttcatcagca 300ttagcaggag cattaaaaaa
tatagatttt gatattataa tagctggaag acaggcgata 360gatggagata
ctgcacaagt tggacctcaa atagctgaac atttaaatct tccatcaata
420acatatgctg aagaaataaa aactgaaggt gaatatgtat tagtaaaaag
acaatttgaa 480gattgttgcc atgacttaaa agttaaaatg ccatgcctta
taacaactct taaagatatg 540aacacaccaa gatacatgaa agttggaaga
atatatgatg ctttcgaaaa tgatgtagta 600gaaacatgga ctgtaaaaga
tatagaagtt gacccttcta atttaggtct taaaggttct 660ccaactagtg
tatttaaatc atttacaaaa tcagttaaac cagctggtac aatatacaat
720gaagatgcga aaacatcagc tggaattatc atagataaat taaaagagaa
gtatatcata 780taa 78351011DNAPeptoclostridium difficile 5atgggtaacg
ttttagtagt aatagaacaa agagaaaatg taattcaaac tgtttcttta 60gaattactag
gaaaggctac agaaatagca aaagattatg atacaaaagt ttctgcatta
120cttttaggta gtaaggtaga aggtttaata gatacattag cacactatgg
tgcagatgag 180gtaatagtag tagatgatga agctttagca gtgtatacaa
ctgaaccata tacaaaagca 240gcttatgaag caataaaagc agctgaccct
atagttgtat tatttggtgc aacttcaata 300ggtagagatt tagcgcctag
agtttctgct agaatacata caggtcttac tgctgactgt 360acaggtcttg
cagtagctga agatacaaaa ttattattaa tgacaagacc tgcctttggt
420ggaaatataa tggcaacaat agtttgtaaa gatttcagac ctcaaatgtc
tacagttaga 480ccaggggtta tgaagaaaaa tgaacctgat gaaactaaag
aagctgtaat taaccgtttc 540aaggtagaat ttaatgatgc tgataaatta
gttcaagttg tacaagtaat aaaagaagct 600aaaaaacaag ttaaaataga
agatgctaag atattagttt ctgctggacg tggaatgggt 660ggaaaagaaa
acttagacat actttatgaa ttagctgaaa ttataggtgg agaagtttct
720ggttctcgtg ccactataga tgcaggttgg ttagataaag caagacaagt
tggtcaaact 780ggtaaaactg taagaccaga cctttatata gcatgtggta
tatctggagc aatacaacat 840atagctggta tggaagatgc tgagtttata
gttgctataa ataaaaatcc agaagctcca 900atatttaaat atgctgatgt
tggtatagtt ggagatgttc ataaagtgct tccagaactt 960atcagtcagt
taagtgttgc aaaagaaaaa ggtgaagttt tagctaacta a
101161176DNAPeptoclostridium difficile 6atgagagaag tagtaattgc
cagtgcagct agaacagcag taggaagttt tggaggagca 60tttaaatcag tttcagcggt
agagttaggg gtaacagcag ctaaagaagc tataaaaaga 120gctaacataa
ctccagatat gatagatgaa tctcttttag ggggagtact tacagcaggt
180cttggacaaa atatagcaag acaaatagca ttaggagcag gaataccagt
agaaaaacca 240gctatgacta taaatatagt ttgtggttct ggattaagat
ctgtttcaat ggcatctcaa 300cttatagcat taggtgatgc tgatataatg
ttagttggtg gagctgaaaa catgagtatg 360tctccttatt tagtaccaag
tgcgagatat ggtgcaagaa tgggtgatgc tgcttttgtt 420gattcaatga
taaaagatgg attatcagac atatttaata actatcacat gggtattact
480gctgaaaaca tagcagagca atggaatata actagagaag aacaagatga
attagctctt 540gcaagtcaaa ataaagctga aaaagctcaa gctgaaggaa
aatttgatga agaaatagtt 600cctgttgtta taaaaggaag aaaaggtgac
actgtagtag ataaagatga atatattaag 660cctggcacta caatggagaa
acttgctaag ttaagacctg catttaaaaa agatggaaca 720gttactgctg
gtaatgcatc aggaataaat gatggtgctg ctatgttagt agtaatggct
780aaagaaaaag ctgaagaact aggaatagag cctcttgcaa ctatagtttc
ttatggaaca 840gctggtgttg accctaaaat aatgggatat ggaccagttc
cagcaactaa aaaagcttta 900gaagctgcta atatgactat tgaagatata
gatttagttg aagctaatga ggcatttgct 960gcccaatctg tagctgtaat
aagagactta aatatagata tgaataaagt taatgttaat 1020ggtggagcaa
tagctatagg acatccaata ggatgctcag gagcaagaat acttactaca
1080cttttatatg aaatgaagag aagagatgct aaaactggtc ttgctacact
ttgtataggc 1140ggtggaatgg gaactacttt aatagttaag agatag
11767846DNAPeptoclostridium difficile 7atgaaattag ctgtaatagg
tagtggaact atgggaagtg gtattgtaca aacttttgca 60agttgtggac atgatgtatg
tttaaagagt agaactcaag gtgctataga taaatgttta 120gctttattag
ataaaaattt aactaagtta gttactaagg gaaaaatgga tgaagctaca
180aaagcagaaa tattaagtca tgttagttca actactaatt atgaagattt
aaaagatatg 240gatttaataa tagaagcatc tgtagaagac atgaatataa
agaaagatgt tttcaagtta 300ctagatgaat tatgtaaaga agatactatc
ttggcaacaa atacttcatc attatctata 360acagaaatag cttcttctac
taagcgccca gataaagtta taggaatgca tttctttaat 420ccagttccta
tgatgaaatt agttgaagtt ataagtggtc agttaacatc aaaagttact
480tttgatacag tatttgaatt atctaagagt atcaataaag taccagtaga
tgtatctgaa 540tctcctggat ttgtagtaaa tagaatactt atacctatga
taaatgaagc tgttggtata 600tatgcagatg gtgttgcaag taaagaagaa
atagatgaag ctatgaaatt aggagcaaac 660catccaatgg gaccactagc
attaggtgat ttaatcggat tagatgttgt tttagctata 720atgaacgttt
tatatactga atttggagat actaaatata gacctcatcc acttttagct
780aaaatggtta gagctaatca attaggaaga aaaactaaga taggattcta
tgattataat 840aaataa 8468798DNAPeptoclostridium difficile
8atgagtacaa gtgatgttaa agtttatgag aatgtagctg ttgaagtaga tggaaatata
60tgtacagtga aaatgaatag acctaaagcc cttaatgcaa taaattcaaa gactttagaa
120gaactttatg aagtatttgt agatattaat aatgatgaaa ctattgatgt
tgtaatattg 180acaggggaag gaaaggcatt tgtagctgga gcagatattg
catacatgaa agatttagat 240gctgtagctg ctaaagattt tagtatctta
ggagcaaaag cttttggaga aatagaaaat 300agtaaaaaag tagtgatagc
tgctgtaaac ggatttgctt taggtggagg atgtgaactt 360gcaatggcat
gtgatataag aattgcatct gctaaagcta aatttggtca gccagaagta
420actcttggaa taactccagg atatggagga actcaaaggc ttacaagatt
ggttggaatg 480gcaaaagcaa aagaattaat ctttacaggt caagttataa
aagctgatga agctgaaaaa 540atagggctag taaatagagt cgttgagcca
gacattttaa tagaagaagt tgagaaatta 600gctaagataa tagctaaaaa
tgctcagctt gcagttagat actctaaaga agcaatacaa 660cttggtgctc
aaactgatat aaatactgga atagatatag aatctaattt atttggtctt
720tgtttttcaa ctaaagacca aaaagaagga atgtcagctt tcgttgaaaa
gagagaagct 780aactttataa aagggtaa 7989903DNAPeptoclostridium
difficile 9atgagaagtt ttgaagaagt aattaagttt gcaaaagaaa gaggacctaa
aactatatca 60gtagcatgtt gccaagataa agaagtttta atggcagttg aaatggctag
aaaagaaaaa 120atagcaaatg ccattttagt aggagatata gaaaagacta
aagaaattgc aaaaagcata 180gacatggata tcgaaaatta tgaactgata
gatataaaag atttagcaga agcatctcta 240aaatctgttg aattagtttc
acaaggaaaa gccgacatgg taatgaaagg cttagtagac 300acatcaataa
tactaaaagc agttttaaat aaagaagtag gtcttagaac tggaaatgta
360ttaagtcacg tagcagtatt tgatgtagag ggatatgata gattattttt
cgtaactgac 420gcagctatga acttagctcc tgatacaaat actaaaaagc
aaatcataga aaatgcttgc 480acagtagcac attcattaga tataagtgaa
ccaaaagttg ctgcaatatg cgcaaaagaa 540aaagtaaatc caaaaatgaa
agatacagtt gaagctaaag aactagaaga aatgtatgaa 600agaggagaaa
tcaaaggttg tatggttggt gggccttttg caattgataa tgcagtatct
660ttagaagcag ctaaacataa aggtataaat catcctgtag caggacgagc
tgatatatta 720ttagccccag atattgaagg tggtaacata ttatataaag
ctttggtatt cttctcaaaa 780tcaaaaaatg caggagttat agttggggct
aaagcaccaa taatattaac ttctagagca 840gacagtgaag aaactaaact
aaactcaata gctttaggtg ttttaatggc agcaaaggca 900taa
903101080DNAPeptoclostridium difficile 10atgagcaaaa tatttaaaat
cttaacaata aatcctggtt cgacatcaac taaaatagct 60gtatttgata atgaggattt
agtatttgaa aaaactttaa gacattcttc agaagaaata 120ggaaaatatg
agaaggtgtc tgaccaattt gaatttcgta aacaagtaat agaagaagct
180ctaaaagaag gtggagtaaa aacatctgaa ttagatgctg tagtaggtag
aggaggactt 240cttaaaccta taaaaggtgg tacttattca gtaagtgctg
ctatgattga agatttaaaa 300gtgggagttt taggagaaca cgcttcaaac
ctaggtggaa taatagcaaa acaaataggt 360gaagaagtaa atgttccttc
atacatagta gaccctgttg ttgtagatga attagaagat 420gttgctagaa
tttctggtat gcctgaaata agtagagcaa gtgtagtaca tgctttaaat
480caaaaggcaa tagcaagaag atatgctaga gaaataaaca agaaatatga
agatataaat 540cttatagttg cacacatggg tggaggagtt tctgttggag
ctcataaaaa tggtaaaata 600gtagatgttg caaacgcatt agatggagaa
ggacctttct ctccagaaag aagtggtgga 660ctaccagtag gtgcattagt
aaaaatgtgc tttagtggaa aatatactca agatgaaatt 720aaaaagaaaa
taaaaggtaa tggcggacta gttgcatact taaacactaa tgatgctaga
780gaagttgaag aaagaattga agctggtgat gaaaaagcta aattagtata
tgaagctatg 840gcatatcaaa tctctaaaga aataggagct agtgctgcag
ttcttaaggg agatgtaaaa 900gcaatattat taactggtgg aatcgcatat
tcaaaaatgt ttacagaaat gattgcagat 960agagttaaat ttatagcaga
tgtaaaagtt tatccaggtg aagatgaaat gattgcatta 1020gctcaaggtg
gacttagagt tttaactggt gaagaagagg ctcaagttta tgataactaa
1080111194DNATreponema denticola 11atgatcgtaa aacctatggt acgcaacaat
atctgcctga acgcccatcc tcagggctgc 60aagaagggag tggaagatca gattgaatat
accaagaaac gcattaccgc agaagtcaaa 120gctggcgcaa aagctccaaa
aaacgttctg gtgcttggct gctcaaatgg ttacggcctg 180gcgagccgca
ttactgctgc gttcggatac ggggctgcga ccatcggcgt gtcctttgaa
240aaagcgggtt cagaaaccaa atatggtaca ccgggatggt acaataattt
ggcatttgat 300gaagcggcaa aacgcgaggg tctttatagc gtgacgatcg
acggcgatgc gttttcagac 360gagatcaagg cccaggtaat tgaggaagcc
aaaaaaaaag gtatcaaatt tgatctgatc 420gtatacagct tggccagccc
agtacgtact gatcctgata caggtatcat gcacaaaagc 480gttttgaaac
cctttggaaa aacgttcaca ggcaaaacag tagatccgtt tactggcgag
540ctgaaggaaa tctccgcgga accagcaaat gacgaggaag cagccgccac
tgttaaagtt 600atggggggtg aagattggga acgttggatt aagcagctgt
cgaaggaagg cctcttagaa 660gaaggctgta ttaccttggc ctatagttat
attggccctg aagctaccca agctttgtac 720cgtaaaggca caatcggcaa
ggccaaagaa cacctggagg ccacagcaca ccgtctcaac 780aaagagaacc
cgtcaatccg tgccttcgtg agcgtgaata aaggcctggt aacccgcgca
840agcgccgtaa tcccggtaat ccctctgtat ctcgccagct tgttcaaagt
aatgaaagag 900aagggcaatc atgaaggttg tattgaacag atcacgcgtc
tgtacgccga gcgcctgtac 960cgtaaagatg gtacaattcc agttgatgag
gaaaatcgca ttcgcattga tgattgggag 1020ttagaagaag acgtccagaa
agcggtatcc gcgttgatgg agaaagtcac gggtgaaaac 1080gcagaatctc
tcactgactt agcggggtac cgccatgatt tcttagctag taacggcttt
1140gatgtagaag gtattaatta tgaagcggaa gttgaacgct tcgaccgtat ctga
1194128650DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 12gtaaaacgac ggccagtgaa ttcgttaaga
cccactttca catttaagtt gtttttctaa 60tccgcatatg atcaattcaa ggccgaataa
gaaggctggc tctgcacctt ggtgatcaaa 120taattcgata gcttgtcgta
ataatggcgg catactatca gtagtaggtg tttccctttc 180ttctttagcg
acttgatgct cttgatcttc caatacgcaa cctaaagtaa aatgccccac
240agcgctgagt gcatataatg cattctctag tgaaaaacct tgttggcata
aaaaggctaa 300ttgattttcg agagtttcat actgtttttc tgtaggccgt
gtacctaaat gtacttttgc 360tccatcgcga tgacttagta aagcacatct
aaaactttta gcgttattac gtaaaaaatc 420ttgccagctt tccccttcta
aagggcaaaa gtgagtatgg tgcctatcta acatctcaat 480ggctaaggcg
tcgagcaaag cccgcttatt ttttacatgc caatacaatg taggctgctc
540tacacctagc ttctgggcga gtttacgggt tgttaaacct tcgattccga
cctcattaag 600cagctctaat gcgctgttaa tcactttact tttatctaat
ctagacatca ttaattccta 660atttttgttg acactctatc attgatagag
ttattttacc actccctatc agtgatagag 720aaaagtgaac tctagaaata
attttgttta actttaagaa ggagatatac atatggattt 780aaattctaaa
aaatatcaga tgcttaaaga gctatatgta agcttcgctg aaaatgaagt
840taaaccttta gcaacagaac ttgatgaaga agaaagattt ccttatgaaa
cagtggaaaa 900aatggcaaaa gcaggaatga tgggtatacc atatccaaaa
gaatatggtg gagaaggtgg 960agacactgta ggatatataa tggcagttga
agaattgtct agagtttgtg gtactacagg 1020agttatatta tcagctcata
catctcttgg ctcatggcct atatatcaat atggtaatga 1080agaacaaaaa
caaaaattct taagaccact agcaagtgga gaaaaattag gagcatttgg
1140tcttactgag cctaatgctg gtacagatgc gtctggccaa caaacaactg
ctgttttaga 1200cggggatgaa tacatactta atggctcaaa aatatttata
acaaacgcaa tagctggtga 1260catatatgta gtaatggcaa tgactgataa
atctaagggg aacaaaggaa tatcagcatt 1320tatagttgaa aaaggaactc
ctgggtttag ctttggagtt aaagaaaaga aaatgggtat 1380aagaggttca
gctacgagtg aattaatatt tgaggattgc agaataccta aagaaaattt
1440acttggaaaa gaaggtcaag gatttaagat agcaatgtct actcttgatg
gtggtagaat 1500tggtatagct gcacaagctt taggtttagc acaaggtgct
cttgatgaaa ctgttaaata 1560tgtaaaagaa agagtacaat ttggtagacc
attatcaaaa ttccaaaata cacaattcca 1620attagctgat atggaagtta
aggtacaagc ggctagacac cttgtatatc aagcagctat 1680aaataaagac
ttaggaaaac cttatggagt agaagcagca atggcaaaat tatttgcagc
1740tgaaacagct atggaagtta ctacaaaagc tgtacaactt catggaggat
atggatacac 1800tcgtgactat ccagtagaaa gaatgatgag agatgctaag
ataactgaaa tatatgaagg 1860aactagtgaa gttcaaagaa tggttatttc
aggaaaacta ttaaaatagt aagaaggaga 1920tatacatatg gaggaaggat
ttatgaatat agtcgtttgt ataaaacaag ttccagatac 1980aacagaagtt
aaactagatc ctaatacagg tactttaatt agagatggag taccaagtat
2040aataaaccct gatgataaag caggtttaga agaagctata aaattaaaag
aagaaatggg 2100tgctcatgta actgttataa caatgggacc tcctcaagca
gatatggctt taaaagaagc 2160tttagcaatg ggtgcagata gaggtatatt
attaacagat agagcatttg cgggtgctga 2220tacttgggca acttcatcag
cattagcagg agcattaaaa aatatagatt ttgatattat 2280aatagctgga
agacaggcga tagatggaga tactgcacaa gttggacctc aaatagctga
2340acatttaaat cttccatcaa taacatatgc tgaagaaata aaaactgaag
gtgaatatgt 2400attagtaaaa agacaatttg aagattgttg ccatgactta
aaagttaaaa tgccatgcct 2460tataacaact cttaaagata tgaacacacc
aagatacatg aaagttggaa gaatatatga 2520tgctttcgaa aatgatgtag
tagaaacatg gactgtaaaa gatatagaag ttgacccttc 2580taatttaggt
cttaaaggtt ctccaactag tgtatttaaa tcatttacaa aatcagttaa
2640accagctggt acaatataca atgaagatgc gaaaacatca gctggaatta
tcatagataa 2700attaaaagag aagtatatca tataataaga aggagatata
catatgggta acgttttagt 2760agtaatagaa caaagagaaa atgtaattca
aactgtttct ttagaattac taggaaaggc 2820tacagaaata gcaaaagatt
atgatacaaa agtttctgca ttacttttag gtagtaaggt 2880agaaggttta
atagatacat tagcacacta tggtgcagat gaggtaatag tagtagatga
2940tgaagcttta gcagtgtata caactgaacc atatacaaaa gcagcttatg
aagcaataaa 3000agcagctgac cctatagttg tattatttgg tgcaacttca
ataggtagag atttagcgcc 3060tagagtttct gctagaatac atacaggtct
tactgctgac tgtacaggtc ttgcagtagc 3120tgaagataca aaattattat
taatgacaag acctgccttt ggtggaaata taatggcaac 3180aatagtttgt
aaagatttca gacctcaaat gtctacagtt agaccagggg ttatgaagaa
3240aaatgaacct gatgaaacta aagaagctgt aattaaccgt ttcaaggtag
aatttaatga 3300tgctgataaa ttagttcaag ttgtacaagt aataaaagaa
gctaaaaaac aagttaaaat 3360agaagatgct aagatattag tttctgctgg
acgtggaatg ggtggaaaag aaaacttaga 3420catactttat gaattagctg
aaattatagg tggagaagtt tctggttctc gtgccactat 3480agatgcaggt
tggttagata aagcaagaca agttggtcaa actggtaaaa ctgtaagacc
3540agacctttat atagcatgtg gtatatctgg agcaatacaa catatagctg
gtatggaaga 3600tgctgagttt atagttgcta taaataaaaa tccagaagct
ccaatattta aatatgctga 3660tgttggtata gttggagatg ttcataaagt
gcttccagaa cttatcagtc agttaagtgt 3720tgcaaaagaa aaaggtgaag
ttttagctaa ctaataagaa ggagatatac atatgagaga 3780agtagtaatt
gccagtgcag ctagaacagc agtaggaagt tttggaggag catttaaatc
3840agtttcagcg gtagagttag gggtaacagc agctaaagaa gctataaaaa
gagctaacat 3900aactccagat atgatagatg aatctctttt agggggagta
cttacagcag gtcttggaca 3960aaatatagca agacaaatag cattaggagc
aggaatacca gtagaaaaac cagctatgac 4020tataaatata gtttgtggtt
ctggattaag atctgtttca atggcatctc aacttatagc 4080attaggtgat
gctgatataa tgttagttgg tggagctgaa aacatgagta tgtctcctta
4140tttagtacca agtgcgagat atggtgcaag aatgggtgat gctgcttttg
ttgattcaat 4200gataaaagat ggattatcag acatatttaa taactatcac
atgggtatta ctgctgaaaa 4260catagcagag caatggaata taactagaga
agaacaagat gaattagctc ttgcaagtca 4320aaataaagct gaaaaagctc
aagctgaagg aaaatttgat gaagaaatag ttcctgttgt 4380tataaaagga
agaaaaggtg acactgtagt agataaagat gaatatatta agcctggcac
4440tacaatggag aaacttgcta agttaagacc tgcatttaaa aaagatggaa
cagttactgc 4500tggtaatgca tcaggaataa atgatggtgc tgctatgtta
gtagtaatgg ctaaagaaaa 4560agctgaagaa ctaggaatag agcctcttgc
aactatagtt tcttatggaa cagctggtgt 4620tgaccctaaa ataatgggat
atggaccagt tccagcaact aaaaaagctt tagaagctgc 4680taatatgact
attgaagata tagatttagt tgaagctaat gaggcatttg ctgcccaatc
4740tgtagctgta ataagagact taaatataga tatgaataaa gttaatgtta
atggtggagc 4800aatagctata ggacatccaa taggatgctc aggagcaaga
atacttacta cacttttata 4860tgaaatgaag agaagagatg ctaaaactgg
tcttgctaca ctttgtatag gcggtggaat 4920gggaactact ttaatagtta
agagatagta agaaggagat atacatatga aattagctgt
4980aataggtagt ggaactatgg gaagtggtat tgtacaaact tttgcaagtt
gtggacatga 5040tgtatgttta aagagtagaa ctcaaggtgc tatagataaa
tgtttagctt tattagataa 5100aaatttaact aagttagtta ctaagggaaa
aatggatgaa gctacaaaag cagaaatatt 5160aagtcatgtt agttcaacta
ctaattatga agatttaaaa gatatggatt taataataga 5220agcatctgta
gaagacatga atataaagaa agatgttttc aagttactag atgaattatg
5280taaagaagat actatcttgg caacaaatac ttcatcatta tctataacag
aaatagcttc 5340ttctactaag cgcccagata aagttatagg aatgcatttc
tttaatccag ttcctatgat 5400gaaattagtt gaagttataa gtggtcagtt
aacatcaaaa gttacttttg atacagtatt 5460tgaattatct aagagtatca
ataaagtacc agtagatgta tctgaatctc ctggatttgt 5520agtaaataga
atacttatac ctatgataaa tgaagctgtt ggtatatatg cagatggtgt
5580tgcaagtaaa gaagaaatag atgaagctat gaaattagga gcaaaccatc
caatgggacc 5640actagcatta ggtgatttaa tcggattaga tgttgtttta
gctataatga acgttttata 5700tactgaattt ggagatacta aatatagacc
tcatccactt ttagctaaaa tggttagagc 5760taatcaatta ggaagaaaaa
ctaagatagg attctatgat tataataaat aataagaagg 5820agatatacat
atgagtacaa gtgatgttaa agtttatgag aatgtagctg ttgaagtaga
5880tggaaatata tgtacagtga aaatgaatag acctaaagcc cttaatgcaa
taaattcaaa 5940gactttagaa gaactttatg aagtatttgt agatattaat
aatgatgaaa ctattgatgt 6000tgtaatattg acaggggaag gaaaggcatt
tgtagctgga gcagatattg catacatgaa 6060agatttagat gctgtagctg
ctaaagattt tagtatctta ggagcaaaag cttttggaga 6120aatagaaaat
agtaaaaaag tagtgatagc tgctgtaaac ggatttgctt taggtggagg
6180atgtgaactt gcaatggcat gtgatataag aattgcatct gctaaagcta
aatttggtca 6240gccagaagta actcttggaa taactccagg atatggagga
actcaaaggc ttacaagatt 6300ggttggaatg gcaaaagcaa aagaattaat
ctttacaggt caagttataa aagctgatga 6360agctgaaaaa atagggctag
taaatagagt cgttgagcca gacattttaa tagaagaagt 6420tgagaaatta
gctaagataa tagctaaaaa tgctcagctt gcagttagat actctaaaga
6480agcaatacaa cttggtgctc aaactgatat aaatactgga atagatatag
aatctaattt 6540atttggtctt tgtttttcaa ctaaagacca aaaagaagga
atgtcagctt tcgttgaaaa 6600gagagaagct aactttataa aagggtaata
agaaggagat atacatatga gaagttttga 6660agaagtaatt aagtttgcaa
aagaaagagg acctaaaact atatcagtag catgttgcca 6720agataaagaa
gttttaatgg cagttgaaat ggctagaaaa gaaaaaatag caaatgccat
6780tttagtagga gatatagaaa agactaaaga aattgcaaaa agcatagaca
tggatatcga 6840aaattatgaa ctgatagata taaaagattt agcagaagca
tctctaaaat ctgttgaatt 6900agtttcacaa ggaaaagccg acatggtaat
gaaaggctta gtagacacat caataatact 6960aaaagcagtt ttaaataaag
aagtaggtct tagaactgga aatgtattaa gtcacgtagc 7020agtatttgat
gtagagggat atgatagatt atttttcgta actgacgcag ctatgaactt
7080agctcctgat acaaatacta aaaagcaaat catagaaaat gcttgcacag
tagcacattc 7140attagatata agtgaaccaa aagttgctgc aatatgcgca
aaagaaaaag taaatccaaa 7200aatgaaagat acagttgaag ctaaagaact
agaagaaatg tatgaaagag gagaaatcaa 7260aggttgtatg gttggtgggc
cttttgcaat tgataatgca gtatctttag aagcagctaa 7320acataaaggt
ataaatcatc ctgtagcagg acgagctgat atattattag ccccagatat
7380tgaaggtggt aacatattat ataaagcttt ggtattcttc tcaaaatcaa
aaaatgcagg 7440agttatagtt ggggctaaag caccaataat attaacttct
agagcagaca gtgaagaaac 7500taaactaaac tcaatagctt taggtgtttt
aatggcagca aaggcataat aagaaggaga 7560tatacatatg agcaaaatat
ttaaaatctt aacaataaat cctggttcga catcaactaa 7620aatagctgta
tttgataatg aggatttagt atttgaaaaa actttaagac attcttcaga
7680agaaatagga aaatatgaga aggtgtctga ccaatttgaa tttcgtaaac
aagtaataga 7740agaagctcta aaagaaggtg gagtaaaaac atctgaatta
gatgctgtag taggtagagg 7800aggacttctt aaacctataa aaggtggtac
ttattcagta agtgctgcta tgattgaaga 7860tttaaaagtg ggagttttag
gagaacacgc ttcaaaccta ggtggaataa tagcaaaaca 7920aataggtgaa
gaagtaaatg ttccttcata catagtagac cctgttgttg tagatgaatt
7980agaagatgtt gctagaattt ctggtatgcc tgaaataagt agagcaagtg
tagtacatgc 8040tttaaatcaa aaggcaatag caagaagata tgctagagaa
ataaacaaga aatatgaaga 8100tataaatctt atagttgcac acatgggtgg
aggagtttct gttggagctc ataaaaatgg 8160taaaatagta gatgttgcaa
acgcattaga tggagaagga cctttctctc cagaaagaag 8220tggtggacta
ccagtaggtg cattagtaaa aatgtgcttt agtggaaaat atactcaaga
8280tgaaattaaa aagaaaataa aaggtaatgg cggactagtt gcatacttaa
acactaatga 8340tgctagagaa gttgaagaaa gaattgaagc tggtgatgaa
aaagctaaat tagtatatga 8400agctatggca tatcaaatct ctaaagaaat
aggagctagt gctgcagttc ttaagggaga 8460tgtaaaagca atattattaa
ctggtggaat cgcatattca aaaatgttta cagaaatgat 8520tgcagataga
gttaaattta tagcagatgt aaaagtttat ccaggtgaag atgaaatgat
8580tgcattagct caaggtggac ttagagtttt aactggtgaa gaagaggctc
aagtttatga 8640taactaataa 8650136862DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
13gtaaaacgac ggccagtgaa ttcgttaaga cccactttca catttaagtt gtttttctaa
60tccgcatatg atcaattcaa ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa
120taattcgata gcttgtcgta ataatggcgg catactatca gtagtaggtg
tttccctttc 180ttctttagcg acttgatgct cttgatcttc caatacgcaa
cctaaagtaa aatgccccac 240agcgctgagt gcatataatg cattctctag
tgaaaaacct tgttggcata aaaaggctaa 300ttgattttcg agagtttcat
actgtttttc tgtaggccgt gtacctaaat gtacttttgc 360tccatcgcga
tgacttagta aagcacatct aaaactttta gcgttattac gtaaaaaatc
420ttgccagctt tccccttcta aagggcaaaa gtgagtatgg tgcctatcta
acatctcaat 480ggctaaggcg tcgagcaaag cccgcttatt ttttacatgc
caatacaatg taggctgctc 540tacacctagc ttctgggcga gtttacgggt
tgttaaacct tcgattccga cctcattaag 600cagctctaat gcgctgttaa
tcactttact tttatctaat ctagacatca ttaattccta 660atttttgttg
acactctatc attgatagag ttattttacc actccctatc agtgatagag
720aaaagtgaac tctagaaata attttgttta actttaagaa ggagatatac
atatgatcgt 780aaaacctatg gtacgcaaca atatctgcct gaacgcccat
cctcagggct gcaagaaggg 840agtggaagat cagattgaat ataccaagaa
acgcattacc gcagaagtca aagctggcgc 900aaaagctcca aaaaacgttc
tggtgcttgg ctgctcaaat ggttacggcc tggcgagccg 960cattactgct
gcgttcggat acggggctgc gaccatcggc gtgtcctttg aaaaagcggg
1020ttcagaaacc aaatatggta caccgggatg gtacaataat ttggcatttg
atgaagcggc 1080aaaacgcgag ggtctttata gcgtgacgat cgacggcgat
gcgttttcag acgagatcaa 1140ggcccaggta attgaggaag ccaaaaaaaa
aggtatcaaa tttgatctga tcgtatacag 1200cttggccagc ccagtacgta
ctgatcctga tacaggtatc atgcacaaaa gcgttttgaa 1260accctttgga
aaaacgttca caggcaaaac agtagatccg tttactggcg agctgaagga
1320aatctccgcg gaaccagcaa atgacgagga agcagccgcc actgttaaag
ttatgggggg 1380tgaagattgg gaacgttgga ttaagcagct gtcgaaggaa
ggcctcttag aagaaggctg 1440tattaccttg gcctatagtt atattggccc
tgaagctacc caagctttgt accgtaaagg 1500cacaatcggc aaggccaaag
aacacctgga ggccacagca caccgtctca acaaagagaa 1560cccgtcaatc
cgtgccttcg tgagcgtgaa taaaggcctg gtaacccgcg caagcgccgt
1620aatcccggta atccctctgt atctcgccag cttgttcaaa gtaatgaaag
agaagggcaa 1680tcatgaaggt tgtattgaac agatcacgcg tctgtacgcc
gagcgcctgt accgtaaaga 1740tggtacaatt ccagttgatg aggaaaatcg
cattcgcatt gatgattggg agttagaaga 1800agacgtccag aaagcggtat
ccgcgttgat ggagaaagtc acgggtgaaa acgcagaatc 1860tctcactgac
ttagcggggt accgccatga tttcttagct agtaacggct ttgatgtaga
1920aggtattaat tatgaagcgg aagttgaacg cttcgaccgt atctgataag
aaggagatat 1980acatatgaga gaagtagtaa ttgccagtgc agctagaaca
gcagtaggaa gttttggagg 2040agcatttaaa tcagtttcag cggtagagtt
aggggtaaca gcagctaaag aagctataaa 2100aagagctaac ataactccag
atatgataga tgaatctctt ttagggggag tacttacagc 2160aggtcttgga
caaaatatag caagacaaat agcattagga gcaggaatac cagtagaaaa
2220accagctatg actataaata tagtttgtgg ttctggatta agatctgttt
caatggcatc 2280tcaacttata gcattaggtg atgctgatat aatgttagtt
ggtggagctg aaaacatgag 2340tatgtctcct tatttagtac caagtgcgag
atatggtgca agaatgggtg atgctgcttt 2400tgttgattca atgataaaag
atggattatc agacatattt aataactatc acatgggtat 2460tactgctgaa
aacatagcag agcaatggaa tataactaga gaagaacaag atgaattagc
2520tcttgcaagt caaaataaag ctgaaaaagc tcaagctgaa ggaaaatttg
atgaagaaat 2580agttcctgtt gttataaaag gaagaaaagg tgacactgta
gtagataaag atgaatatat 2640taagcctggc actacaatgg agaaacttgc
taagttaaga cctgcattta aaaaagatgg 2700aacagttact gctggtaatg
catcaggaat aaatgatggt gctgctatgt tagtagtaat 2760ggctaaagaa
aaagctgaag aactaggaat agagcctctt gcaactatag tttcttatgg
2820aacagctggt gttgacccta aaataatggg atatggacca gttccagcaa
ctaaaaaagc 2880tttagaagct gctaatatga ctattgaaga tatagattta
gttgaagcta atgaggcatt 2940tgctgcccaa tctgtagctg taataagaga
cttaaatata gatatgaata aagttaatgt 3000taatggtgga gcaatagcta
taggacatcc aataggatgc tcaggagcaa gaatacttac 3060tacactttta
tatgaaatga agagaagaga tgctaaaact ggtcttgcta cactttgtat
3120aggcggtgga atgggaacta ctttaatagt taagagatag taagaaggag
atatacatat 3180gaaattagct gtaataggta gtggaactat gggaagtggt
attgtacaaa cttttgcaag 3240ttgtggacat gatgtatgtt taaagagtag
aactcaaggt gctatagata aatgtttagc 3300tttattagat aaaaatttaa
ctaagttagt tactaaggga aaaatggatg aagctacaaa 3360agcagaaata
ttaagtcatg ttagttcaac tactaattat gaagatttaa aagatatgga
3420tttaataata gaagcatctg tagaagacat gaatataaag aaagatgttt
tcaagttact 3480agatgaatta tgtaaagaag atactatctt ggcaacaaat
acttcatcat tatctataac 3540agaaatagct tcttctacta agcgcccaga
taaagttata ggaatgcatt tctttaatcc 3600agttcctatg atgaaattag
ttgaagttat aagtggtcag ttaacatcaa aagttacttt 3660tgatacagta
tttgaattat ctaagagtat caataaagta ccagtagatg tatctgaatc
3720tcctggattt gtagtaaata gaatacttat acctatgata aatgaagctg
ttggtatata 3780tgcagatggt gttgcaagta aagaagaaat agatgaagct
atgaaattag gagcaaacca 3840tccaatggga ccactagcat taggtgattt
aatcggatta gatgttgttt tagctataat 3900gaacgtttta tatactgaat
ttggagatac taaatataga cctcatccac ttttagctaa 3960aatggttaga
gctaatcaat taggaagaaa aactaagata ggattctatg attataataa
4020ataataagaa ggagatatac atatgagtac aagtgatgtt aaagtttatg
agaatgtagc 4080tgttgaagta gatggaaata tatgtacagt gaaaatgaat
agacctaaag cccttaatgc 4140aataaattca aagactttag aagaacttta
tgaagtattt gtagatatta ataatgatga 4200aactattgat gttgtaatat
tgacagggga aggaaaggca tttgtagctg gagcagatat 4260tgcatacatg
aaagatttag atgctgtagc tgctaaagat tttagtatct taggagcaaa
4320agcttttgga gaaatagaaa atagtaaaaa agtagtgata gctgctgtaa
acggatttgc 4380tttaggtgga ggatgtgaac ttgcaatggc atgtgatata
agaattgcat ctgctaaagc 4440taaatttggt cagccagaag taactcttgg
aataactcca ggatatggag gaactcaaag 4500gcttacaaga ttggttggaa
tggcaaaagc aaaagaatta atctttacag gtcaagttat 4560aaaagctgat
gaagctgaaa aaatagggct agtaaataga gtcgttgagc cagacatttt
4620aatagaagaa gttgagaaat tagctaagat aatagctaaa aatgctcagc
ttgcagttag 4680atactctaaa gaagcaatac aacttggtgc tcaaactgat
ataaatactg gaatagatat 4740agaatctaat ttatttggtc tttgtttttc
aactaaagac caaaaagaag gaatgtcagc 4800tttcgttgaa aagagagaag
ctaactttat aaaagggtaa taagaaggag atatacatat 4860gagaagtttt
gaagaagtaa ttaagtttgc aaaagaaaga ggacctaaaa ctatatcagt
4920agcatgttgc caagataaag aagttttaat ggcagttgaa atggctagaa
aagaaaaaat 4980agcaaatgcc attttagtag gagatataga aaagactaaa
gaaattgcaa aaagcataga 5040catggatatc gaaaattatg aactgataga
tataaaagat ttagcagaag catctctaaa 5100atctgttgaa ttagtttcac
aaggaaaagc cgacatggta atgaaaggct tagtagacac 5160atcaataata
ctaaaagcag ttttaaataa agaagtaggt cttagaactg gaaatgtatt
5220aagtcacgta gcagtatttg atgtagaggg atatgataga ttatttttcg
taactgacgc 5280agctatgaac ttagctcctg atacaaatac taaaaagcaa
atcatagaaa atgcttgcac 5340agtagcacat tcattagata taagtgaacc
aaaagttgct gcaatatgcg caaaagaaaa 5400agtaaatcca aaaatgaaag
atacagttga agctaaagaa ctagaagaaa tgtatgaaag 5460aggagaaatc
aaaggttgta tggttggtgg gccttttgca attgataatg cagtatcttt
5520agaagcagct aaacataaag gtataaatca tcctgtagca ggacgagctg
atatattatt 5580agccccagat attgaaggtg gtaacatatt atataaagct
ttggtattct tctcaaaatc 5640aaaaaatgca ggagttatag ttggggctaa
agcaccaata atattaactt ctagagcaga 5700cagtgaagaa actaaactaa
actcaatagc tttaggtgtt ttaatggcag caaaggcata 5760ataagaagga
gatatacata tgagcaaaat atttaaaatc ttaacaataa atcctggttc
5820gacatcaact aaaatagctg tatttgataa tgaggattta gtatttgaaa
aaactttaag 5880acattcttca gaagaaatag gaaaatatga gaaggtgtct
gaccaatttg aatttcgtaa 5940acaagtaata gaagaagctc taaaagaagg
tggagtaaaa acatctgaat tagatgctgt 6000agtaggtaga ggaggacttc
ttaaacctat aaaaggtggt acttattcag taagtgctgc 6060tatgattgaa
gatttaaaag tgggagtttt aggagaacac gcttcaaacc taggtggaat
6120aatagcaaaa caaataggtg aagaagtaaa tgttccttca tacatagtag
accctgttgt 6180tgtagatgaa ttagaagatg ttgctagaat ttctggtatg
cctgaaataa gtagagcaag 6240tgtagtacat gctttaaatc aaaaggcaat
agcaagaaga tatgctagag aaataaacaa 6300gaaatatgaa gatataaatc
ttatagttgc acacatgggt ggaggagttt ctgttggagc 6360tcataaaaat
ggtaaaatag tagatgttgc aaacgcatta gatggagaag gacctttctc
6420tccagaaaga agtggtggac taccagtagg tgcattagta aaaatgtgct
ttagtggaaa 6480atatactcaa gatgaaatta aaaagaaaat aaaaggtaat
ggcggactag ttgcatactt 6540aaacactaat gatgctagag aagttgaaga
aagaattgaa gctggtgatg aaaaagctaa 6600attagtatat gaagctatgg
catatcaaat ctctaaagaa ataggagcta gtgctgcagt 6660tcttaaggga
gatgtaaaag caatattatt aactggtgga atcgcatatt caaaaatgtt
6720tacagaaatg attgcagata gagttaaatt tatagcagat gtaaaagttt
atccaggtga 6780agatgaaatg attgcattag ctcaaggtgg acttagagtt
ttaactggtg aagaagaggc 6840tcaagtttat gataactaat aa
6862141420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 14ttattatcgc accgcaatcg ggattttcga
ttcataaagc aggtcgtagg tcggcttgtt 60gagcaggtct tgcagcgtga aaccgtccag
atacgtgaaa aacgacttca ttgcaccgcc 120gagtatgccc gtcagccggc
aggacggcgt aatcaggcat tcgttgttcg ggcccataca 180ctcgaccagc
tgcatcggtt cgaggtggcg gacgaccgcg ccgatattga tgcgttcggg
240cggcgcggcc agcctcagcc cgccgccttt cccgcgtacg ctgtgcaaga
acccgccttt 300gaccagcgcg gtaaccactt tcatcaaatg gcttttggaa
atgccgtagg tcgaggcgat 360ggtggcgata ttgaccagcg cgtcgtcgtt
gacggcggtg tagatgagga cgcgcagccc 420gtagtcggta tgttgggtca
gatacataca acctccttag tacatgcaaa attatttcta 480gagcaacata
cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagttgagtt
540gaggaattat aacaggaaga aatattcctc atacgcttgt aattcctcta
tggttgttga 600caattaatca tcggctcgta taatgtataa cattcatatt
ttgtgaattt taaactctag 660aaataatttt gtttaacttt aagaaggaga
tatacatatg gctagcaaag gcgaagaatt 720gttcacgggc gttgttccta
ttttggttga attggatggc gatgttaatg gccataaatt 780cagcgttagc
ggcgaaggcg aaggcgatgc tacgtatggc aaattgacgt tgaaattcat
840ttgtacgacg ggcaaattgc ctgttccttg gcctacgttg gttacgacgt
tcagctatgg 900cgttcaatgt ttcagccgtt atcctgatca tatgaaacgt
catgatttct tcaaaagcgc 960tatgcctgaa ggctatgttc aagaacgtac
gattagcttc aaagatgatg gcaattataa 1020aacgcgtgct gaagttaaat
tcgaaggcga tacgttggtt aatcgtattg aattgaaagg 1080cattgatttc
aaagaagatg gcaatatttt gggccataaa ttggaatata attataatag
1140ccataatgtt tatattacgg ctgataaaca aaaaaatggc attaaagcta
atttcaaaat 1200tcgtcataat attgaagatg gcagcgttca attggctgat
cattatcaac aaaatacgcc 1260tattggcgat ggccctgttt tgttgcctga
taatcattat ttgagcacgc aaagcgcttt 1320gagcaaagat cctaatgaaa
aacgtgatca tatggttttg ttggaattcg ttacggctgc 1380tggcattacg
catggcatgg atgaattgta taaataataa 142015967DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
15caaatatcac ataatcttaa catatcaata aacacagtaa agtttcatgt gaaaaacatc
60aaacataaaa tacaagctcg gaatacgaat cacgctatac acattgctaa caggaatgag
120attatctaaa tgaggattga tatattaatt ggacatacta gtttttttca
tcaaaccagt 180agagataact tccttcacta tctcaatgag gaagaaataa
aacgctatga tcagtttcat 240tttgtgagtg ataaagaact ctatatttta
agccgtatcc tgctcaaaac agcactaaaa 300agatatcaac ctgatgtctc
attacaatca tggcaattta gtacgtgcaa atatggcaaa 360ccatttatag
tttttcctca gttggcaaaa aagatttttt ttaacctttc ccatactata
420gatacagtag ccgttgctat tagttctcac tgcgagcttg gtgtcgatat
tgaacaaata 480agagatttag acaactctta tctgaatatc agtcagcatt
tttttactcc acaggaagct 540actaacatag tttcacttcc tcgttatgaa
ggtcaattac ttttttggaa aatgtggacg 600ctcaaagaag cttacatcaa
atatcgaggt aaaggcctat ctttaggact ggattgtatt 660gaatttcatt
taacaaataa aaaactaact tcaaaatata gaggttcacc tgtttatttc
720tctcaatgga aaatatgtaa ctcatttctc gcattagcct ctccactcat
cacccctaaa 780ataactattg agctatttcc tatgcagtcc caactttatc
accacgacta tcagctaatt 840cattcgtcaa atgggcagaa ttgaatcgcc
acggataatc tagacacttc tgagccgtcg 900ataatattga ttttcatatt
ccgtcggtgg tgtaagtatc ccgcataatc gtgccattca 960catttag
96716424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 16ggatgggggg aaacatggat aagttcaaag
aaaaaaaccc gttatctctg cgtgaaagac 60aagtattgcg catgctggca caaggtgatg
agtactctca aatatcacat aatcttaaca 120tatcaataaa cacagtaaag
tttcatgtga aaaacatcaa acataaaata caagctcgga 180atacgaatca
cgctatacac attgctaaca ggaatgagat tatctaaatg aggattgatg
240tgtaggctgg agctgcttcg aagttcctat actttctaga gaataggaac
ttcggaatag 300gaacttcgga ataggaacta aggaggatat tcatatgtcg
tcaaatgggc agaattgaat 360cgccacggat aatctagaca cttctgagcc
gtcgataata ttgattttca tattccgtcg 420gtgg 424
* * * * *
References