U.S. patent application number 12/723035 was filed with the patent office on 2010-09-16 for antisense antibacterial method and compound.
Invention is credited to Bruce L. Geller, Patrick L. Iversen, Lucas D. Tilley.
Application Number | 20100234280 12/723035 |
Document ID | / |
Family ID | 38923531 |
Filed Date | 2010-09-16 |
United States Patent
Application |
20100234280 |
Kind Code |
A1 |
Geller; Bruce L. ; et
al. |
September 16, 2010 |
ANTISENSE ANTIBACTERIAL METHOD AND COMPOUND
Abstract
An antibacterial antisense conjugate and method of using the
same for treating a bacterial infection in a mammalian host are
disclosed. The conjugate includes an antisense oligonucleotide
conjugated to a carrier peptide that significantly enhances the
antibacterial activity of the oligonucleotide. The antisense
oligonucleotide contains 10-20 nucleotide bases and has a targeting
nucleic acid sequence complementary to a target sequence containing
or within 10 bases, in a downstream direction, of the translational
start codon of a bacterial mRNA that encodes a bacterial protein
essential for bacterial replication, where the compound binds to a
target mRNA with a T.sub.m of between 50.degree. to 60.degree. C.
The carrier peptide is an arginine-rich peptide containing between
6 and 12 amino acids.
Inventors: |
Geller; Bruce L.;
(Corvallis, OR) ; Iversen; Patrick L.; (Corvallis,
OR) ; Tilley; Lucas D.; (Burlington, VT) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE, SUITE 5400
SEATTLE
WA
98104
US
|
Family ID: |
38923531 |
Appl. No.: |
12/723035 |
Filed: |
March 12, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11487009 |
Jul 13, 2006 |
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12723035 |
|
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60699280 |
Jul 13, 2005 |
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Current U.S.
Class: |
514/2.4 ;
514/2.8 |
Current CPC
Class: |
C12N 15/113 20130101;
A61P 31/04 20180101; C12N 2310/3513 20130101; C12N 2310/11
20130101; C12N 2310/3233 20130101; A61K 48/00 20130101; C12N
2320/50 20130101; C12N 15/111 20130101 |
Class at
Publication: |
514/8 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 31/04 20060101 A61P031/04 |
Claims
1-22. (canceled)
23. A method of treating a bacterial infection in a mammalian host,
comprising administering to the host, a therapeutically effective
amount of an antisense conjugate composed of: (a) a substantially
uncharged antisense oligonucleotide having between 10-20 bases and
a targeting sequence of at least 10 contiguous bases complementary
to a target region of the infecting bacteria's mRNA for acyl
carrier protein (acpP), where the target region contains the
translational start codon of the bacterial mRNA, or a sequence that
is within 20 bases, in a downstream direction, of the translational
start codon, thereby to inhibit replication of the bacteria, and
(b) conjugated to the oligonucleotide, a carrier peptide that is
(i) represented by the sequence (RXX).sub.n--, where X is an
uncharged amino acid selected from the group consisting of alanine,
.beta.-alanine, valine, leucine, isoleucine, serine, threonine,
phenylalanine, and tryptophan, and n=2 or 3, and (ii) coupled to
the oligonucleotide at the peptide's C terminus.
24. The method of claim 23, wherein the carrier peptide has the
form (RFF).sub.n.
25. The method of claim 24, wherein the carrier peptide has the
form (RFF).sub.nR--.
26. The method of claim 23, wherein the carrier peptide is linked
at its C-terminus to the 5' end of the oligonucleotide through a
one- or two-amino acid linker.
27. The method of claim 23, wherein the carrier peptide is linked
at its C-terminus to the 3' end of the oligonucleotide through a
one- or two-amino acid linker.
28. The method of claim 26, wherein the linker is Ahx.beta.Ala,
where Ahx is 6-aminohexanoic acid and .beta.Ala is
.beta.-alanine.
29. The method of claim 23, wherein the oligonucleotide of the
conjugate is composed of morpholino subunits and
phosphorus-containing intersubunit linkages joining a morpholino
nitrogen of one subunit to a 5' exocyclic carbon of an adjacent
subunit.
30. The method of claim 29, wherein the morpholino subunits in the
oligonucleotide are joined by phosphorodiamidate linkages, in
accordance with the structure: ##STR00002## where Y.sub.1.dbd.O,
Z.dbd.O, Pj is a purine or pyrimidine base-pairing moiety effective
to bind, by base-specific hydrogen bonding, to a base in a
polynucleotide, and X is alkyl, alkoxy, thioalkoxy, amino or alkyl
amino, including dialkylamino.
31. The method of claim 29, wherein the oligonucleotide contains 1
positively charged linkage per every 3-10 linkages.
32. The method of claim 23, for treating a gram-negative bacterial
infection.
33. The method of claim 32, wherein the gram-negative bacterial
infection is a Burkholderia infection.
34. The method of claim 33, wherein the Burkholderia infection is a
Burkholderia cepacia, Burkholderia mallei, or Burkholderia
pseudomallei infection.
35. The method of claim 34, wherein the Burkholderia cepacia
infection is a Burkholderia multivorans, Burkholderia
vietnamiensis, Burkholderia stabilis, Burkholderia cenocepacia, or
Burkholderia ambifaria infection.
36. The method of claim 23, for treating a gram-positive bacterial
infection.
37. The method of claim 36, wherein the gram-positive infection is
a Clostridium infection.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/699,280, filed Jul. 13, 2005, incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to peptide-conjugated
oligonucleotide compounds that are antisense to bacterial genes and
methods for use of such compounds in inhibiting bacterial growth,
e.g., In an infected mammalian subject.
REFERENCES
[0003] Anderson, K. P., M. C. Fox, et al. (1996). Antimicrob Agents
Chemother 40(9): 2004-11. [0004] Blommers, M. J., U. Pieles, et al.
(1994). Nucleic Acids Res 22(20): 4187-94. [0005] Bramhill, D.
(1997). Annu Rev Cell Dev Biol 13: 395-424. [0006] Cross, C. W., J.
S. Rice, of al. (1997). Biochemistry 36(14): 4096-107. [0007]
Donachie, W. D. (1993). Annu Rev Microbiol 47: 199-230. [0008]
Gait, M. J., A. S. Jones, et al. (1974). J Chem Soc [Perkin 1]
0(14): 1684-6. [0009] Geller, B. L., J. D. Deere, et al. (2003).
Antimicrob Agents Chemother 47(10): 3233-9. [0010] Geller, B. L.
and H. M. Green (1989). J Biol Chem 264(28): 16465-9. [0011]
Gerdes, S. Y., M. D. Scholle, et al. (2003). J Bacteriol 185(19):
5673-84. [0012] Good, L., S. K. Awasthi, et al., (2001). Nat
Biotechnol 19(4): 360-4. [0013] Good, L. and P. E. Nielsen (1998).
Proc Natl Acad Sci USA 95(5): 2073-6. [0014] Hale, C. A. and P. A.
de Boer (1999). J Bacteriol 181(1): 167-76. [0015] Lesnikowski, Z.
J., M. Jaworska, et al., (1990). Nucleic Acids Res 18(8): 2109-15.
[0016] Lutkenhaus, J. and S. G. Addinall (1997). Annu Rev Biochem
66: 93-116. [0017] Mertes, M. P. and E. A. Coats (1969). J Med Chem
12(1): 154-7. [0018] Pari, G. S., A. K. Field, et al., (1995).
Antimicrob Agents Chemother 39(5): 1157-61. [0019] Rahman, M. A.,
J. Summerton, et al. (1991). Antisense Res Dev 1(4): 319-27. [0020]
Summerton, J., D. Stein, et al. (1997). Antisense Nucleic Acid Drug
Dev 7(2): 63-70. [0021] Summerton, J. and D. Weller (1997).
Antisense Nucleic Acid Drug Dev 7(3): 187-95. [0022] Zhang, Y. and
J. E. Cronan, Jr. (1996). J Bacteriol 178(12): 3614-20.
BACKGROUND OF THE INVENTION
[0023] Currently, there are several types of antibiotic compounds
in use against bacterial pathogens, and these compounds act through
a variety of anti-bacterial mechanisms. For example, beta-lactam
antibiotics, such as penicillin and cephalosporin, act to inhibit
the final step in peptidoglycan synthesis. Glycopeptide
antibiotics, including vancomycin and teichoplanin, inhibit both
transglycosylation and transpeptidation of muramyl-pentapeptide,
again interfering with peptidoglycan synthesis. Other well-known
antibiotics include the quinolones, which inhibit bacterial DNA
replication, inhibitors of bacterial RNA polymerase, such as
rifampin, and inhibitors of enzymes in the pathway for production
of tetrahydrofolate, including the sulfonamides.
[0024] Some classes of antibiotics act at the level of protein
synthesis. Notable among these are the aminoglycosides, such as
kanamycin and gentamycin. This class of compounds targets the
bacterial 30S ribosome subunit, preventing the association with the
50S subunit to form functional ribosomes. Tetracyclines, another
important class of antibiotics, also target the 30S ribosome
subunit, acting by preventing alignment of aminoacylated tRNA's
with the corresponding mRNA codon. Macrolides and lincosamides,
another class of antibiotics, inhibit bacterial synthesis by
binding to the 50S ribosome subunit, and inhibiting peptide
elongation or preventing ribosome translocation.
[0025] Despite impressive successes in controlling or eliminating
bacterial infections by antibiotics, the widespread use of
antibiotics both in human medicine and as a feed supplement in
poultry and livestock production has led to drug resistance in many
pathogenic bacteria. Antibiotic resistance mechanisms can take a
variety of forms. One of the major mechanisms of resistance to beta
lactams, particularly in Gram-negative bacteria, is the enzyme
beta-lactamase, which renders the antibiotic inactive. Likewise,
resistance to aminoglycosides often involves an enzyme capable of
inactivating the antibiotic, in this case by adding a phosphoryl,
adenyl, or acetyl group. Active efflux of antibiotics is another
way that many bacteria develop resistance. Genes encoding efflux
proteins, such as the tetA, tetG, tetL, and tetK genes for
tetracycline efflux, have been identified. A bacterial target may
develop resistance by altering the target of the drug. For example,
the so-called penicillin binding proteins (PBPs) in many
beta-lactam resistant bacteria are altered to inhibit the critical
antibiotic binding to the target protein. Resistance to
tetracycline may involve, in addition to enhanced efflux, the
appearance of cytoplasmic proteins capable of competing with
ribosomes for binding to the antibiotic. For those antibiotics that
act by inhibiting a bacterial enzyme, such as for sulfonamides,
point mutations in the target enzyme may confer resistance.
[0026] The appearance of antibiotic resistance in many pathogenic
bacteria--in many cases involving multi-drug resistance--has raised
the specter of a pre-antibiotic era in which many bacterial
pathogens are simply untreatable by medical intervention. There are
two main factors that could contribute to this scenario. The first
is the rapid spread of resistance and multi-resistance genes across
bacterial strains, species, and genera by conjugative elements, the
most important of which are self-transmissible plasmids. The second
factor is a lack of current research efforts to find new types of
antibiotics, due in part to the perceived investment in time and
money needed to find new antibiotic agents and bring them through
clinical trials, a process that may require a 20-year research
effort in some cases.
[0027] In addressing the second of these factors, some
drug-discovery approaches that may accelerate the search for new
antibiotics have been proposed. For example, efforts to screen for
and identify new antibiotic compounds by high-throughput screening
have been reported, but to date no important lead compounds have
been discovered by this route.
[0028] Several approaches that involve antisense agents designed to
block the expression of bacterial resistance genes or to target
cellular RNA targets, such as the rRNA in the 30S ribosomal
subunit, have been proposed (Rahman, Summerton at al. 1991; Good
and Nielsen 1998). In general, these approaches have been
successful only in a limited number of cases, or have required high
antisense concentrations (e.g., (Summerton, Stein et al. 1997), or
the requirement that the treated cells show high permeability for
antibiotics (Good and Nielsen 1998; Geller, Deere at al. 2003).
[0029] There is thus a growing need for new antibiotics that (i)
are not subject to the principal types of antibiotic resistance
currently hampering antibiotic treatment of bacteria, (ii) can be
developed rapidly and with some reasonable degree of predictability
as to target-bacteria specificity, (iii) can also be designed for
broad-spectrum activity, (iv) are effective at low doses, meaning,
in part, that they are efficiently taken up by wild-type bacteria
or even bacteria that have reduced permeability for antibiotics,
and (v) show few side effects.
SUMMARY OF THE INVENTION
[0030] The invention includes, in one aspect, an antibacterial
antisense conjugate for use in treating a bacterial infection in a
mammalian host. The conjugate includes a substantially uncharged
antisense oligonucleotide having between 10-20 bases and a
targeting sequence of at least 10 contiguous bases complementary to
a target region of the infecting bacteria's mRNA for acyl carrier
protein (acpP), gyrase A subunit (gyrA), or ftsZ gene, where the
target region contains the translational start codon of the
bacterial mRNA, or a sequence that is within 20 bases, in a
downstream direction, of the translational start codon, and where
oligonucleotide binds to the mRNA to form a heteroduplex having a
T.sub.m of at least 50.degree., thereby to inhibit replication of
the bacteria.
[0031] Also included in the conjugate, and conjugated to the
oligonucleotide, is an arginine-rich carrier protein coupled to the
oligonucleotide at the peptide's C terminus, and preferably
represented by the peptide sequence (RXX).sub.n--, where X is an
uncharged amino acid selected from the group consisting of alanine,
.beta.-alanine, valine, leucine, isoleucine, serine, threonine,
phenyalanine, and tryptophan, and n=2 or 3.
[0032] In exemplary embodiments, the carrier peptide has the
sequence (RFF).sub.n or (RFF).sub.nR, where n=2 or 3. The carrier
peptide may be linked at its C-terminus to one end of the
oligonucleotide, e.g., the 5'-end, through a one- or two-amino acid
linker, such as the linker is Ahx.beta.Ala, where Ahx is
6-aminohexanoic acid and .beta.Ala is .beta.-alanine.
[0033] The arginine-rich carrier peptide in the conjugate
preferably has the ability, when conjugated to the 5' end of a
phosphorodiamidate-linked morpholino oligomer (PMO) having SEQ ID
NO:66, to enhance the anti-bacterial activity of the PMO by a
factor of at least 10, preferably at least 10.sup.2, as measured by
the reduction in bacterial colony-forming units/ml (CFU/ml) when
the peptide-conjugated PMO compound is added at a concentration of
20 .mu.M in a culture to E coli, strain W3110 at 5.times.10.sup.7
CFU/ml in Luria broth for a period of 8 hours at 37.degree. C. with
aeration, relative to the same activity of the PMO oligonucleotide
alone.
[0034] The oligonucleotide of the conjugate may be composed of
morpholino subunits and phosphorus-containing intersubunit linkages
joining a morpholino nitrogen of one subunit to a 5' exocyclic
carbon of an adjacent subunit. The morpholino subunits in the
oligonucleotide may be joined by phosphorodiamidate linkages, in
accordance with the structure:
##STR00001##
where Y.sub.1.dbd.O, Z.dbd.O, Pj is a purine or pyrimidine
base-pairing moiety effective to bind, by base-specific hydrogen
bonding, to a base in a polynucleotide, and X is alkyl, alkoxy,
thioalkoxy, amino or alkyl amino, including dialkylamino.
[0035] The targeting sequence of the oligonucleotide may be
complementary to a target sequence containing or within 20 bases,
in a downstream direction, of the translational start codon of a
bacterial mRNA that encodes acyl carrier protein (acpP). In various
embodiments, particularly for use in treating a gram-negative
bacterial infection, the targeting sequence may be complementary to
at least ten contiguous bases in a sequence selected from the group
consisting of SEQ ID NOS: 2, 5, 8, 11, 14, 17, 20, 23, 28, 31, 34,
36, 39, 42, 45, 48, 51, 54, 57 and 60.
[0036] Alternatively, the targeting sequence of the oligonucleotide
may be complementary to a target sequence containing or within 20
bases, in a downstream direction, of the translational start codon
of a bacterial mRNA that encodes gyrase A subunit (gyrA). In
various embodiments, particularly for use in treating a
gram-negative bacterial infection, the targeting sequence may be
complementary to at least ten contiguous bases in a sequence
selected from the group consisting of SEQ ID NOS: 3, 6, 9, 12, 15,
18, 24, 29, 32, 35, 37, 40, 43, 46, 49, 52, 55, 58 and 61.
[0037] Where, the conjugate targets a bacterial mRNA encoding a
bacterial ftsZ protein, the compound targeting sequence may be
complementary to at least ten contiguous bases in a sequence
selected from the group consisting of SEQ ID NOS: 1, 4, 7, 10, 13,
16, 19, 22, 27, 30, 33, 38, 41, 44, 47, 50, 53, 56 and 59.
[0038] In accordance with another aspect of the invention, the
conjugate is used in treating a bacterial infection, by
administering a therapeutically effective amount of the conjugate
to a mammalian host.
[0039] These and other objects and features of the claimed subject
matter will become more fully apparent when the following detailed
description is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0040] FIGS. 1A-D show the repeating subunit segment of exemplary
morpholino oligonucleotides, designated A through D, constructed
using subunits having 5-atom (A), six-atom (B) and seven-atom (C-D)
linking groups suitable for forming polymers.
[0041] FIGS. 2A-2G show examples of uncharged linkage types in
oligonucleotide analogs and FIG. 2H shows a cationic linkage
group.
[0042] FIGS. 3A and 3B show the effect of AcpP antisense length on
growth of E. coli AS19. Cultures of E. coli AS19 were grown
(37.degree. C.) with various lengths (6 to 20 bases) of overlapping
PMO (20 .mu.M) targeted to the region around the start codon of the
E. coli acpP (Table 2, SEQ ID NO:2). Optical density (OD) was
monitored over time (FIG. 3A) and open squares indicate culture
with 11-base PMO 169 (SEQ ID NO:66) and viable cells (CFU/ml)
measured after 8 hours (FIG. 3B).
[0043] FIG. 4 shows the effect of antisense length on
AcpP-luciferase expression in cell-free translation reactions. PMOs
of various lengths and targeted around the start codon of acpP
(Table 2) were added individually (100 nM) to bacterial cell-free
translation reactions programmed to make AcpP-luc.
[0044] FIG. 5 shows CFU/ml in peritoneal lavages from mice infected
with permeable E. coli strain AS19 and treated with acpP PMO
(.box-solid.; SEQ ID NO:66), nonsense PMO (.tangle-solidup.), or
PBS () at 0 hours. At each time indicated, peritoneal lavage was
collected and analyzed for bacteria (CFU/ml) from 3 mice in each
treatment group.
[0045] FIG. 6 shows CFU/ml in peritoneal lavages from mice infected
E. coli strain SM105 and treated with PMO as described in FIG.
13.
[0046] FIGS. 7 to 10 show the growth as measured by optical density
(OD.sub.600) of four strains of E. coli grown for 24 hours in the
presence of the peptide-conjugated PMO (P-PMO) RFF-AcpP11 (SEQ ID
NO:79) compared to no treatment and treatment with a mixture of the
AcpP11 PMO and the RFF peptide (SEQ ID NOS:66 and 79,
respectively).
[0047] FIG. 11 shows the colony-forming units per milliliter
(CFU/ml) of four strains of E. coli after eight hours incubation in
the presence of the RFF-AcpP11 P-PMO compared to no treatment and
treatment with a mixture of the AcpP11 PMO and the RFF peptide.
[0048] FIG. 12 shows the antibacterial activity of three P-PMOs as
measured by CFU/ml after 8 hours of treatment.
[0049] FIG. 13 shows the effect of treatment with a dilution series
of RFF peptide, free RFF peptide mixed with AcpP11 PMO, REF-AcpP11
P-PMO, ampicillin or no treatment.
[0050] FIG. 14 shows the dose response curves for each of two PPMOs
compared to ampicillin and the associated IC.sub.50 values for
RFF-AcpP11, RTR-AcpP11 (SEQ ID NOS:88 and 89) and ampicillin.
[0051] FIGS. 15 and 16 show the effect of RFF-AcpP11 P-PMO on S.
typhimurium and an enterpathogenic strain of E. coli (O127:H6) as
measured by CFU/ml after eight hours of treatment compared to no
treatment, AcpP11 alone, REF peptide alone, scrambled controls, and
a mixture of AcpP11 and RFF peptide.
[0052] FIGS. 17 and 18 show the effect of RFFR-conjugated PMOs on
the growth of Burkholderia cenocepacia and Pseudomonas aeruginosa,
respectively.
DETAILED DESCRIPTION
I. Definitions
[0053] The terms below, as used herein, have the following
meanings, unless indicated otherwise:
[0054] As used herein, the terms "compound", "agent", "oligomer"
and "oligonucleotide" may be used interchangeably with respect to
the antisense oligonucleotides of the claimed subject matter.
[0055] As used herein, the terms "antisense oligonucleotide" and
"oligonucleotide" or "antisense compound" or "oligonucleotide
compound" are used interchangeably and refer to a sequence of
subunits, each having a base carried on a backbone subunit composed
of ribose or other pentose sugar or morpholino group, and where the
backbone groups are linked by intersubunit linkages (the majority
of which are uncharged) that allow the bases in the compound to
hybridize to a target sequence in an RNA by Watson-Crick base
pairing, to form an RNA:oligonucleotide heteroduplex within the
target sequence. The oligonucleotide may have exact sequence
complementarity to the target sequence or near complementarity.
Such antisense oligonucleotides are designed to block or inhibit
translation of the mRNA containing the target sequence, and may be
said to be "directed to" a sequence with which it hybridizes.
Exemplary structures for antisense oligonucleotides for use in the
claimed subject matter include the .beta.-morpholino subunit types
shown in FIGS. 1A-D. It will be appreciated that a polymer may
contain more than one linkage type.
[0056] The term "oligonucleotide" or "antisense oligonucleotide"
also encompasses an oligonucleotide having one or more additional
moieties conjugated to the oligonucleotide, e.g., at its 3'- or
5'-end, such as a polyethyleneglycol moiety or other hydrophilic
polymer, e.g., one having 10-100 monomeric subunits, which may be
useful in enhancing solubility, or a moiety such as a lipid or
peptide moiety that is effective to enhance the uptake of the
compound into target bacterial cells and/or enhance the activity of
the compound within the cell, e.g., enhance its binding to a target
polynucleotide.
[0057] A carrier peptide conjugated to an antisense
oligonucleotide, e.g., by covalent linkage between the peptide's C
terminal end and the 5' end of the oligonucleotide, is separately
named, i.e., not included within the term "oligonucleotide." The
carrier peptide and covalently attached antisense oligonucleotide
are also referred to herein as a conjugate or conjugate compound.
More generally, a "peptide-conjugated morpholino antisense
oligonucleotide" is a morpholino antisense oligonucleotide
conjugated at either its 5' or 3' termini to an arginine-rich
peptide carrier.
[0058] By "arginine-rich carrier peptide" is meant that the carrier
peptide that has at least 2, and preferably 2-4 arginine residues,
each preferably separated by one or more uncharged, hydrophobic
residues (at least as hydrophobic as threonine), and preferably
containing 6-12 amino acid residues. Exemplary arginine rich
peptides are listed as SEQ ID NOS:79-82, and 85-87. In exemplary
embodiments, the carrier peptide has the sequence (RXX).sub.n,
where X is an uncharged amino acid selected from the group
consisting of alanine, .beta.-alanine, valine, leucine, isoleucine,
serine, threonine, phenyalanine, and tryptophan, and n=2 or 3. In
preferred embodiments, the carrier peptide has the form (RFF).sub.n
or (RFF).sub.nR, where n=2 or 3. The carrier peptide may be linked
at its C-terminus to one end of the oligonucleotide, e.g., the
5'-end, through a one- or two-amino acid linker, such as the linker
is Ahx.beta.Ala, where Ahx is 6-aminohexanoic acid and .beta.Ala is
.beta.-alanine, and where the linker forms part of the carrier
peptide.
[0059] FIG. 1A shows a 1-atom phosphorous-containing linkage which
forms the five atom repeating-unit backbone where the morpholino
rings are linked by a 1-atom phosphonamide linkage.
[0060] FIG. 1B shows a six atom repeating-unit backbone where the
atom Y linking the 5' morpholino carbon to the phosphorous group
may be sulfur, nitrogen, carbon or, preferably, oxygen. The X
moiety pendant from the phosphorous may be any of the following:
fluorine; an alkyl or substituted alkyl; an alkoxy or substituted
alkoxy; a thioalkoxy or substituted thioalkoxy; or, an
unsubstituted, monosubstituted, or disubstituted nitrogen,
including cyclic structures.
[0061] FIGS. 1C-D show 7-atom unit-length backbones. In FIG. 1C the
X moiety is as in Structure B of FIG. 1 and the moiety Y may be a
methylene, sulfur, or preferably oxygen. In the structure shown in
FIG. 1D the X and Y moieties are as in FIG. 1B. In all subunits
depicted in FIGS. 1A-D, Z is O or S, and P.sub.i or P.sub.j is
adenine, cytosine, guanine, thymine, uracil or inosine.
[0062] As used herein, a "morpholino oligomer" or "morpholino
oligonucleotide" refers to an antisense oligonucleotide having a
backbone which supports bases capable of hydrogen bonding to
natural polynucleotides, e.g., DNA or RNA, is composed of
morpholino subunit structures of the form shown in FIG. 1B, where
(i) the structures are linked together by phosphorous-containing
linkages, one to three atoms long, joining the morpholino nitrogen
of one subunit to the 5' exocyclic carbon of an adjacent subunit,
and (ii) P.sub.i and P.sub.j are purine or pyrimidine base-pairing
moieties effective to bind, by base-specific hydrogen bonding, to a
base in a polynucleotide.
[0063] This preferred aspect of the claimed subject matter is
illustrated in FIG. 2G, which shows two such subunits joined by a
phosphorodiamidate linkage. Morpholino oligonucleotides (including
antisense oligonucleotides) are detailed, for example, in co-owned
U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,
5,166,315, 5,185,444, 5,521,063, and 5,506,337, all of which are
expressly incorporated by reference herein. FIG. 2H shows a
cationic linkage group.
[0064] As used herein, a "nuclease-resistant" oligonucleotide
molecule (oligonucleotide) is one whose backbone is not susceptible
to nuclease cleavage of a phosphodiester bond. Exemplary nuclease
resistant antisense oligonucleotides are oligonucleotide analogs,
such as phosphorothioate and phosphate-amine DNA (pnDNA), both of
which have a charged backbone, and methyl-phosphonate, and
morpholino oligonucleotides, all of which may have uncharged
backbones.
[0065] As used herein, an antisense oligonucleotide "specifically
hybridizes" to a target polynucleotide if the oligonucleotide
hybridizes to the target under physiological conditions, with a Tm
greater than 37.degree. C. As will be seen below, the antisense
oligonucleotides of the claimed subject matter have a preferred Tm
values with respect to their target mRNAs of at least 50.degree.
C., typically between 50.degree.-60.degree. C. or greater.
[0066] The "Tm" of an oligonucleotide compound, with respect to its
target mRNA, is the temperature at which 50% of a target sequence
hybridizes to a complementary polynucleotide. Tm is determined
under standard conditions in physiological saline, as described,
for example, in Miyada C. G. and Wallace R. B. 1987,
Oligonucleotide hybridization techniques, Methods Enzymol.,
154:94-107.
[0067] Polynucleotides are described as "complementary" to one
another when hybridization occurs in an antiparallel configuration
between two single-stranded polynucleotides. A double-stranded
polynucleotide can be "complementary" to another polynucleotide, if
hybridization can occur between one of the strands of the first
polynucleotide and the second. Complementarity (the degree that one
polynucleotide is complementary with another) is quantifiable in
terms of the proportion of bases in opposing strands that are
expected to form hydrogen bonds with each other, according to
generally accepted base-pairing rules.
[0068] As used herein, a first sequence is an "antisense sequence"
with respect to a second sequence if a polynucleotide whose
sequence is the first sequence specifically binds to, or
specifically hybridizes with, the second polynucleotide sequence
under physiological conditions.
[0069] As used herein, a "base-specific intracellular binding event
involving a target RNA" refers to the sequence specific binding of
an oligonucleotide to a target RNA sequence inside a cell. For
example, a single-stranded polynucleotide can specifically bind to
a single-stranded polynucleotide that is complementary in
sequence.
[0070] As used herein, "nuclease-resistant heteroduplex" refers to
a heteroduplex formed by the binding of an antisense
oligonucleotide to its complementary target, which is resistant to
in vivo degradation by ubiquitous intracellular and extracellular
nucleases.
[0071] As used herein, "essential bacterial genes" are those genes
whose products play an essential role in an organism's functional
repertoire as determined using genetic footprinting or other
comparable techniques to identify gene essentiality.
[0072] An agent is "actively taken up by bacterial cells" when the
agent can enter the cell by a mechanism other than passive
diffusion across the cell membrane. The agent may be transported,
for example, by "active transport", referring to transport of
agents across a mammalian cell membrane by e.g. an ATP-dependent
transport mechanism, or by "facilitated transport", referring to
transport of antisense agents across the cell membrane by a
transport mechanism that requires binding of the agent to a
transport protein, which then facilitates passage of the bound
agent across the membrane. For both active and facilitated
transport, the oligonucleotide compound preferably has a
substantially uncharged backbone, as defined below.
[0073] As used herein, the terms "modulating expression" and
"antisense activity" relative to an oligonucleotide refers to the
ability of an antisense oligonucleotide to either enhance or reduce
the expression of a given protein by interfering with the
expression, or translation of RNA. In the case of reduced protein
expression, the antisense oligonucleotide may directly block
expression of a given gene, or contribute to the accelerated
breakdown of the RNA transcribed from that gene.
[0074] As used herein, the term "inhibiting bacterial growth,
refers to blocking or inhibiting replication and/or reducing the
rate of replication of bacterial cells in a given environment, for
example, in an infective mammalian host.
[0075] As used herein, the term "pathogenic bacterium," or
"pathogenic bacteria," or "pathogenic bacterial cells," refers to
bacterial cells capable of infecting and causing disease in a
mammalian host, as well as producing infection-related symptoms in
the infected host, such as fever or other signs of inflammation,
intestinal symptoms, respiratory symptoms, dehydration, and the
like.
[0076] As used herein, the terms "Gram-negative pathogenic
bacteria" or "Gram-negative bacteria" refer to the phylum of
proteobacteria, which have an outer membrane composed largely of
lipopolysaccharides. All proteobacteria are gram negative, and
include, but are not limited to Escherichia coli, Salmonella, other
Enterobacteriaceae, Pseudomonas, Burkholderi, Moraxella,
Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria,
and Legionella. Other notable groups of gram negative bacteria
include Haemophilus influenzae, the cyanobacteria, spirochaetes,
green sulfur and green non-sulfur bacteria. The pathogenic
capability of gram negative bacteria is usually associated with
components of the bacterial cell wall, in particular the
lipopolysaccharide (also known as LPS or endotoxin) layer.
[0077] As used herein, the terms "Gram-positive pathogenic
bacteria" or "Gram-positive bacteria" refer to those bacteria that
are stained dark blue or violet by Gram staining, in contrast to
Gram-negative bacteria, which cannot retain the stain, instead
taking up the counterstain and appearing red or pink. The stain is
caused by a high amount of peptidoglycan in the cell wall, which
typically, but not always, lacks the secondary membrane and
lipopolysaccharide layer found in Gram-negative bacteria.
[0078] Gram-positive bacteria include many well-known genera such
as Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus,
and Clostridium. It has also been expanded to include the
Mollicutes, and bacteria such as Mycoplasma, which lack cell walls
and so cannot be stained by Gram, but are derived from such
forms.
[0079] As used herein, "effective amount" or "therapeutically
effective amount" or "growth-inhibiting amount" relative to an
antisense oligonucleotide refers to the amount of antisense
oligonucleotide administered to a mammalian subject, either as a
single dose or as part of a series of doses and which is effective
to inhibit bacterial replication in an infected host, by inhibiting
translation of a selected bacterial target nucleic acid sequence.
The ability to block or inhibit bacterial replication in an
infected host may be evidenced by a reduction in infection-related
symptoms.
[0080] As used herein "treatment" of an individual or a cell is any
type of intervention used in an attempt to alter the natural course
of the individual or cell. Treatment includes, but is not limited
to, administration of e.g., a pharmaceutical composition, and may
be performed either prophylactically, or subsequent to the
initiation of a pathologic event or contact with an etiologic
agent.
[0081] A "substantially uncharged", phosphorus containing backbone
in an oligonucleotide analog is one in which a majority of the
subunit linkages, e.g., between 50-100%, are uncharged at
physiological pH, and contain a single phosphorous atom, For
example, the backbone may contain only uncharged linkages or 1
positively charged linkage per every 3-10 linkages.
II. Exemplary Oligonucleotide Backbones
[0082] Examples of nonionic linkages that may be used in
oligonucleotide analogs are shown in FIGS. 2A-2G. In these figures,
B represents a purine or pyrimidine base-pairing moiety effective
to bind, by base-specific hydrogen bonding, to a base in a
polynucleotide, preferably selected from adenine, cytosine,
guanine, thymidine, uracil and inosine. Suitable backbone
structures include carbonate (2A, R.dbd.O) and carbamate (2A,
R.dbd.NH.sub.2) linkages (Mertes and Coats 1969; Gait, Jones et al.
1974); alkyl phosphonate and phosphotriester linkages (2B, R=alkyl
or --O-alkyl)(Lesnikowski, Jaworska et al. 1990); amide linkages
(2C) (Blommers, Pieles et al. 1994); sulfone and sulfonamide
linkages (2D, R1, R2=CH.sub.2); and a thioformacetyl linkage (2E)
(Cross, Rice et al. 1997). The latter is reported to have enhanced
duplex and triplex stability with respect to phosphorothioate
antisense compounds (Cross, Rice et al. 1997). Also reported are
the 3'-methylene-N-methylhydroxyamino compounds of structure 2F.
The structure shown in FIG. 2H is an exemplary cationic linkage
type.
[0083] A preferred oligonucleotide structure employs
morpholino-based subunits bearing base-pairing moieties as
illustrated in FIGS. 1A-1D, joined by uncharged linkages, as
described above. Especially preferred is a substantially uncharged
phosphorodiamidate-linked morpholino oligonucleotide, such as
illustrated in FIG. 2G. Morpholino oligonucleotides, including
antisense oligonucleotides, are detailed, for example, in
(Summerton and Weller 1997) and in co-owned U.S. Pat. Nos.
5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444,
5,521,063, and 5,506,337, all of which are expressly incorporated
by reference herein.
[0084] Important properties of the morpholino-based subunits
include: 1) the ability to be linked in a oligomeric form by
stable, uncharged backbone linkages; 2) the ability to support a
nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil
and inosine) such that the polymer formed can hybridize with a
complementary-base target nucleic acid, including target RNA, Tm
values above about 50.degree. C. in relatively short
oligonucleotides (e.g., 10-15 bases); 3) the ability of the
oligonucleotide to be actively or passively transported into
bacterial cells; and 4) the ability of the oligonucleotide:RNA
heteroduplex to resist RNAse degradation.
[0085] A. Exemplary Antisense Oligonucleotides
[0086] Exemplary backbone structures for antisense oligonucleotides
of the claimed subject matter include the .beta.-morpholino subunit
types shown in FIGS. 1A-1D, each linked by an uncharged,
phosphorus-containing subunit linkage. FIG. 1A shows a
phosphorus-containing linkage which forms the five atom
repeating-unit backbone, where the morpholino rings are linked by a
1-atom phosphoamide linkage. FIG. 1B shows a linkage which produces
a 6-atom repeating-unit backbone. In this structure, the atom Y
linking the 5' morpholino carbon to the phosphorus group may be
sulfur, nitrogen, carbon or, preferably, oxygen. The X moiety
pendant from the phosphorus may be fluorine, an alkyl or
substituted alkyl, an alkoxy or substituted alkoxy, a thioalkoxy or
substituted thioalkoxy, or unsubstituted, monosubstituted, or
disubstituted nitrogen, including cyclic structures, such as
morpholines or piperidines. Alkyl, alkoxy and thioalkoxy preferably
include 1-6 carbon atoms. The Z moieties are sulfur or oxygen, and
are preferably oxygen.
[0087] The linkages shown in FIGS. 1C and 1D are designed for
7-atom unit-length backbones. In Structure 1C, the X moiety is as
in Structure 1B, and the moiety Y may be methylene, sulfur, or,
preferably, oxygen. In Structure 1D, the X and Y moieties are as in
Structure 1B. Particularly preferred morpholino oligonucleotides
include those composed of morpholino subunit structures of the form
shown in FIG. 1B, where X.dbd.NH.sub.2 or N(CH.sub.3).sub.2,
Y.dbd.O, and Z.dbd.O.
[0088] As noted above, the substantially uncharged oligonucleotide
may include a one or more of charged linkages, e.g. up to about 1
per every 3-10 uncharged linkages, such as about 1 per every 10
uncharged linkages. Therefore, a small number of charged linkages,
e.g. charged phosphoramidate or phosphorothioate, may also be
incorporated into the oligomers. An exemplary cationic linkage is
shown in FIG. 2H.
[0089] The antisense compounds can be prepared by stepwise
solid-phase synthesis, employing methods detailed in the references
cited above. In some cases, it may be desirable to add additional
chemical moieties to the antisense compound, e.g. to enhance
pharmacokinetics or to facilitate capture or detection of the
compound. Such a moiety may be covalently attached, typically to a
terminus of the oligomer, according to standard synthetic methods.
For example, addition of a polyethyleneglycol moiety or other
hydrophilic polymer, e.g., one having 10-100 monomeric subunits,
may be useful in enhancing solubility. One or more charged groups,
e.g., anionic charged groups such as an organic acid, may enhance
cell uptake. A reporter moiety, such as fluorescein or a
radiolabeled group, may be attached for purposes of detection.
Alternatively, the reporter label attached to the oligomer may be a
ligand, such as an antigen or biotin, capable of binding a labeled
antibody or streptavidin. In selecting a moiety for attachment or
modification of an antisense oligomer, it is generally of course
desirable to select chemical compounds of groups that are
biocompatible and likely to be tolerated by a subject without
undesirable side effects.
[0090] B. Antibacterial Antisense Oligonucleotides
[0091] In addition to the structural features described above, the
antisense compound of the claimed subject matter contains no more
than 20 nucleotide bases, and has a targeting nucleic acid sequence
(the sequence which is complementary to the target sequence) of no
fewer than 10 contiguous bases. The targeting sequence is
complementary to a target sequence containing or within 15 bases,
in a downstream direction, of the translational start codon of a
bacterial mRNA that encodes a bacterial protein essential for
bacterial replication. The compound has a T.sub.m, when hybridized
with the target sequence, of at least about 50.degree. C.,
typically between about 50.degree. to 60.degree. C., although the
Tm may be higher, e.g., 65.degree. C. The selection of bacterial
targets, and bacterial mRNA target sequences and complementary
targeting sequences are considered in the two sections below.
[0092] The antisense compound is covalently conjugated to an 8 to
12 residue arginine-rich peptide at either the 5' or 3' end of the
antisense oligomer of the type described above.
III. Bacterial Targets
[0093] This section considers a number of bacterial targets,
including pathogenic bacteria, and specific bacterial protein
targets against which the antisense compound can be directed.
[0094] A. Bacterial Targets
[0095] Escherichia coli (E. coli) is a Gram-negative bacterium that
is part of the normal flora of the gastrointestinal tract. There
are hundreds of strains of E. coli, most of which are harmless and
live in the gastrointestinal tract of healthy humans and animals.
Currently, there are four recognized classes of enterovirulent E.
coli (the "EEC group") that cause gastroenteritis in humans. Among
these are the enteropathogenic (EPEC) strains and those whose
virulence mechanism is related to the excretion of typical E. coli
enterotoxins. Such strains of E. coli can cause various diseases
including those associated with infection of the gastrointestinal
tract and urinary tract, septicemia, pneumonia, and meningitis.
Antibiotics are not effective against some strains and do not
necessarily prevent recurrence of infection.
[0096] For example, E. coli strain 0157:H7 is estimated to cause
10,000 to 20,000 cases of infection in the United States annually
(Federal Centers for Disease Control and Prevention). Hemorrhagic
colitis is the name of the acute disease caused by E. coil O157:H7.
Preschool children and the elderly are at the greatest risk of
serious complications. E. coli strain 0157:H7 was recently reported
as the cause the death of four children who ate under cooked
hamburgers from a fast-food restaurant in the Pacific Northwest.
(See, e.g., Jackson et al., Epidemiol. Infect. 120(1):17-20,
1998.)
[0097] Exemplary sequences for enterovirulent E. coli strains
include GenBank Accession Numbers AB011549, X97542, AF074613,
Y11275 and AJ007716.
[0098] Salmonella thyphimurium, are Gram-negative bacteria which
cause various conditions that range clinically from localized
gastrointestinal infections, gastroenterits (diarrhea, abdominal
cramps, and fever) to enteric fevers (including typhoid fever)
which are serious systemic illnesses. Salmonella infection also
causes substantial losses of livestock.
[0099] Typical of Gram-negative bacilli, the cell wall of
Salmonella spp. contains a complex lipopolysaccharide (LPS)
structure that is liberated upon lysis of the cell and may function
as an endotoxin, which contributes to the virulence of the
organism.
[0100] Contaminated food is the major mode of transmission for
non-typhoidal salmonella infection, due to the fact that Salmonella
survive in meats and animal products that are not thoroughly
cooked. The most common animal sources are chickens, turkeys, pigs,
and cows; in addition to numerous other domestic and wild animals.
The epidemiology of typhoid fever and other enteric fevers caused
by Salmonella spp. is associated with water contaminated with human
feces.
[0101] Vaccines are available for typhoid fever and are partially
effective; however, no vaccines are available for non-typhoidal
Salmonella infection. Non-typhoidal salmonellosis is controlled by
hygienic slaughtering practices and thorough cooking and
refrigeration of food. Antibiotics are indicated for systemic
disease, and Ampicillin has been used with some success. However,
in patients under treatment with excessive amounts of antibiotics,
patients under treatment with immunsuppressive drugs, following
gastric surgery, and in patients with hemolytic anemia, leukemia,
lymphoma, or AIDS, Salmonella infection remains a medical
problem.
[0102] Pseudomonas spp. are motile, Gram-negative rods which are
clinically important because they are resistant to most
antibiotics, and are a major cause of hospital acquired
(nosocomial) infections. Infection is most common in:
immunocompromised individuals, burn victims, individuals on
respirators, individuals with indwelling catheters, IV narcotic
users and individual with chronic pulmonary disease (e.g., cystic
fibrosis). Although infection is rare in healthy individuals, it
can occur at many sites and lead to urinary tract infections,
sepsis, pneumonia, pharyngitis, and numerous other problems, and
treatment often fails with greater significant mortality.
[0103] Pseudomonas aeruginosa is a Gram-negative, aerobic,
rod-shaped bacterium with unipolar motility. An opportunistic human
pathogen, P. aeruginosa is also an opportunistic pathogen of
plants. Like other Pseudomonads, P. aeruginosa secretes a variety
of pigments. Definitive clinical identification of P. aeruginosa
can include identifying the production of both pyocyanin and
fluorescein as well as the organism's ability to grow at 42.degree.
C. P. aeruginosa is also capable of growth in diesel and jet fuel,
for which it is known as a hydrocarbon utilizing microorganism (or
"HUM bug"), causing microbial corrosion.
[0104] Vibrio cholera is a Gram-negative rod which infects humans
and causes cholera, a disease spread by poor sanitation, resulting
in contaminated water supplies. Vibrio cholerae can colonize the
human small intestine, where it produces a toxin that disrupts ion
transport across the mucosa, causing diarrhea and water loss.
Individuals infected with Vibrio cholerae require rehydration
either intravenously or orally with a solution containing
electrolytes. The illness is generally self-limiting; however,
death can occur from dehydration and loss of essential
electrolytes. Antibiotics such as tetracycline have been
demonstrated to shorten the course of the illness, and oral
vaccines are currently under development. Neisseria gonorrhoea is a
Gram-negative coccus, which is the causative agent of the common
sexually transmitted disease, gonorrhea. Neisseria gonorrhoea can
vary its surface antigens, preventing development of immunity to
reinfection. Nearly 750,000 cases of gonorrhea are reported
annually in the United States, with an estimated 750,000 additional
unreported cases annually, mostly among teenagers and young adults.
Ampicillin, amoxicillin, or some type of penicillin used to be
recommended for the treatment of gonorrhea. However, the incidence
of penicillin-resistant gonorrhea is increasing, and new
antibiotics given by injection, e.g., ceftriaxone or spectinomycin,
are now used to treat most gonococcal infections. Staphylococcus
aureus is a Gram-positive coccus which normally colonizes the human
nose and is sometimes found on the skin. Staphylococcus can cause
bloodstream infections, pneumonia, and surgical-site infections in
the hospital setting (i.e., nosocomial infections). Staph. aureus
can cause severe food poisoning, and many strains grow in food and
produce exotoxins. Staphylococcus resistance to common antibiotics,
e.g., vancomycin, has emerged in the United States and abroad as a
major public health challenge both in community and hospital
settings. Recently, a vancomycin-resistant Staph. aureus isolate
has also been identified in Japan.
[0105] Mycobacterium tuberculosis is a Gram positive bacterium
which is the causative agent of tuberculosis, a sometimes crippling
and deadly disease. Tuberculosis is on the rise and globally and
the leading cause of death from a single infectious disease (with a
current death rate of three million people per year). It can affect
several organs of the human body, including the brain, the kidneys
and the bones, however, tuberculosis most commonly affects the
lungs.
[0106] In the United States, approximately ten million individuals
are infected with Mycobacterium tuberculosis, as indicated by
positive skin tests, with approximately 26,000 new cases of active
disease each year. The increase in tuberculosis (TB) cases has been
associated with HIV/AIDS, homelessness, drug abuse and immigration
of persons with active infections. Current treatment programs for
drug-susceptible TB involve taking two or four drugs (e.g.,
isoniazid, rifampin, pyrazinamide, ethambutol or streptomycin), for
a period of from six to nine months, because all of the TB germs
cannot be destroyed by a single drug. In addition, the observation
of drug-resistant and multiple drug resistant strains of
Mycobacterium tuberculosis is on the rise.
[0107] Helicobacter pylori (H. pylori) is a micro-aerophilic,
Gram-negative, slow-growing, flagellated organism with a spiral or
S-shaped morphology which infects the lining of the stomach. H.
pylori is a human gastric pathogen associated with chronic
superficial gastritis, peptic ulcer disease, and chronic atrophic
gastritis leading to gastric adenocarcinoma. H. pylori is one of
the most common chronic bacterial infections in humans and is found
in over 90% of patients with active gastritis. Current treatment
includes triple drug therapy with bismuth, metronidazole, and
either tetracycline or amoxicillin which eradicates H. pylori in
most cases. Problems with triple therapy include patient
compliance, side effects, and metronidazole resistance. Alternate
regimens of dual therapy which show promise are amoxicillin plus
metronidazole or omeprazole plus amoxicillin.
[0108] Streptococcus pneumoniae is a Gram-positive coccus and one
of the most common causes of bacterial pneumonia as well as middle
ear infections (otitis media) and meningitis. Each year in the
United States, pneumococcal diseases account for approximately
50,000 cases of bacteremia; 3,000 cases of meningitis;
100,000-135,000 hospitalizations; and 7 million cases of otitis
media. Pneumococcal infections cause an estimated 40,000 deaths
annually in the United States. Children less than 2 years of age,
adults over 65 years of age and persons of any age with underlying
medical conditions, including, e.g., congestive heart disease,
diabetes, emphysema, liver disease, sickle cell, HIV, and those
living in special environments, e.g., nursing homes and long-term
care facilities, at highest risk for infection.
[0109] Drug-resistant S. pneumoniae strains have become common in
the United States, with many penicillin-resistant pneumococci also
resistant to other antimicrobial drugs, such as erythromycin or
trimethoprim-sulfamethoxazole.
[0110] Treponema pallidium is a spirochete which causes syphilis.
T. pallidum is exclusively a pathogen which causes syphilis, yaws
and non-venereal endemic syphilis or pinta. Treponema pallidum
cannot be grown in vitro and does replicate in the absence of
mammalian cells. The initial infection causes an ulcer at the site
of infection; however, the bacteria move throughout the body,
damaging many organs over time. In its late stages, untreated
syphilis, although not contagious, can cause serious heart
abnormalities, mental disorders, blindness, other neurologic
problems, and death.
[0111] Syphilis is usually treated with penicillin, administered by
injection. Other antibiotics are available for patients allergic to
penicillin, or who do not respond to the usual doses of penicillin.
In all stages of syphilis, proper treatment will cure the disease,
but in late syphilis, damage already done to body organs cannot be
reversed.
[0112] Chlamydia trachomatis is the most common bacterial sexually
transmitted disease in the United States and it is estimated that 4
million new cases occur each year. The highest rates of infection
are in 15 to 19 year olds. Chlamydia is a major cause of
non-gonococcal urethritis (NGU), cervicitis, bacterial vaginitis,
and pelvic inflammatory disease (PID). Chlamydia infections may
have very mild symptoms or no symptoms at all; however, if left
untreated Chlamydia infections can lead to serious damage to the
reproductive organs, particularly in women. Antibiotics such as
azithromycin, erythromycin, oflloxacin, amoxicillin or doxycycline
are typically prescribed to treat Chlamydia infection.
[0113] Bartonella henselae Cat Scratch Fever (CSF) or cat scratch
disease (CSD), is a disease of humans acquired through exposure to
cats, caused by a Gram-negative rod originally named Rochalimaea
henselae, and currently known as Bartonella henselae. Symptoms
include fever and swollen lymph nodes and CSF is generally a
relatively benign, self-limiting disease in people, however,
infection with Bartonella henselae can produce distinct clinical
symptoms in immunocompromised people, including, acute febrile
illness with bacteremia, bacillary angiomatosis, peliosis hepatis,
bacillary splenitis, and other chronic disease manifestations such
as AIDS encephalopathy.
[0114] The disease is treated with antibiotics, such as
doxycycline, erythromycin, rifampin, penicillin, gentamycin,
ceftriaxone, ciprofloxacin, and azithromycin.
[0115] Haemophilus influenzae (H. influenza) is a family of
Gram-negative bacteria; six types of which are known, with most H.
influenza-related disease caused by type B, or "HIB". Until a
vaccine for HIB was developed, HIB was a common causes of otitis
media, sinus infections, bronchitis, the most common cause of
meningitis, and a frequent culprit in cases of pneumonia, septic
arthritis (joint infections), cellulitis (infections of soft
tissues), and pericarditis (infections of the membrane surrounding
the heart). The H. influenza type B bacterium is widespread in
humans and usually lives in the throat and nose without causing
illness. Unvaccinated children under age 5 are at risk for HIB
disease. Meningitis and other serious infections caused by H.
influenza infection can lead to brain damage or death.
[0116] Shigella dysenteriae (Shigella dys.) is a Gram-negative rod
which causes dysentary. In the colon, the bacteria enter mucosal
cells and divide within mucosal cells, resulting in an extensive
inflammatory response. Shigella infection can cause severe diarrhea
which may lead to dehydration and can be dangerous for the very
young, very old or chronically ill. Shigella dys. forms a potent
toxin (shiga toxin), which is cytotoxic, enterotoxic, neurotoxic
and acts as a inhibitor of protein synthesis. Resistance to
antibiotics such as ampicillin and TMP-SMX has developed, however,
treatment with newer, more expensive antibiotics such as
ciprofloxacin, norfloxacin and enoxacin, remains effective.
[0117] Listeria is a genus of Gram-positive, motile bacteria found
in human and animal feces. Listeria monocytogenes causes such
diseases as listeriosis, meningoencephalitis and meningitis. This
organism is one of the leading causes of death from food-borne
pathogens especially in pregnant women, newborns, the elderly, and
immunocompromised individuals. It is found in environments such as
decaying vegetable matter, sewage, water, and soil, and it can
survive extremes of both temperatures and salt concentration making
it an extremely dangerous food-born pathogen, especially on food
that is not reheated. The bacterium can spread from the site of
infection in the intestines to the central nervous system and the
fetal-placental unit. Meningitis, gastroenteritis, and septicemia
can result from infection. In cattle and sheep, listeria infection
causes encephalitis and spontaneous abortion.
[0118] Proteus mirabilis is an enteric, Gram-negative commensal
organism, distantly related to E. coli. It normally colonizes the
human urethra, but is an opportunistic pathogen that is the leading
cause of urinary tract infections in catheterized individuals. P.
mirabilis has two exceptional characteristics: 1) it has very rapid
motility, which manifests itself as a swarming phenomenon on
culture plates; and 2) it produce urease, which gives it the
ability to degrade urea and survive in the genitourinary tract.
[0119] Yersinia pestis is the causative agent of plague (bubonic
and pulmonary) a devastating disease which has killed millions
worldwide. The organism can be transmitted from rats to humans
through the bite of an infected flea or from human-to-human through
the air during widespread infection. Yersinia pestis is an
extremely pathogenic organism that requires very few numbers in
order to cause disease, and is often lethal if left untreated. The
organism is enteroinvasive, and can survive and propagate in
macrophages prior to spreading systemically throughout the
host.
[0120] Bacillus anthracis is also known as anthrax. Humans become
infected when they come into contact with a contaminated animal.
Anthrax is not transmitted due to person-to-person contact. The
three forms of the disease reflect the sites of infection which
include cutaneous (skin), pulmonary (lung), and intestinal.
Pulmonary and intestinal infections are often fatal if left
untreated. Spores are taken up by macrophages and become
internalized into phagolysozomes (membranous compartment) whereupon
germination initiates. Bacteria are released into the bloodstream
once the infected macrophage lyses whereupon they rapidly multiply,
spreading throughout the circulatory and lymphatic systems, a
process that results in septic shock, respiratory distress and
organ failure. The spores of this pathogen have been used as a
terror weapon.
[0121] Burkholderia mallei is a Gram-negative aerobic bacterium
that causes Glanders, an infectious disease that occurs primarily
in horses, mules, and donkeys. It is rarely associated with human
infection and is more commonly seen in domesticated animals. This
organism is similar to B. pseudomallei and is differentiated by
being nonmotile. The pathogen is host-adapted and is not found in
the environment outside of its host. Glanders is often fatal if not
treated with antibiotics, and transmission can occur through the
air, or more commonly when in contact with infected animals.
Rapid-onset pneumonia, bacteremia (spread of the organism through
the blood), pustules, and death are common outcomes during
infection. The virulence mechanisms are not well understood,
although a type III secretion system similar to the one from
Salmonella typhimurium is necessary. No vaccine exists for this
potentially dangerous organism which is thought to have potential
as a biological terror agent. The genome of this organism carries a
large number of insertion sequences as compared to the related
Bukholderia pseudomallei (below), and a large number of simple
sequence repeats that may function in antigenic variation of cell
surface proteins.
[0122] Burkholderia pseudomallei is a Gram-negative bacterium that
causes meliodosis in humans and animals. Meliodosis is a disease
found in certain parts of Asia, Thailand, and Australia. B.
pseudomallei is typically a soil organism and has been recovered
from rice paddies and moist tropical soil, but as an opportunistic
pathogen can cause disease in susceptible individuals such as those
that suffer from diabetes mellitus. The organism can exist
intracellularly, and causes pneumonia and bacteremia (spread of the
bacterium through the bloodstream). The latency period can be
extremely long, with infection preceding disease by decades, and
treatment can take months of antibiotic use, with relapse a
commonly observed phenomenon. Intercellular spread can occur via
induction of actin polymerization at one pole of the cell, allowing
movement through the cytoplasm and from cell-to-cell. This organism
carries a number of small sequence repeats which may promoter
antigenic variation, similar to what was found with the B. mallei
genome.
[0123] Burkholderia cepacia is a Gram-negative bacterium composed
of at least seven different sub-species, including Burkholderia
multivorans, Burkholderia vietnamiensis, Burkholderia stabilis,
Burkholderia cenocepacia and Burkholderia ambifaria. B. cepacia is
an important human pathogen which most often causes pneumonia in
people with underlying lung disease (such as cystic fibrosis or
immune problems (such as (chronic granulomatous disease). B.
cepacia is typically found in water and soil and can survive for
prolonged periods in moist environments. Person-to-person spread
has been documented; as a result, many hospitals, clinics, and
camps for patients with cystic fibrosis have enacted strict
isolation precautions B. cepacia. Individuals with the bacteria are
often treated in a separate area than those without to limit
spread. This is because infection with B. cepacia can lead to a
rapid decline in lung function resulting in death. Diagnosis of B.
cepacia involves isolation of the bacteria from sputum cultures.
Treatment is difficult because B. cepacia is naturally resistant to
many common antibiotics including aminoglycosides (such as
tobramycin) and polymixin B. Treatment typically includes multiple
antibiotics and may include ceftazidime, doxycycline, piperacillin,
chloramphenicol, and co-trimoxazole.
[0124] Francisella tularensis was first noticed as the causative
agent of a plague-like illness that affected squirrels in Tulare
County in California in the early part of the 20th century by
Edward Francis. The organism now bears his namesake. The disease is
called tularemia and has been noted throughout recorded history.
The organism can be transmitted from infected ticks or deerflies to
a human, through infected meat, or via aerosol, and thus is a
potential bioterrorism agent. It is an aquatic organism, and can
be'found living inside protozoans, similar to what is observed with
Legionella. It has a high infectivity rate, and can invade
phagocytic and nonphagocytic cells, multiplying rapidly. Once
within a macrophage, the organism can escape the phagosome and live
in the cytosol.
[0125] Veterinary Applications
[0126] A healthy microflora in the gastro-intestinal tract of
livestock is of vital importance for health and corresponding
production of associated food products. As with humans; the
gastrointestinal tract of a healthy animal contains numerous types
of bacteria (i.e., E. coli, Pseudomonas aeruginosa and Salmonella
spp.), which live in ecological balance with one another. This
balance may be disturbed by a change in diet, stress, or in
response to antibiotic or other therapeutic treatment, resulting in
bacterial diseases in the animals generally caused by bacteria such
as Salmonella, Campylobacter, Enterococci, Tularemia and E. coli.
Bacterial infection in these animals often necessitates therapeutic
intervention, which has treatment costs as well being frequently
associated with a decrease in productivity.
[0127] As a result, livestock are routinely treated with
antibiotics to maintain the balance of flora in the
gastrointestinal tract. The disadvantages of this approach are the
development of antibiotic resistant bacteria and the carry over of
such antibiotics and the resistant bacteria into resulting food
products for human consumption.
[0128] B. Cell Division and Cell Cycle Target Proteins
[0129] The antisense oligomers of the claimed subject matter are
designed to hybridize to a region of a bacterial mRNA that encodes
an essential bacterial gene. Exemplary genes are those required for
cell division, cell cycle proteins, or genes required for lipid
biosynthesis or nucleic acid replication. Any essential bacterial
gene can be targeted once a gene's essentiality is determined. One
approach to determining which genes in an organism are essential is
to use genetic footprinting techniques as described (Gerdes,
Scholle et al. 2003). In this report, 620 E. coli genes were
identified as essential and 3,126 genes as dispensable for growth
under culture conditions for robust aerobic growth. Evolutionary
context analysis demonstrated that a significant number of
essential E. coli genes are preserved throughout the bacterial
kingdom, especially the subset of genes for key cellular processes
such as DNA replication, cell division and protein synthesis.
[0130] In various aspects, the claimed subject matter provides an
antisense oligomer which is a nucleic acid sequence effective to
stably and specifically bind to a nucleic acid target sequence
which encodes an essential bacterial protein including the
following: (1) a sequence specific to a particular strain of a
given species of bacteria, such as a strain of E. coli associated
with food poisoning, e.g., O157:H7 (see Table 1 below); (2) a
sequence common to two or more species of bacteria; (3) a sequence
common to two related genera of bacteria (i.e., bacterial genera of
similar phylogenetic origin); (4) a sequence generally conserved
among Gram-negative bacteria; (5) generally conserved among
Gram-positive bacteria; or (6) a consensus sequence for essential
bacterial protein-encoding nucleic acid sequences in general.
[0131] In general, the target for modulation of gene expression
using the antisense methods of the claimed subject matter comprises
an mRNA expressed during active bacterial growth or replication,
such as an mRNA sequence transcribed from a gene of the cell
division and cell wall synthesis (dcw) gene cluster, including, but
not limited to, zipA, sulA, secA, dicA, dicB, dicC, dicF, ftsA,
ftsI, ftsN, ftsK, ftsL, ftsQ, ftsW, ftsZ, murC, murD, murE, murF,
murG, minC, minD, minE, mraY, mraW, mraZ, seqA and ddlB. See
(Bramhill 1997), and (Donachie 1993), both of which are expressly
incorporated by reference herein, for general reviews of bacterial
cell division and the cell cycle of E. coli, respectively.
Additional targets include genes involved in lipid biosynthesis
(e.g. acpP) and replication (e.g. gyrA).
[0132] Cell division in E. coli involves coordinated invagination
of all 3 layers of the cell envelope (cytoplasmic membrane, rigid
peptidoglycan layer and outer membrane). Constriction of the septum
severs the cell into 2 compartments and segregates the replicated
DNA. At least 9 essential gene products participate in this
process: ftsZ, ftsA, ftsQ, ftsL, ftsI, ftsN, ftsK, ftsW and zipA
(Hale and de Boer 1999). Preferred protein targets are the three
discussed below, and in particular, the GyrA and AcpP targets
described below.
[0133] FtsZ, one of the earliest essential cell division genes in
E. coli, is a soluble, tubulin-like GTPase that forms a
membrane-associated ring at the division site of bacterial cells.
The ring is thought to drive cell constriction, and appears to
affect cell wall invagination. FtsZ binds directly to a novel
integral inner membrane protein in E. coli called zipA, an
essential component of the septal ring structure that mediates cell
division in E. coli (Lutkenhaus and Addinall 1997).
[0134] GyrA refers to subunit A of the bacterial gyrase enzyme, and
the gene therefore. Bacterial gyrase is one of the bacterial DNA
topoisomerases that control the level of supercoiling of DNA in
cells and is required for DNA replication.
[0135] AcpP encodes acyl carrier protein, an essential cofactor in
lipid biosynthesis. The fatty acid biosynthetic pathway requires
that the heat stable cofactor acyl carrier protein binds
intermediates in the pathway.
[0136] For each of these three proteins, Table 1 provides exemplary
bacterial sequences which contain a target sequence for each of a
number of important pathogenic bacteria. The gene sequences are
derived from the GenBank Reference full genome sequence for each
bacterial strain (http://www.ncbi.nlm.nih.gov/genomes/Iproks.cgi).
The gene location on either the positive (+) or negative (-) strand
of the genome is listed under "Strand", it being recognized that
the strand indicated is the coding sequence for the protein, that
is, the sequence corresponding to the mRNA target sequence for that
gene. For example, the two E. coli genes (ftsZ and acpP) in which
the coding sequence is on the positive strand, the sequence is read
5' to 3' in the left-to-right direction. Similarly for the E. coli
gyrA gene having the coding region on the minus genomic strand, the
coding sequence is read as the reverse complement in the right to
left direction (5' to 3').
TABLE-US-00001 TABLE 1 Exemplary Bacterial Target Gene Sequences
GenBank Target Nucleotide Organism Ref. Gene Strand Region
Escherichia coli NC 000913 ftsZ + 105305-106456 acpP +
1150838-1151074 gyrA - 2334813-2337440 Escherichia coli NC 002655
ftsZ + 109911-111062 0157:H7 acpP + 1595796-1596032 gyrA -
3133832-3136459 Salmonella NC 003197 ftsZ + 155683-156834
thyphimurium acpP + 1280113-1280349 gyrA - 2373711-2376347
Pseudomonas NC 002516 ftsZ - 4939299-4940483 aeruginosa acpP -
3324946-3325182 gyrA - 3556426-3559197 Vibrio cholera NC 002505
ftsZ - 2565047-2566243 acpP + 254505-254747 gyrA + 1330207-1332891
Neisseria NC 002946 ftsZ - 1498872-1500050 gonorrhoea acpP +
1724401-1724637 gyrA - 618439-621189 Staphylococcus NC 002745 ftsZ
+ 1165782-1166954 aureus gyrA + 7005-9674 fmhB - 2321156-2322421
Mycobacterium NC 002755 ftsZ - 2407076-2408281 tuberculosis acpP +
1510182-1510502 gyrA + 7302-9818 pimA - 2934009-2935145 cysS2 -
4014534-4015943 Helicobacter pylori NC 000915 ftsZ +
1042237-1043394 acpP - 594037-594273 gyrA + 752512-754995
Streptococcus NC 003028 ftsZ - 1565447-1566706 pneumoniae acp +
396691-396915 gyrA - 1147387-1149855 Treponema NC 000919 ftsZ +
414751-416007 palladium acp + 877632-877868 gyrA + 4391-6832
Chlamydia NC 000117 acpP - 263702-263935 trachomatis gyrA -
755022-756494 Bartonella NC 005956 ftsZ - 1232094-1233839 henselae
acpP + 623143-623379 gyrA - 1120562-1123357 Hemophilis NC 000907
ftsZ + 1212021-1213286 influenza acpP - 170930-171160 gyrA -
1341719-1344361 Listeria NC 002973 ftsZ - 2102307-2101132
monocytogenes acpP - 1860771-1860538 gyrA + 8065-10593 Yersinia
pestis NC 003143 ftsZ + 605874-607025 acpP + 1824120-1824356 gyrA +
1370729-1373404 Bacillus anthracis NC 005945 ftsZ - 3724197-3725357
acpP - 3666663-3666896 gyrA + 6596-9067 Burkholderia NC 006348 ftsZ
- 2649616-2650812 mallei acpP + 559430-559669 gyrA - 459302-461902
Burkholderia NC 006350 ftsZ - 3599162-3600358 pseudomallei acpP -
2944967-2945206 gyrA - 3036533-3039133 Francisella NC 006570 ftsZ +
203748-204893 tularensis acpP + 1421900-1422184 gyrA -
1637300-1639906
[0137] C. Selection of Oligomer Target Sequences and Lengths
[0138] As noted above, the claimed subject matter derives from the
discovery herein that oligomeric antisense compounds having at
least 10 and up to 20 bases complementary to a bacterial RNA
target, e.g., at or just downstream (within 20 bases) of the AUG
start site of the mRNA for an essential bacterial protein or a
critical rRNA target region, have enhanced anti-bacterial activity
as evidence by enhanced inhibition of bacterial growth when they
are conjugated to short arginine- and/or lysine-containing
peptides. Unconjugated antisense compounds having a ribose or
morpholino subunit backbone may be most effective in inhibiting
bacterial growth when the subunit length is between 10-12 bases,
preferably 11 bases, where the compound contains at least 10 bases,
preferably 11-12 bases, that are complementary to the target mRNA
sequence. These studies were carried out on bacterial gene
expression in pure culture, a bacterial cell-free protein
expression system and an in vivo murine peritonitis model, as
discussed in detail in the examples below.
[0139] Antisense compounds directed against the AUG start site
region of the bacterial AcpP gene were tested for their ability to
inhibit bacterial growth in culture. These studies are reported in
Example 1, with reference to FIGS. 3A and 3B. As seen in the latter
figure, a striking inhibition was observed for antisense compounds
having between 10 and 14 bases, with nearly complete inhibition
being observed for the compound with an 11-base length. As with the
expression studies involving marker genes described above, the
results for inhibition of a bacterial gene in bacteria are
unpredictable from the behavior of the same antisense compounds in
a cell-free bacterial system. As seen in FIG. 4, strongest
inhibition was observed for antisense compounds between 11 and 20
bases.
[0140] As described in Example 3 and shown in FIGS. 7 to 16,
conjugation of an arginine-rich peptide to the antisense
oligonucleotides described above greatly enhance their
antibacterial properties. Exemplary amino acid sequences and the
peptides used in experiments in support of the claimed subject
matter are listed in Table 4 below as SEQ ID NOS:79-82 and 85-93.
One exemplary peptide is named RFF (SEQ ID NO:79) and consists of
the sequence N-RFFRFFRFF Ahx.beta.Ala-COOH (using the standard one
letter amino acid code and Ahx for 6-aminohexanoic acid and
.beta.Ala for beta-alanine), and has three repeating Arg-Phe-Phe
residues. This peptide is representative of one model peptide
having at least two, preferably three repeating Arg-Phe-Phe units.
One exemplary peptide-conjugated PMO (P-PMO) derived from the RFF
peptide (RFF-AcpP11, SEQ ID NO:79) demonstrated the ability to
reduce the CFU/ml of E. coli strains by as much as five orders of
magnitude (FIG. 11) and an IC.sub.50 of 3.7 .mu.M that is lower
than the 50% inhibitory concentration (IC.sub.50) observed for
ampicillin (7.7 .mu.M) under the same culture conditions (FIG.
14).
[0141] More generally, the peptide conjugated to the oligomer in
the compound is a peptide of 6-12 residues in length containing at
least two, and preferably 2-4 arginine residues, each preferably
separated by one or more uncharged, hydrophobic residues. In
exemplary embodiments, the carrier peptide has the sequence
(RXX).sub.n, where X is an uncharged amino acid selected from the
group consisting of alanine, .beta.-alanine, valine, leucine,
isoleucine, serine, threonine, phenyalanine, and tryptophan, and
n=2 or 3. In preferred embodiments, the carrier peptide has the
form (RFF).sub.n or (RFF).sub.nR, where n=2 or 3. The carrier
peptide may be linked at its C-terminus to one end of the
oligonucleotide, e.g., the 5'-end, through a one- or two-amino acid
linker, such as the linker is Ahx.beta.Ala, where Ahx is
6-aminohexanoic acid and .beta.Ala is .beta.-alanine, and where the
linker forms part of the carrier peptide.
[0142] The carrier peptides have the ability, when conjugated to
the 5' end of the anti-bacterial antisense PMO compound having SEQ
ID NO:66, to enhance the anti-bacterial activity of the PMO
compound by a factor of at least 10 and typically at least
10.sup.2, as measured by the reduction in bacterial cell-forming
units/ml when the peptide-conjugated PMO compound is added at a
concentration of 20 .mu.M in a culture to E coli, strain W3110 for
a period of 8 hours, relative to the same activity of the PMO
compound alone.
[0143] Thus, to determine whether an arginine-rich peptide having a
given amino acid sequence is a suitable peptide for the compound of
the claimed subject matter, a carrier peptide is synthesized and
coupled to the 5' end of the anti-bacterial antisense PMO compound
having SEQ ID NO:66, employing synthetic methods described and
referenced herein. The conjugate is then added, at a concentration
of 20 .mu.M in a culture to E coli, strain W3110 for a period of 8
hours, with the PMO alone (control) being added at a similar
concentration to a culture of the same bacteria. After an
eight-hour incubation time, the number of colony forming units per
ml (CFU/ml) are measured in both the "conjugate" and control
cultures. If the CFU/ml count for the conjugate culture is more
than 100-fold less, and preferably a 1000-fold less than that of
the control culture, the peptide can be identified as one suitable
for use in the claimed subject matter.
[0144] Based on these considerations, exemplary targeting sequences
for use in practicing the claimed invention are those having
between 10-20 bases, preferably complete but at least 10-base
complementarity with the mRNA target sequence, and complementary to
a region of the mRNA that includes the AUG start site or a region
up to 20 bases downstream of the start site. Where the compound of
the claimed subject matter is used in inhibiting infection by one
of the bacteria identified in the table below, by inhibiting one of
the three identified bacterial proteins, the antisense oligomer
compound has a sequence that is complementary to at least 10
contiguous bases of the corresponding target sequence indicated in
the table, where these target sequences are identified in the
sequence listing below by SEQ ID NOS:1-61.
TABLE-US-00002 TABLE 2 Exemplary bacterial target regions Organism
Target SEQ ID (GenBank Ref.) Gene Nucleotide Region NO. Escherichia
coli ftsZ 105295-105325 1 (NC 000913) acpP 1150828-1150858 2 gyrA
2337422-2337452 3 Escherichia coli 0157:H7 ftsZ 109901-109931 4 (NC
002655) acpP 1595786-1595816 5 gyrA 3136439-3136469 6 Salmonella
thyphimurium ftsZ 155673-155703 7 (NC 003197) acpP 1280103-1280133
8 gyrA 2376327-2376357 9 Pseudomonas aeruginosa ftsZ
4940463-4940493 10 (NC 002516) acpP 3325162-3325192 11 gyrA
3559177-3559207 12 Vibrio cholera ftsZ 2566223-2566253 13 (NC
002505) acpP 254495-254525 14 gyrA 1330197-1330227 15 Neisseria
gonorrhoea ftsZ 1500031-1500060 16 (NC 002946) acpP 1724391-1724420
17 gyrA 621170-621199 18 Staphylococcus aureus ftsZ 1165772-1165802
19 (NC 002745) gyrA 6995-7025 20 fmhB 2322402-2322431 21
Mycobacterium tuberculosis ftsZ 2408265-2408295 22 (NC 002755) acpP
1510172-1510202 23 gyrA 7292-7322 24 pimA 2935126-2935126 25 cysS2
4015924-4015953 26 Helicobacter pylori ftsZ 1042227-1042257 27 (NC
000915) acpP 594253-594283 28 gyrA 752502-752532 29 Streptococcus
pneumoniae ftsZ 1566686-1566716 30 (NC 003028) acpP 396681-396711
31 gyrA 1149835-1149865 32 Treponema palladium ftsZ 414741-414771
33 (NC 000919) acpP 877626-877656 34 gyrA 4381-4411 35 Chlamydia
trachomatis acpP 263915-263945 36 (NC 000117) gyrA 756474-756504 37
Bartonella henselae ftsZ 1232075-1232104 38 (NC 005956) acpP
623133-623162 39 gyrA 1123338-1123367 40 Hemophilis influenza ftsZ
1212011-1212041 41 (NC 000907) acpP 171140-171170 42 gyrA
1344341-1344371 43 Listeria monocytogenes ftsZ 2102288-2102307 44
(NC 002973) acpP 1860519-1860548 45 gyrA 8055-8084 46 Yersinia
pestis ftsZ 605864-605893 47 (NC 003143) acpP 1824110-1824139 48
gyrA 1370719-1370748 49 Bacillus anthracis ftsZ 3725338-3725367 50
(NC 005945) acpP 3666877-3666906 51 gyrA 6586-6615 52 Burkholderia
mallei ftsZ 2650793-2650822 53 (NC 006348) acpP 559420-559449 54
gyrA 461883-461912 55 Burkholderia pseudomallei ftsZ
3600339-3600368 56 (NC 006350) acpP 2945187-2945216 57 gyrA
3039114-3039143 58 Francisella tularensis ftsZ 203738-203767 59 (NC
006570) acpP 1421890-1421919 60 gyrA 1639887-1639916 61
[0145] Any essential bacterial gene can be targeted using the
methods of the claimed subject matter. As described above, an
essential bacterial gene for any bacterial species can be
determined using a variety of methods including those described by
Gerdes for E. coli (Gerdes, Scholle et al., 2003). Many essential
genes are conserved across the bacterial kingdom thereby providing
additional guidance in target selection. Target regions can be
obtained using readily available bioinformatics resources such as
those maintained by the National Center for Biotechnology
Information (NCBI). Complete reference genomic sequences for a
large number of micorbial species can be obtained (e.g., see
http://www.ncbi.nlm.nih.gov/genomes/Iproks.cgi) and sequences for
essential bacterial genes identified. Bacterial strains can be
obtained from the American Type Culture Collection (ATCC). Simple
cell culture methods, such as those described in the Examples,
using the appropriate culture medium and conditions for any given
species, can be established to determine the antibacterial activity
of antisense compounds. Once a suitable targeting antisense
oligomer has been identified, the peptide moieties of the compounds
can be altered to obtain optimal antibacterial activity. An optimal
peptide moiety can then be fixed and alternative antisense moieties
tested for improved antibacterial activity. One or more iterations
of this process can lead to compounds with improved activity but,
in general, no more than two iterations are needed to identify
highly active antibacterial agents.
[0146] The first step in selecting a suitable antisense compound is
to identify, by the methods above, a targeting sequence that
includes the AUG start site and/or contains at least about 10-20
bases downstream of the start site. Table 2 above gives the
base-number locations of 30- to 31-base targeting sequences that
span the AUG start site by about 10 bases on the upstream (5' side)
and about 20 bases (including the start site) in the downstream
coding region. The actual target sequences corresponding to these
target-site locations are given in the sequence listing below,
identified by SEQ ID NOS:1-61.
[0147] For purposes of illustration, assume that the antisense
compound to be prepared is for use in inhibiting an E. coli
bacterial infection in an individual infected with E. coli strain
0157:H7, and that the essential gene being targeted is the E. coli
acpP gene. One suitable target sequence for this gene identified by
the methods above is SEQ ID NO:2 having the sequence
5'-ATTTAAGAGTATGAGCACTATCGAAGAACGC-3' where the sequence gives the
DNA thymine (T) bases rather than the RNA uracil (U) bases, and
where the AUG start site (ATG) is shown in bold.
[0148] Again, for purposes of illustration, four model antisense
targeting sequences, each of them 11 bases in length, are selected:
(i) an antisense sequence that spans the AUG start site with four
bases of each side and has the sequence identified by SEQ ID NO:94;
(ii) an antisense sequence that overlaps the AUG starts at its 5'
end and extends in a 3' direction an additional 8 bases into the
coding region of the gene, identified as SEQ ID NO:95; (iii) an
antisense sequence complementary to bases 5-15 of the gene's coding
region, identified as SEQ ID NO:66, and (iv) an antisense sequence
complementary to bases 11-21 of the gene's coding region,
identified as SEQ ID NO:96.
[0149] Once antisense sequences have been selected and the
antisense compound synthesized and conjugated to arginine and/or
lysine-containing peptides, the compounds may be tested for the
ability to inhibit bacterial growth, in this case growth of an E.
coli strain in culture. Following the protocol in Example 1, for
example, the four 11 mer sequences described above are individually
tested for optimal activity, e.g., maximum drop in CFU/ml at a
given dose, e.g., 5-20 .mu.M, against an E. coli culture.
Compound(s) showing optimal activity are then tested in animal
models, as described in Example 2, or veterinary animals, prior to
use for treating human infection.
IV. Method for Inhibiting Bacteria
[0150] In one aspect, the claimed subject matter includes a method
of inhibiting bacterial infection, by exposing the infecting
bacteria to a 10-20 base peptide-conjugated oligomeric antisense
compound of the type characterized above. This general method is
demonstrated by the study reported in Example 1 and described above
with respect to FIGS. 3A and 3B.
[0151] In one aspect, the method is applied to inhibiting a
bacterial infection in a mammalian subject, including a human
subject, by administering the antisense compound to the subject in
a therapeutic amount. To demonstrate the method, groups of 12 mice
were injected IP with E. coli AS19, which has a genetic defect that
makes it abnormally permeable to high MW solutes. Immediately
following infection, each mouse was injected IP with 300 .mu.g of
an 11-base PMO complementary to acpP (SEQ ID NO:66), an 11-base
nonsense sequence PMO, or PBS, as detailed in Example 2. As seen in
FIG. 5, mice treated with the target antisense showed a reduction
in bacterial CFUs of about 600 at 23 hours, compared with control
treatment.
[0152] The same PMOs were again tested, except with E. coli SM105,
which has a normal outer membrane. In this method, acpP PMO reduced
CFU by 84% compared to nonsense PMO at 12 hours post-infection.
There was no reduction of CFU at 2, 6, or 24 hours (FIG. 6). Mice
were injected with a second dose at 24 hours post-infection. By 48
hours post-infection the CFU of acpP PMO-treated mice were 70%
lower than the CFU of nonsense PMO-treated mice (FIG. 6).
[0153] To demonstrate that the effect on bacterial infection was
sequence specific, a luciferase reporter gene whose expression
would not affect growth was used, and luciferase expression was
measured directly by two independent criteria, luciferase activity
and luciferase protein abundance. As detailed in Example 2, the
study demonstrated that an antisense compound complementary to the
luciferase mRNA inhibited luciferase expression at two different
times after administration of the PMO. Moreover, inhibition was
quantitatively similar with both methods of measurement. These
results show directly that PMO inhibit bacterial target gene
expression in vivo in a sequence-specific manner.
[0154] An improved antibacterial PMO can be obtained by conjugating
a short 6-12 amino acid peptide that enhances either intracellular
delivery or antisense activity or both. Exemplary peptides and
peptide-conjugated PMO (P-PMO) are listed in Table 4 as SEQ ID
NOS:88-93. As described in Example 3, enhanced antibacterial
activity was observed in pure culture experiments using a variety
of E. coli and S. typhimurium strains including the clinically
isolated enterpathogenic E. coli (EPEC) strain O127:H6. Example 4
describes the antibacterial activity of peptide-conjugated PMO
targeted to B. cenocepacia and P. aeruginosa.
[0155] It will be understood that the in vivo efficacy of such a
peptide-conjugated antisense oligomer in a subject using the
methods of the claimed invention is dependent upon numerous factors
including, but not limited to, (1) the target sequence; (2) the
duration, dose and frequency of antisense administration; and (3)
the general condition of the subject.
[0156] In other cases, the antisense oligonucleotides of the
claimed subject matter find utility in the preparation of
anti-bacterial vaccines. In this aspect of the claimed subject
matter, a culture of a particular type of bacteria is incubated in
the presence of a morpholino-based, peptide-conjugated antisense
oligomer of the type described above, in an amount effective to
produce replication-crippled and/or morphologically abnormal
bacterial cells. Such replication-crippled and/or morphologically
abnormal bacterial cells are administered to a subject and act as a
vaccine.
[0157] The efficacy of an in vivo administered antisense oligomer
of the claimed subject matter in inhibiting or eliminating the
growth of one or more types of bacteria may be determined by in
vitro culture or microscopic examination of a biological sample
(tissue, blood, etc.) taken from a subject prior to, during and
subsequent to administration of the peptide-conjugated antisense
oligomer. (See, for example, (Pari, Field et al. 1995); and
(Anderson, Fox et al. 1996). The efficacy of an in vivo
administered vaccine of peptide-conjugated, antisense
oligomer-treated bacteria may be determined by standard
immunological techniques for detection of an immune response, e.g.,
ELISA, Western blot, radioimmunoassay (RIA), mixed lymphoctye
reaction (MLR), assay for bacteria-specific cytotoxic T lymphocytes
(CTL), etc.
[0158] A. Administration Methods
[0159] Effective delivery of the peptide-conjugated antisense
oligomer to the target nucleic acid is an important aspect of
treatment. In accordance with the claimed invention, such routes of
delivery include, but are not limited to, various systemic routes,
including oral and parenteral routes, e.g., intravenous,
subcutaneous, intraperitoneal, and intramuscular, as well as
inhalation, transdermal and topical delivery. The appropriate route
may be determined by one of skill in the art, as appropriate to the
condition of the subject under treatment. For example, an
appropriate route for delivery of a peptide-conjugated antisense
oligomer in the treatment of a bacterial infection of the skin is
topical delivery; while delivery of a peptide conjugated antisense
oligomer in the treatment of a bacterial respiratory infection is
by inhalation. Methods effective to deliver the oligomer to the
site of bacterial infection or to introduce the compound into the
bloodstream are also contemplated.
[0160] Transdermal delivery of peptide-conjugated antisense
oligomers may be accomplished by use of a pharmaceutically
acceptable carrier adapted for e.g., topical administration. One
example of morpholino oligomer delivery is described in PCT patent
application WO 97/40854, incorporated herein by reference.
[0161] In one preferred embodiment, the compound is a
peptide-conjugated morpholino oligomer, contained in a
pharmaceutically acceptable carrier, and is delivered orally.
[0162] The peptide-conjugated antisense oligonucleotide may be
administered in any convenient vehicle which is physiologically
acceptable. Such a composition may include any of a variety of
standard pharmaceutically accepted carriers employed by those of
ordinary skill in the art. Examples of such pharmaceutical carriers
include, but are not limited to, saline, phosphate buffered saline
(PBS), water, aqueous ethanol, emulsions such as oil/water
emulsions, triglyceride emulsions, wetting agents, tablets and
capsules. It will be understood that the choice of suitable
physiologically acceptable carrier will vary dependent upon the
chosen mode of administration.
[0163] In some instances liposomes may be employed to facilitate
uptake of the antisense oligonucleotide into cells. (See, e.g.,
Williams, S. A., Leukemia 10(12):1980-1989, 1996; Lappalainen et
al., Antiviral Res. 23:119, 1994; Uhlmann et al., ANTISENSE
OLIGONUCLEOTIDES: A NEW THERAPEUTIC PRINCIPLES, Chemical Reviews,
Volume 90, No. 4, pages 544-584, 1990; Gregoriadis, G., Chapter 14,
Liposomes, Drug Carriers in Biology and Medicine, pp. 287-341,
Academic Press, 1979). Hydrogels may also be used as vehicles for
antisense oligomer administration, for example, as described in WO
93/01286. Alternatively, the oligonucleotides may be administered
in microspheres or microparticles. (See, e.g., Wu, G. Y. and Wu, C.
H., J. Biol. Chem. 262:4429-4432, 1987.)
[0164] Sustained release compositions are also contemplated within
the scope of this application. These may include semipermeable
polymeric matrices in the form of shaped articles such as films or
microcapsules.
[0165] Typically, one or more doses of peptide-conjugated antisense
oligomer are administered, generally at regular intervals for a
period of about one to two weeks.
[0166] Preferred doses for oral administration are from about 10 mg
oligomer/patient to about 250 mg oligomer/patient (based on a
weight of 70 kg). In some cases, doses of greater than 250 mg
oligomer/patient may be necessary. For IV administration, the
preferred doses are from about 1.0 mg oligomer/patient to about 100
mg oligomer/patient (based on an adult weight of 70 kg). The
peptide-conjugated antisense compound is generally administered in
an amount and manner effective to result in a peak blood
concentration of at least 200-400 nM oligomer.
[0167] In a further aspect of this embodiment, a peptide-conjugated
morpholino antisense oligonucleotide is administered at regular
intervals for a short time period, e.g., daily for two weeks or
less. However, in some cases the peptide-conjugated antisense
oligomer is administered intermittently over a longer period of
time. Administration of a peptide-conjugated morpholino antisense
oligomer to a subject may also be followed by, or concurrent with,
administration of an antibiotic or other therapeutic treatment.
[0168] In one aspect of the method, the subject is a human subject,
e.g., a patient diagnosed as having a localized or systemic
bacterial infection. The condition of a patient may also dictate
prophylactic administration of a peptide-conjugated antisense
oligomer of the claimed subject matter or a peptide-conjugated
antisense oligomer treated bacterial vaccine, e.g. in the case of a
patient who (1) is immunocompromised; (2) is a burn victim; (3) has
an indwelling catheter; or (4) is about to undergo or has recently
undergone surgery.
[0169] In another application of the method, the subject is a
livestock animal, e.g., a chicken, turkey, pig, cow or goat, etc,
and the treatment is either prophylactic or therapeutic. The
invention also includes a livestock and poultry food composition
containing a food grain supplemented with a subtherapeutic amount
of an antibacterial antisense compound of the type described above.
Also contemplated is in a method of feeding livestock and poultry
with a food grain supplemented with subtherapeutic levels of an
antibiotic, an improvement in which the food grain is supplemented
with a subtherapeutic amount of an antibacterial oligonucleotide
composition as described above.
[0170] The methods of the invention are applicable, in general, to
treatment of any condition wherein inhibiting or eliminating the
growth of bacteria would be effective to result in an improved
therapeutic outcome for the subject under treatment.
[0171] One aspect of the invention is a method for treatment of a
bacterial infection which includes the administration of a
morpholino antisense oligomer to a subject, followed by or
concurrent with administration of an antibiotic or other
therapeutic treatment to the subject.
[0172] B. Treatment Monitoring Methods
[0173] It will be understood that an effective in vivo treatment
regimen using the peptide-conjugated antisense oligonucleotide
compounds of the invention will vary according to the frequency and
route of administration, as well as the condition of the subject
under treatment (i.e., prophylactic administration versus
administration in response to localized or systemic infection).
Accordingly, such in vivo therapy will generally require monitoring
by tests appropriate to the particular type of bacterial infection
under treatment and a corresponding adjustment in the dose or
treatment regimen in order to achieve an optimal therapeutic
outcome.
[0174] The efficacy of a given therapeutic regimen involving the
methods described herein may be monitored, e.g., by general
indicators of infection, such as complete blood count (CBC),
nucleic acid detection methods, immunodiagnostic tests, or
bacterial culture.
[0175] Identification and monitoring of bacterial infection
generally involves one or more of (1) nucleic acid detection
methods, (2) serological detection methods, i.e., conventional
immunoassay, (3) culture methods, and (4) biochemical methods. Such
methods may be qualitative or quantitative.
[0176] Nucleic acid probes may be designed based on publicly
available bacterial nucleic acid sequences, and used to detect
target genes or metabolites (i.e., toxins) indicative of bacterial
infection, which may be specific to a particular bacterial type,
e.g., a particular species or strain, or common to more than one
species or type of bacteria (i.e., Gram positive or Gram negative
bacteria). Nucleic amplification tests (e.g., PCR) may also be used
in such detection methods.
[0177] Serological identification may be accomplished using a
bacterial sample or culture isolated from a biological specimen,
e.g., stool, urine, cerebrospinal fluid, blood, etc. Immunoassay
for the detection of bacteria is generally carried out by methods
routinely employed by those of skill in the art, e.g., ELISA or
Western blot. In addition, monoclonal antibodies specific to
particular bacterial strains or species are often commercially
available.
[0178] Culture methods may be used to isolate and identify
particular types of bacteria, by employing techniques including,
but not limited to, aerobic versus anaerobic culture, growth and
morphology under various culture conditions. Exemplary biochemical
tests include Gram stain (Gram, 1884; Gram positive bacteria stain
dark blue, and Gram negative stain red), enzymatic analyses (i.e.,
oxidase, catalase positive for Pseudomonas aeruginosa), and phage
typing.
[0179] It will be understood that the exact nature of such
diagnostic, and quantitative tests as well as other physiological
factors indicative of bacterial infection will vary dependent upon
the bacterial target, the condition being treated and whether the
treatment is prophylactic or therapeutic.
[0180] In cases where the subject has been diagnosed as having a
particular type of bacterial infection, the status of the bacterial
infection is also monitored using diagnostic techniques typically
used by those of skill in the art to monitor the particular type of
bacterial infection under treatment.
[0181] The peptide-conjugated antisense oligomer treatment regimen
may be adjusted (dose, frequency, route, etc.), as indicated, based
on the results of immunoassays, other biochemical tests and
physiological examination of the subject under treatment.
[0182] From the foregoing, it will be appreciated how various
objects and features of the invention are met. The method provides
an improvement in therapy against bacterial infection, using
peptide-conjugated antisense oligonucleotide sequences to achieve
enhanced cell uptake and anti-bacterial action. As a result, drug
therapy is more effective and less expensive, both in terms of cost
and amount of compound required.
[0183] An important advantage of the invention is that compounds
effective against virtually any pathogenic bacterium can be readily
designed and tested, e.g., for rapid response against new
drug-resistant bacteria, or in cases of bioterrorism. Once a target
bacterium is identified, the sequence selection methods described
allow one to readily identify one or more likely gene targets,
among a number of essential genes, and prepare antisense compounds
directed against the identified target. Because clinical testing on
the safety and efficacy, once established for a small group of
compounds, can be extrapolated to virtually any new target,
relatively little time is needed in addressing new
bacterial-infection challenges as they arise.
[0184] The following examples are intended to illustrate but not to
limit the invention.
Materials and Methods
Phosphorodiamidate Morpholino Oligomers
[0185] PMO were synthesized and purified at AVI BioPharma, Inc.
(Corvallis, Oreg.) as previously described (Summerton and Weller
1997; Geller, Deere et al. 2003), dissolved in water, filtered
through a 0.2 .mu.M membrane (HT Tuffryn.RTM., Gelman Sciences,
Inc., Ann Arbor, Mich.), and stored at 4.degree. C. Sequences of
PMO used in this study are shown in Table 3. The concentration of
PMO was determined spectrophotometrically by measuring the
absorbance at 260 nm and calculating the molarity using the
appropriate extinction coefficient.
Peptide Synthesis and Conjugation to PMO
[0186] Peptide synthesis of RFF (CPO4073, (RFF).sub.3Ahx.beta.Ala),
SEQ ID NO:79 and RTR(CP04074, RTRTRFLRRTAhx.beta.Ala, SEQ ID NO:80)
and conjugation to PMO were performed using the following
techniques. All the peptides of the present invention can be
synthesized and conjugated to PMO using these synthetic
techniques.
[0187] Peptides were synthesized by Fmoc Solid Phase Peptide
Synthesis, referred to herein as SPPS. A p-benzyloxybenzyl alcohol
resin was used for synthesis of peptides (Novabiochem, San Diego,
Calif.). A typical synthesis cycle began with N-terminal
deprotection via 20% piperidine. Then, N-.alpha.-Fmoc-protected
amino acids were coupled to the growing peptide chain by activation
with 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) in the presence of
N,N-diisopropylethylamine (DIEA). Arginine side chains were
protected with the
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) protecting
group and the t-Butyl (tBu) group for tyrosine side chains. The
cycle was repeated until all of the amino acids were added, in a
carboxy-to-amino direction, in the desired sequence. Cleavage from
the synthesis resin and side chain deprotection of the CP04073
peptide were carried out simultaneously by treating the
peptidyl-resin with a solution of 5% H.sub.2O and 95%
trifluoroacetic acid (TFA). For the CP04074 peptide residue, a
cleavage cocktail of 81.5% TFA, 5% Thioanisole, 5% Phenol, 5%
H.sub.2O, 2.5% 1,2-ethanedithiol (EDT) and 1% triisopropyl silane
(TIS) was used for simultaneous cleavage and side chain
deprotection. Crude peptides were isolated by precipitation using a
tenfold excess of diethyl ether. Strong cation exchange HPLC
utilizing Source 15S resin (Amersham Biosciences, Piscataway, N.J.)
was used for purification, followed by a reversed phase desalt
employing Amberchrom 300M resin (Tosoh Bioscience, Montgomeryville,
Pa.). Desalted peptides were lyophilized and analyzed for identity
and purity by matrix assisted laser desorption ionization time of
flight mass spectroscopy (MALDI-TOF MS) and strong cation exchange
high performance liquid chromatograph (SCX HPLC).
[0188] Attachment of the peptides at the 5' termini of the PMO was
performed via an amide bond as follows. A C-terminally reactive
peptide-benzotriazolyl ester was prepared by dissolving the
peptide-acid (15 .mu.mol), HBTU (14.25 .mu.mol), and HOBt (15
.mu.mol) in 200 .mu.L NMP and adding DIEA (22.5 .mu.mol).
Immediately after addition of DIEA, the peptide solution was added
to 1 mL of a 12 mM solution of 5'-piperazine-functionalized,
3'-acetyl-PMO in DMSO. After 180 minutes at 30.degree. C., the
reaction was diluted with a four-fold excess of water. The crude
CP04074 conjugate was purified first through a CM-Sepharose weak
cation exchange column (Sigma, St. Louis, Mo.) to remove
unconjugated PMO, and then through a reversed phase column
(Amberchrom 300M resin (Tosoh Bioscience, Montgomeryville, Pa.).
The crude CP04073 conjugate was purified by resolving
chromatography using strong cation exchange resin (Source 30S,
Amersham Biosciences, Piscataway, N.J.) to remove unconjugated PMO
and excess peptide. The SCX chromatography was followed by a
reverse phase column (Amberchrom 300M resin (Tosoh Bioscience,
Montgomeryville, Pa.). The conjugates were lyophilized and analyzed
by MALDI-TOF MS and SCX HPLC.
Bacteria and Growth Conditions
[0189] Bacterial strains were obtained from the American Type
Culture Collection (ATCC) or the E. coli Genetic Stock Center at
Yale University. E. coli strain AS19 was obtained from Dr. Pete
Nielsen (University of Copenhagen, Denmark). All pure culture
experiments were done in 96-well plates. OD600 readings and plating
of cells for CFU/ml determinations were done in triplicate. E. coli
AS19 and SM101, which have defects in lipopolysaccharide synthesis
that result in outer membrane permeability to high MW solutes, were
grown aerobically in LB broth at 37.degree. C., and 30.degree. C.,
respectively.
[0190] E. coli AS19 and SM105 were grown in LB broth (supplemented
with 100 .mu.g/ml ampicillin for transformants that expressed
luciferase) to OD.sub.600=0.12, centrifuged (4,000.times.g, 10 min,
20.degree. C.), and resuspended in 5% mucin (type II, Sigma
Chemical Co., St. Louis,)/PBS to final concentrations as follows:
AS19, 1.5.times.10.sup.8 CFU/ml; SM105, 5.7.times.10.sup.7 CFU/ml;
AS19 (pT7myc-luc), 7.2.times.10.sup.9 CFU/ml.
Reporter Gene
[0191] Standard molecular biology procedures were used for all
constructions. All constructs were sequenced. The acpP-luc reporter
(pCNacpP-luc) was made by ligating a SalI-NotI restriction fragment
of luc with the SalI-NotI fragment of pCiNeo (Promega Corp.,
Madison, Wis.), removing the adenosine from the start codon by
site-directed mutagenesis, then directionally cloning a synthetic
fragment of E. coli acpP (bp -17 to +23, inclusive, where +1 is
adenosine of the start codon) between the NheI-SalI sites.
Luciferase enzyme activity was measured in bacteria as described
(Geller, Deere at al. 2003).
Cell-Free Protein Synthesis
[0192] Bacterial, cell-free protein synthesis reactions were
performed by mixing reactants on ice according to the
manufacturer's instruction (Promega Corp.). Reactions were
programmed with mRNA synthesized in a cell-free RNA synthesis
reaction (Ambion, Inc., Austin, Tex., MEGAscript T7 High Yield
Transcription Kit) programmed with pCNacpP-luc. Where indicated,
cell-free reactions were composed with rabbit reticulocyte lysate
as described by the manufacturer (Promega Corp.). PMO was added to
a final concentration of either 100 nM or 200 nM as indicated.
After 1 hour at 37.degree. C., the reactions were cooled on ice and
luciferase was measured as described (Geller, Deere et al.,
2003).
Mammalian Tissue Culture
[0193] HeLa cells were transfected in T75 tissue culture flasks
(Nalge Nunc, Inc., Rochester, N.Y.) with a luciferase reporter
plasmid (pCNmyc-luc) using Lipofectamine Reagent (Gibco BRL, Grand
Island, N.Y.,) according to user's manual in serum-free media
(Gibco, Inc., Carlsbad, Calif., Opti-MEM1) for 5 hours before
re-addition of growth medium (Hyclone, Inc., Logan, Utah, HyQ
DME/F12 supplemented with 10% Fetal Bovine Serum and Gibco
Antibiotic-Antimycotic 15240-062) at 37.degree. C. in 5% CO.sub.2.
After 24 hours, the cells were pooled and 1.times.10.sup.6 were
added to each well of a 6-well plate (BD Biosciences, San Jose,
Calif.) in 2 ml of growth media. After an additional 24 hours, PMO
was added to a final concentration of 10 .mu.M in 2 ml fresh growth
media and the cells were scraped from the plate surface with a
rubber policeman to deliver the PMO to the cell as previously
described (Partridge, Vincent et al. 1996). After scrape-loading,
the cells were transferred to fresh 6-well plates and incubated at
37.degree. C. until the time of assay. At 7 and 24 hours the cells
were examined by microscopy to verify that each culture had the
same number of cells, harvested by centrifugation, and lysed in
Promega Cell Culture Lysis Reagent (Promega Corp.). Luciferase was
measured by mixing the cell lysate with Luciferase Assay Reagent
(Promega Corp.) and reading light emission in a Model TD-20e
luminometer (Turner Designs, Inc., Mountain View, Calif.).
Animals
[0194] Female, 6 to 8 week old Swiss Webster mice (Simonsen Labs,
Inc., Gilroy, Calif.) were used in all but one experiment, but
identical results were obtained with males. Infection was
established as described in (Frimodt-Moller, Knudsen et al. 1999).
Each mouse was injected IP with 0.1 ml of bacteria resuspended in
5% mucin/PBS, then immediately injected IP with 0.1 ml of PMO (3.0
mg/ml) or PBS. At various times after infection (as indicated in
the figures), groups (n=3 to 5) of mice were injected IP with 2.0
ml PBS, and their abdomens gently massaged for 2 min. Peritoneal
lavage was removed and stored on ice for .about.1 hour. The lavages
were diluted in PBS and plated in triplicate on LB to determine
CFU.
Luciferase and Western Blot
[0195] Peritoneal lavages (1.00 ml) from mice infected with AS19
(pT7myc-luc) were centrifuged (10,000.times.g, 2 min, 4.degree. C.)
and the supernatants discarded. The pellets were resuspended in 50
.mu.l PBS. An aliquot of resuspended cells was mixed with an equal
volume of 2.times. cell culture lysis reagent (Promega, Inc.,
Madison, Wis.) and frozen at -85.degree. C. Frozen lysates were
thawed and luciferase light production was measured in duplicate in
a luminometer as described (Geller, Deere et al. 2003). A second
aliquot of the cell suspension was mixed with 2.times.SDS sample
buffer and analyzed by western blot using 4-20% gradient Gene Mate
Express Gels (ISC BioExpress, Inc., Kaysville, Utah). Blots were
prepared with primary antibody to luciferase (Cortex Biochmical,
San Leandro, Calif.) or antisera to OmpA (Geller and Green 1989),
secondary goat anti-rabbit IgG-horse radish peroxidase conjugate
(Santa Cruz Biotechnology, Inc., Santa, Cruz, Calif.), and ECL
Western Blotting Reagent (Amersham Biosciences, Buckinghamshire,
England). Film negatives were scanned and digitized on an Kodak
Image Station 440 CF. The net intensity of each band was calculated
by subtracting the mean background intensity. Luciferase protein
was normalized to OmpA by dividing the net intensity of the
luciferase band by the net intensity of the OmpA band in the same
sample. The % inhibition was calculated by subtracting the mean
luciferase/OmpA of luc PMO-treated mice from mean luciferase/OmpA
of nonsense PMO-treated mice, dividing the difference by mean
luciferase/OmpA of nonsense PMO-treated mice, then multiplying by
100%.
Statistical Analysis
[0196] Spearman's rank-order correlation was used to analyze
correlations between the inhibitory effects of PMO and either G+C
content or secondary structure score of each PMO. Individual mouse
CFU/ml values were transformed logarithmically for statistical
analysis using InStat statistical software (GraphPad Software, San
Diego, Calif.). Differences in treatment group means were analysed
with unpaired t test, not assuming equal variances, with Welch
correction. Treatment group values were analysed for Gaussian
distributions using the method of Kolmogorov and Smirnov, which
confirmed in all analyses the normality test of the data. A
one-tailed t test was applied to differences in means between AcpP
PMO and either PBS or scrambled PMO treatment groups, whereas
two-sided t test was applied in all other analyses.
Oligomer Sequences
[0197] Exemplary targeting oligomers used in describing the present
invention are listed below in Table 3. The listed oligomers all
target E. coli, the experimental bacterial strain used in
experiments in support of the invention. Table 4 lists the peptides
of the invention and the peptide-PMO conjugates used in experiments
in support of the invention.
TABLE-US-00003 TABLE 3 PMO Sequences PMO SEQ ID # Sequence (5' to
3') Target NO 62-1 TTC TTC GAT AGT GCT CAT AC acpP - 20mer 62 62-2
TC TTC GAT AGT GCT CAT A acpP - 18mer 63 62-3 C TTC GAT AGT GCT CAT
acpP - 16mer 64 62-4 TC GAT AGT GCT CAT acpP - 14mer 65 169 C TTC
GAT AGT G acpP - 11mer 66 379 TTC GAT AGT G acpP - 10mer 67 380 TTC
GAT AGT acpP - 9mer 68 381 TC GAT AGT acpP - 8mer 69 382 TC GAT AG
acpP - 7mer 70 383 C GAT AG acpP - 6mer 71 62-5 TTG TCC TGA ATA TCA
CTT CG Nonsense control-acpP 72 62-7 G TCC TGA ATA TCA CTT Nonsense
control-acpP 73 62-8 TCG TGA GTA TCA CT Nonsense control-acpP 74
170 TCT CAG ATG GT Nonsense control-acpP 75 384 AAT CGG A Nonsense
control-acpP 76 ACG TTG AGG C luc 77 TCC ACT TGC C luc nonsense
78
TABLE-US-00004 TABLE 4 Peptide and Peptide-PMO Sequences Sequence
SEQ ID Name (Amino to Carboxy Terminus, 5' to 3') NO RFF
N-RFFRFFRFFAhx.beta.Ala-COOH 79 RTR N-RTRTRFLRRTAhx.beta.Ala-COOH
80 RFFR N-RFFRFFRFFRAhx.beta.Ala-COOH 81 KTR
N-KTRTKFLKKTAhx.beta.Ala-COOH 82 KFF N-KFFKFFKFFAhx.beta.Ala-COOH
83 KFFK N-KFFKFFKFFKAhx.beta.Ala-COOH 84 (RFF).sub.2
N-RFFRFFAhx.beta.Ala-COOH 85 (RFF).sub.2R
N-RFFRFFRAhx.beta.Ala-COOH 86 RAhx
N-RAhxAhxRAhxAhxRAhxAhx.beta.Ala-COOH 87 RFF-AcpP11
N-RFFRFFRFFAhx.beta.Ala-CTTCGATAGTG-3' 88 RTR-AcpP11
N-RTRTRFLRRTAhx.beta.Ala-CTTCGATAGTG-3' 89 RFFR-AcpP11
N-RFFRFFRFFRAhx.beta.Ala-CTTCGATAGTG-3' 90 (RFF).sub.2-AcpP11
N-RFFRFFAhx.beta.Ala-CTTCGATAGTG 91 (RFF).sub.2R-AcpP11
N-RFFRFFRAhx.beta.Ala-CTTCGATAGTG 92 RAhx-AcpP11
N-RAhxAhxRAhxAhxRAhxAhx.beta.Ala-CTTCGATAGTG 93
Example 1
Acyl Carrier Protein as an Endogenous Bacterial Gene Target
[0198] The effect of PMO was tested on an endogenous bacterial gene
that encodes acyl carrier protein, acpP, which is essential for
viability (Zhang and Cronan 1996) and has been used previously to
inhibit bacterial growth (Good, Awasthi et al., 2001; Geller, Deere
et al., 2003). PMO from 6 to 20 bases in length and complementary
to the region around the start codon in mRNA for acpP (Table 3, SEQ
ID NOS:62-71, respectively) were added to growing cultures of AS19
and growth at 37.degree. C. was monitored by optical density and
viable cell counts. Growth curves were normal for all cultures
except for that with the 11 base PMO, which caused significant
inhibition (FIG. 3A). Slight and reproducible, but statistically
insignificant inhibitions of OD occurred in cultures with the 10
and 14 base PMO. Viable cells were significantly reduced in 8 hour
cultures that contained PMO of 10, 11 or 14 bases (FIG. 3B). No
reduction in CFU was apparent in cultures treated with PMO of less
than 10 or more than 14 bases in length. Cultures without PMO, or
with nonsense base sequences (7c, 14c and 20c; SEQ ID NOS:76, 74
and 72, respectively) did not demonstrate growth inhibition.
[0199] PMO of various lengths (from 6 to 20 bases; SEQ ID
NOS:62-71, respectively) and targeted to acpP were added to
bacterial, cell-free protein synthesis reactions programmed to
express an acpP-luc fusion reporter. The results (FIG. 4) show that
PMO 11 to 20 bases in length inhibited reporter expression to about
the same extent. PMO shorter than 11 bases in length, or nonsense
sequence controls (16c and 20c; SEQ ID NOS:73 and 72, respectively)
did not inhibit luciferase expression significantly.
Example 2
In Vivo Antisense Antibacterial Activity
[0200] Groups of 12 mice in each of three treatment groups were
injected IP with E. coli strain AS19, which has a genetic defect
that makes it abnormally permeable to high MW solutes. Immediately
following infection, each mouse was injected IP with 300 .mu.g of
an 11-base PMO complementary to acpP (PMO 169; SEQ ID NO:66), an
11-base nonsense sequence control PMO (PMO 170; SEQ ID NO:75), or
PBS. Peritoneal lavages were collected at 2, 7, 13, and 23 hours
post-infection, and plated for bacteria. The results show that at
all times analyzed, the acpP PMO-treated mice had significantly
(P<0.05) lower CFU than the mice treated with either nonsense
PMO or PBS (FIG. 5). The differences between the acpP PMO-treated
group and the nonsense PMO control ranges from 39-fold at 2 hours
to 600-fold at 23 hours.
[0201] The same PMOs were again tested, except with E. coli strain
SM105, which has a normal outer membrane. AcpP PMO reduced CFU by
84% compared to nonsense PMO at 12 hours post-infection. There was
no reduction of CFU at 2, 6, or 24 hours (FIG. 6). Mice were
injected with a second dose at 24 hours post-infection. By 4 hours
post-infection the CFU of acpP PMO-treated mice were 70% lower than
the CFU of nonsense PMO-treated mice (FIG. 6).
[0202] The above results with acpP and nonsense PMOs suggest that
inhibition was sequence specific. To demonstrate directly a
sequence-specific effect, mice were infected with an E. coli AS19
that expresses firefly luciferase, then treated at 0 and 13 hours
post-infection with a PMO (luc; SEQ ID NO:77) complementary to the
region around the start codon of the luciferase transcript, or a
nonsense PMO (luc nonsense; SEQ ID NO:78). Peritoneal lavages were
collected at 13 and 22 hours post-infection and analyzed for CFU,
luciferase activity, and luciferase and OmpA protein by western
immuno-blot analysis. As expected, the results show no inhibition
of growth with the luc PMO treatment compared to nonsense PMO
treatment (Table 5). Luciferase activity in samples from luc
PMO-treated mice was inhibited 53% and 46% at 13 and 22 hours,
respectively, compared to samples from nonsense PMO-treated mice
(Table 5).
[0203] Western blot analysis agreed closely with the results of
luciferase activity. In samples from luc PMO-treated mice, there
was a 68% and 47% reduction in the amount of luciferase protein at
13 and 22 hours, respectively, compared to samples from nonsense
PMO-treated mice (Table 5).
TABLE-US-00005 TABLE 5 Gene Specific Inhibition Luciferase Activity
Western Blot Time after RLU/CFU Luc/OmpA PMO treatment CFU/ml Mean
(SEM) % Mean (SEM) % Treatment (h) (.times.10.sup.6) n = 8 P
Inhibition n = 7-8 P Inhibition Luc 13 6.3 2.90 (0.629) .0035 53
0.122 (.0312) .0002 68 Nonsense 13 4.3 6.19 (0.773) 0 0.382 (.0296)
0 Luc 22 0.96 3.20 (0.582) .0093 46 0.147 (.0363) .0145 57 Nonsense
22 0.39 8.12* (1.94) 0 0.339 (.0668) 0
Example 3
Enhanced Anti-Bacterial Properties of Peptide-Conjugated PMO
[0204] PMO conjugated at the 5' terminus with a series of three
different peptides were evaluated for their antibacterial
properties. The 11 mer PMO that targets the E. coli acpP gene (SEQ
ID NO:66) was used as the antisense oligomer moiety and conjugated
to either the RFF, RTR or RAhx peptides (SEQ ID NOS:79-80 and SEQ
ID NO:87, respectively) to produce peptide-conjugated PMOs (P-PMOs)
RFF-AcpP11, RTR-AcpP11 and RAhx-AcpP11 (SEQ ID NOS:88-89 and SEQ ID
NO:93, respectively).
[0205] Four laboratory strains of E. coli (W3110, MIC2067,
DH5.alpha., SM105) were treated with 20 .mu.M RFF-AcpP11 PPMO (SEQ
ID NO:88), 20 .mu.M free RFF peptide (SEQ ID NO:79) with
unconjugated AcpP11 PMO (SEQ ID NO:66), or received no treatment
(control) in LB broth, then were incubated at 37.degree. C. for 24
hours. Every hour for 8 hours then at 24 hours the OD600 values
(turbidity measurement) of the cultures were measured using a
spectrophotometer. After 8 hours, aliquots of the cultures were
diluted then plated onto LB agar plates and incubated for 24 hours
at 37.degree. C. After incubation, the colonies were counted by
hand and the colony formation units/mL (CFU/mL) for each treatment
were calculated. FIGS. 7 to 10 show the 24 hour growth curves for
strains W3110, MIC2067, DH5.alpha. and SM105, respectively, in the
presence of the RFF-AcpP11 P-PMO. FIG. 11 shows the CFU/ml after 8
hours treatment for the four strains of E. coli.
[0206] Using identical conditions as described above, E. coli W3110
was treated with three different P-PMOs (RAhx-AcpP11, RTR-AcpP11
and RFF-AcpP11) or received no treatment. FIG. 12 shows the CFU/ml
after 8 hours of treatment with the three different P-PMOs compared
to no treatment.
[0207] E. coli W3110 was treated with a short dilution series of
RFF peptide at 5 .mu.M, 20 .mu.M, 50 .mu.M and 100 .mu.M, 20 .mu.M
RFF free peptide mixed with AcpP11 PMO, 20 .mu.M RFF-AcpP11 P-PMO,
10 .mu.M ampicillin or no treatment. FIG. 13 shows the CFU/ml after
8 hours for each treatment. This data strongly supports the
conclusion that only the P-PMO and ampicillin showed antibacterial
activity and that the delivery peptide must be conjugated to the
PMO for this effect. Furthermore, the RFF-AcpP11 P-PMO demonstrates
an approximately 10 fold improved antibacterial activity at 10
.mu.M compared to ampicillin at the same concentration.
[0208] A series of dose-response experiments were performed where
E. coli W3110 was exposed to RFF-AcpP11, RTR-AcpP11, and ampicillin
in pure culture. E. coli W3110 was treated with a dilution series
of RFF-AcpP11, RTR-AcpP11 or ampicillin (80 .mu.M, 40 .mu.M, 20
.mu.M, 10 .mu.M, 5 .mu.M, 2.5 .mu.M) or received no treatment.
Determination of 50% inhibitory concentration values (IC.sub.50)
for RFF-AcpP11, RTR-AcpP11, and ampicillin were made using standard
methods. FIG. 14 shows the dose response curves for each of the two
PPMOs compared to ampicillin and the associated IC.sub.50 values of
3.7, 12.1 and 7.7 mM for RFF-AcpP11, RTR-AcpP11 and ampicillin,
respectively.
[0209] The sensitivity of Salmonella typhimurium 1535 and a
clinically isolated enterpathogenic strain of E. coli (EPEC strain
O127:H6) to RFF-AcpP11 P-PMO in culture was determined. The target
sequences for AcpP11 (SEQ ID NOS:2 and 8) in S. typhimurium and E.
coli are identical. Both strains were treated with 20 .mu.M of
RFF-AcpP11 P-PMO, RFF free peptide mixed with unconjugated AcpP11
PMO, AcpP11scr PMO, RFF-Scr P-PMO, RFF free peptide mixed with
unconjugate AcpP11 PMO or no treatment. FIGS. 15 and 16 show the
CFU/mL after 8 hours treatment for S. typhimurium and EPEC strain
O127:H6. This data clearly demonstrate the utility of the
RFF-AcpP11 compound as an antibacterial agent against clinically
relevant bacterial isolates.
Example 4
Antibacterial Activity of Peptide-Conjugated PMO Targeting
Burkholderia and Pseudomonas species
[0210] Burkholderia cenocepacia growth in the presence of
peptide-conjugated PMO was determined using the compounds of the
invention. A stationary phase culture of B. cenocepacia was diluted
to 5.times.10.sup.5 cfu/ml in Mueller-Hinton broth. The
peptide-conjugated PMO that target the acpP and gyrA genes were
added to identical cultures to a final concentration of 200
.mu.mol/L. The cultures were grown aerobically at 37.degree. C. and
optical density was monitored. After 36 hours, each culture was
diluted and plated to determine viable cell count as colony forming
units per ml (CFU/ml). All peptide-conjugated PMOs had the same
peptide attached to the 5' end, which was (RFF).sub.3RAhx.beta.Ala
(SEQ ID NO:81). All PMOs were 11 bases in length and targeted to
regions around the start codon of acyl carrier protein (acpP) or
the DNA gyrase subunit A (gyrA) as described above. Scr is a
negative control, scrambled base sequence peptide-conjugated PMO
that has no complementary target in B. cenocepacia. FIG. 17 shows
the inhibition of Burkholderia cenocepacia growth, as measured by
optical density, by the gyrA and acpP peptide-conjugated PMO
compared to the Scr control. The viable cell count after 36 hours
treatment was less than 1.times.10.sup.4 CFU/ml for the
peptide-conjugated PMOs targeting the acpP and gyrA genes whereas
for the Scr control peptide-conjugated PMO the viable cell count
was 8.5.times.10.sup.7 CFU/ml. Similar results were obtained using
these PMO against B. multivorans and B. gen.II.
[0211] Similar experiments targeting Pseudomonas aeruginosa using
RFFR (SEQ ID NO:81) conjugated to a PMO that targets the P.
aeruginosa acpP gene. The RFFR-AcpP PMO targeted to acpP was added
to a growing culture of P. aeruginosa in Mueller-Hinton broth at a
20 micromolar concentration. Aerobic growth at 37.degree. C. was
measured by optical density at 600 nm. The results shown in FIG. 18
show complete inhibition of growth and the scrambled base sequence
control had no effect on growth.
Sequence CWU 1
1
111131DNAEscherichia coli 1gagagaaact atgtttgaac caatggaact t
31231DNAEscherichia coli 2atttaagagt atgagcacta tcgaagaacg c
31331DNAEscherichia coli 3tagcggttag atgagcgacc ttgcgagagaa
31431DNAEscherichia coli 4gagagaaact atgtttgaac caatggaact t
31531DNAEscherichia coli 5atttaagagt atgagcacta tcgaagaacg c
31631DNAEscherichia coli 6tagcggttag atgagcgacc ttgcgagagaa
31731DNASalmonella typhimurium 7gagagagatt atgtttgaac ctatggaact a
31831DNASalmonella typhimurium 8atttaagagt atgagcacta tcgaagaacg c
31931DNASalmonella typhimurium 9tagcggttag atgagcgacc ttgcgagagaa
311031DNAPseudomonas aeruginosa 10gagaggggaa atgtttgaac tggtcgataac
311131DNAPseudomonas aeruginosa 11aaaacaaggt atgagcacca tcgaagaacgc
311231DNAPseudomonas aeruginosa 12caggcttctc atgggcgaac tggccaaagaa
311331DNAVibrio cholera 13gagataacac atgtttgaac cgatgatgga a
311431DNAVibrio cholera 14actatattgg atggtttata tgtctatctc t
311531DNAVibrio cholera 15taatggctct atgagcgatc tagctaaaga g
311630DNANeisseria gonorrhoea 16gagtttttga atggaatttg tttacgacgt
301730DNANeisseria gonorrhoea 17aacgactgat atgtcaaaca tcgaacaaca
301830DNANeisseria gonorrhoea 18cattgaaacc atgaccgacg caaccatccg
301931DNAStaphylococcus aureus 19ggaaatttaa atgttagaat ttgaacaagg a
312031DNAStaphylococcus aureus 20ggaactcttg atggctgaat tacctcaatc a
312130DNAStaphylococcus aureus 21atcataaatc atggaaaaga tgcatatcac
302231DNAMycobacterium tuberculosis 22ctctaagcct atggttgagg
ttgagagttt g 312331DNAMycobacterium tuberculosis 23cccgggcgcg
atgtggcgat atccactaagt 312431DNAMycobacterium tuberculosis
24cgaggaatag atgacagaca cgacgttgccg 312530DNAMycobacterium
tuberculosis 25ggaaagcctg atgcggatcg gcatgatttg
302630DNAMycobacterium tuberculosis 26ctggcacgtc gtgaccgatc
gggctcgctt 302731DNAHelicobacter pylori 27gaatgtggct atggttcatc
aatcagagat g 312831DNAHelicobacter pylori 28agttttaatt atggctttat
ttgaagatat t 312931DNAHelicobacter pylori 29agggagacac atgcaagata
attcagtcaa t 313031DNAStreptococcus pneumoniae 30aaaataaatt
atgacatttt catttgatac a 313131DNAStreptococcus pneumoniae
31gagtcctatc atggcagtat ttgaaaaagt a 313231DNAStreptococcus
pneumoniae 32gcatttatta atgcaggata aaaatttagt g 313331DNATreponema
palladium 33tgggagggga atgatgaata tagagcttgca 313431DNATreponema
palladium 34tgccccgtgg atgagttgtt cttaagaatg a 313531DNATreponema
palladium 35tgcccgccct atggaagaaa ttagcacccca 313631DNAChlamydia
trachomatis 36ggatcatagg atgagtttag aagatgatgt a 313731DNAChlamydia
trachomatis 37aaacgaactt atgagcgacc tctcggacct a
313829DNABartonella henselae 38aggcaaatta attggtaaaa aattagaga
293930DNABartonella henselae 39ggatttcaac atgagtgata cagtagagcg
304030DNABartonella henselae 40gtctaaagct gtgacagatc taaacccgca
304131DNAHaemophilus influenzae 41gagaacatca atgctatacc cagagtaccc
t 314231DNAHaemophilus influenzae 42ggaaaaacaa atgagtattg
aagaacgcgtg 314331DNAHaemophilus influenzae 43aggaatacca atgacggatt
caatccaatc a 314430DNAListeria monocytogenes 44aggcaataat
atgttagaat ttgacactag 304530DNAListeria monocytogenes 45cgaacgcata
aaactttatg tgaccggata 304630DNAListeria monocytogenes 46ttctctaaca
atggcagaaa caccaaatca 304730DNAYersinia pestis 47gagagaaact
atgtttgaac ctatggaact 304830DNAYersinia pestis 48atttaagagt
atgagcacta tcgaagaacg 304930DNAYersinia pestis 49tagcggctca
atgagcgacc ttgccagaga 305030DNABacillus anthracis 50ggatttcgac
atgttagagt ttgatactac 305130DNABacillus anthracis 51ggtgaatgga
atggcagatg ttttagagcg 305230DNABacillus anthracis 52gtgctcgttg
atgtcagaca atcaacaaca 305330DNABurkholderia mallei 53ggaggcaaca
atggaattcg aaatgctgga 305430DNABurkholderia mallei 54cggaggggta
atggacaaca tcgaacaacg 305530DNABurkholderia mallei 55atacggatac
atggatcaat tcgccaaaga 305630DNABurkholderia pseudomallei
56ggaggcaaca atggaattcg aaatgctgga 305730DNABurkholderia
pseudomallei 57cggaggggta atggacaaca tcgaacaacg
305830DNABurkholderia pseudomallei 58atacggatac atggatcaat
tcgccaaaga 305930DNAFrancisella tularensis 59ggagtaaaat atgtttgatt
ttaacgattc 306030DNAFrancisella tularensis 60aggaaaaaat atgagtacac
ataacgaaga 306130DNAFrancisella tularensis 61gcgataacta atgtctataa
ttactaaaga 306220DNAArtificial SequenceSynthetic Oligomer
62ttcttcgata gtgctcatac 206318DNAArtificial SequenceSynthetic
Oligomer 63tcttcgatag tgctcata 186416DNAArtificial
SequenceSynthetic Oligomer 64cttcgatagt gctcat 166514DNAArtificial
SequenceSynthetic Oligomer 65tcgatagtgc tcat 146611DNAArtificial
SequenceSynthetic Oligomer 66cttcgatagt g 116710DNAArtificial
SequenceSynthetic Oligomer 67ttcgatagtg 10689DNAArtificial
SequenceSynthetic Oligomer 68ttcgatagt 9698DNAArtificial
SequenceSynthetic Oligomer 69tcgatagt 8707DNAArtificial
SequenceSynthetic Oligomer 70tcgatag 7716DNAArtificial
SequenceSynthetic Oligomer 71cgatag 67220DNAArtificial
SequenceSynthetic Oligomer 72ttgtcctgaa tatcacttcg
207316DNAArtificial SequenceSynthetic Oligomer 73gtcctgaata tcactt
167414DNAArtificial SequenceSynthetic Oligomer 74tcgtgagtat cact
147511DNAArtificial SequenceSynthetic Oligomer 75tctcagatgg t
11767DNAArtificial SequenceSynthetic Oligomer 76aatcgga
77710DNAArtificial SequenceSynthetic Oligomer 77acgttgaggc
107810DNAArtificial SequenceSynthetic Oligomer 78tccacttgcc
107911PRTArtificial SequenceSynthetic Peptide 79Arg Phe Phe Arg Phe
Phe Arg Phe Phe Xaa Xaa1 5 108012PRTArtificial SequenceSynthetic
Peptide 80Arg Thr Arg Thr Arg Phe Leu Arg Arg Thr Xaa Xaa1 5
108112PRTArtificial SequenceSynthetic Peptide 81Arg Phe Phe Arg Phe
Phe Arg Phe Phe Arg Xaa Xaa1 5 108212PRTArtificial
SequenceSynthetic Peptide 82Lys Thr Arg Thr Lys Phe Leu Lys Lys Thr
Xaa Xaa1 5 108311PRTArtificial SequenceSynthetic Peptide 83Lys Phe
Phe Lys Phe Phe Lys Phe Phe Xaa Xaa1 5 108412PRTArtificial
SequenceSynthetic Peptide 84Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys
Xaa Xaa1 5 10858PRTArtificial SequenceSynthetic Peptide 85Arg Phe
Phe Arg Phe Phe Xaa Xaa1 5869PRTArtificial SequenceSynthetic
Peptide 86Arg Phe Phe Arg Phe Phe Arg Xaa Xaa1 58710PRTArtificial
SequenceSynthetic Peptide 87Arg Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa
Xaa1 5 108811PRTArtificial SequenceSynthetic Peptide 88Arg Phe Phe
Arg Phe Phe Arg Phe Phe Xaa Xaa1 5 108912PRTArtificial
SequenceSynthetic Peptide 89Arg Thr Arg Thr Arg Phe Leu Arg Arg Thr
Xaa Xaa1 5 109012PRTArtificial SequenceSynthetic Peptide 90Arg Phe
Phe Arg Phe Phe Arg Phe Phe Arg Xaa Xaa1 5 10918PRTArtificial
SequenceSynthetic Peptide 91Arg Phe Phe Arg Phe Phe Xaa Xaa1
5929PRTArtificial SequenceSynthetic Peptide 92Arg Phe Phe Arg Phe
Phe Arg Xaa Xaa1 59310PRTArtificial SequenceSynthetic Peptide 93Arg
Xaa Xaa Arg Xaa Xaa Arg Xaa Xaa Xaa1 5 109411DNAArtificial
SequenceSynthetic Oligomer 94tgctcatact c 119511DNAArtificial
SequenceSynthetic Oligomer 95atagtgctca t 119611DNAArtificial
SequenceSynthetic Oligomer 96gcgttcttcc g 119714PRTArtificial
SequenceSynthetic Peptide 97Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg
Xaa Arg Xaa Xaa1 5 109814PRTArtificial SequenceSynthetic Peptide
98Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa1 5
109913PRTArtificial SequenceSynthetic Peptide 99Arg Arg Xaa Arg Arg
Xaa Arg Arg Xaa Arg Arg Xaa Xaa1 5 1010014PRTArtificial
SequenceSynthetic Peptide 100Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg
Arg Xaa Arg Xaa Xaa1 5 1010114PRTArtificial SequenceSynthetic
Peptide 101Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Xaa1
5 1010213PRTArtificial SequenceSynthetic Peptide 102Arg Xaa Arg Xaa
Arg Xaa Arg Xaa Arg Xaa Arg Xaa Xaa1 5 1010315PRTArtificial
SequenceSynthetic Peptide 103Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg
Xaa Arg Xaa Arg Xaa Xaa1 5 10 1510411PRTArtificial
SequenceSynthetic Peptide 104Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg
Xaa Xaa1 5 1010514PRTArtificial SequenceSynthetic Peptide 105Arg
Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Xaa Xaa1 5
1010617PRTArtificial SequenceSynthetic Peptide 106Arg Xaa Arg Xaa
Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa Arg Xaa1 5 10
15Xaa10719DNAArtificial SequenceSynthetic Oligomer 107gtgctcatgg
tgcacggtc 1910819DNAArtificial SequenceSynthetic Oligomer
108gccatggttt tttctcagg 1910918DNAArtificial SequenceSynthetic
Oligomer 109tgggtatgtt gtagccat 1811020DNAArtificial
SequenceSynthetic Oligomer 110cctgcccttt gttctagttg
2011120DNAArtificial SequenceSynthetic Oligomer 111ctgggatgag
atccatcact 20
* * * * *
References