Patent application title: ANTISENSE ANTIBACTERIAL METHOD AND COMPOUND
Bruce L. Geller (Corvallis, OR, US)
Patrick L. Iversen (Corvallis, OR, US)
Lucas D. Tilley (Burlington, VT, US)
IPC8 Class: AA61K3816FI
Class name: Designated organic active ingredient containing (doai) peptide containing (e.g., protein, peptones, fibrinogen, etc.) doai glycoprotein (carbohydrate containing)
Publication date: 2010-09-16
Patent application number: 20100234280
Patent application title: ANTISENSE ANTIBACTERIAL METHOD AND COMPOUND
Patrick L. Iversen
Bruce L. Geller
Lucas D. Tilley
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
Origin: SEATTLE, WA US
IPC8 Class: AA61K3816FI
Publication date: 09/16/2010
Patent application number: 20100234280
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 Tm of between 50° to 60° C. The
carrier peptide is an arginine-rich peptide containing between 6 and 12
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)n--, where X is an uncharged amino acid selected from the group consisting of alanine, β-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)n.
25. The method of claim 24, wherein the carrier peptide has the form (RFF)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 AhxβAla, where Ahx is 6-aminohexanoic acid and βAla is β-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═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.
CROSS-REFERENCE TO RELATED APPLICATIONS
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
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.
Anderson, K. P., M. C. Fox, et al. (1996). Antimicrob Agents Chemother 40(9): 2004-11. Blommers, M. J., U. Pieles, et al. (1994). Nucleic Acids Res 22(20): 4187-94. Bramhill, D. (1997). Annu Rev Cell Dev Biol 13: 395-424. Cross, C. W., J. S. Rice, of al. (1997). Biochemistry 36(14): 4096-107. Donachie, W. D. (1993). Annu Rev Microbiol 47: 199-230. Gait, M. J., A. S. Jones, et al. (1974). J Chem Soc [Perkin 1] 0(14): 1684-6. Geller, B. L., J. D. Deere, et al. (2003). Antimicrob Agents Chemother 47(10): 3233-9. Geller, B. L. and H. M. Green (1989). J Biol Chem 264(28): 16465-9. Gerdes, S. Y., M. D. Scholle, et al. (2003). J Bacteriol 185(19): 5673-84. Good, L., S. K. Awasthi, et al., (2001). Nat Biotechnol 19(4): 360-4. Good, L. and P. E. Nielsen (1998). Proc Natl Acad Sci USA 95(5): 2073-6. Hale, C. A. and P. A. de Boer (1999). J Bacteriol 181(1): 167-76. Lesnikowski, Z. J., M. Jaworska, et al., (1990). Nucleic Acids Res 18(8): 2109-15. Lutkenhaus, J. and S. G. Addinall (1997). Annu Rev Biochem 66: 93-116. Mertes, M. P. and E. A. Coats (1969). J Med Chem 12(1): 154-7. Pari, G. S., A. K. Field, et al., (1995). Antimicrob Agents Chemother 39(5): 1157-61. Rahman, M. A., J. Summerton, et al. (1991). Antisense Res Dev 1(4): 319-27. Summerton, J., D. Stein, et al. (1997). Antisense Nucleic Acid Drug Dev 7(2): 63-70. Summerton, J. and D. Weller (1997). Antisense Nucleic Acid Drug Dev 7(3): 187-95. Zhang, Y. and J. E. Cronan, Jr. (1996). J Bacteriol 178(12): 3614-20.
BACKGROUND OF THE INVENTION
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.
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.
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.
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.
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.
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).
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
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 Tm of at least 50°, thereby to inhibit replication of the bacteria.
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)n--, where X is an uncharged amino acid selected from the group consisting of alanine, β-alanine, valine, leucine, isoleucine, serine, threonine, phenyalanine, and tryptophan, and n=2 or 3.
In exemplary embodiments, the carrier peptide has the sequence (RFF)n or (RFF)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 AhxβAla, where Ahx is 6-aminohexanoic acid and βAla is β-alanine.
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 102, 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 μM in a culture to E coli, strain W3110 at 5×107 CFU/ml in Luria broth for a period of 8 hours at 37° C. with aeration, relative to the same activity of the PMO oligonucleotide alone.
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:
where Y1═O, Z═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.
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.
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.
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.
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.
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
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.
FIGS. 2A-2G show examples of uncharged linkage types in oligonucleotide analogs and FIG. 2H shows a cationic linkage group.
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° C.) with various lengths (6 to 20 bases) of overlapping PMO (20 μM) 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).
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.
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.
FIG. 6 shows CFU/ml in peritoneal lavages from mice infected E. coli strain SM105 and treated with PMO as described in FIG. 13.
FIGS. 7 to 10 show the growth as measured by optical density (OD600) 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).
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.
FIG. 12 shows the antibacterial activity of three P-PMOs as measured by CFU/ml after 8 hours of treatment.
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.
FIG. 14 shows the dose response curves for each of two PPMOs compared to ampicillin and the associated IC50 values for RFF-AcpP11, RTR-AcpP11 (SEQ ID NOS:88 and 89) and ampicillin.
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.
FIGS. 17 and 18 show the effect of RFFR-conjugated PMOs on the growth of Burkholderia cenocepacia and Pseudomonas aeruginosa, respectively.
The terms below, as used herein, have the following meanings, unless indicated otherwise:
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.
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 β-morpholino subunit types shown in FIGS. 1A-D. It will be appreciated that a polymer may contain more than one linkage type.
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.
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.
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)n, where X is an uncharged amino acid selected from the group consisting of alanine, β-alanine, valine, leucine, isoleucine, serine, threonine, phenyalanine, and tryptophan, and n=2 or 3. In preferred embodiments, the carrier peptide has the form (RFF)n or (RFF)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 AhxβAla, where Ahx is 6-aminohexanoic acid and βAla is β-alanine, and where the linker forms part of the carrier peptide.
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.
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.
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 Pi or Pj is adenine, cytosine, guanine, thymine, uracil or inosine.
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) Pi and Pj are purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.
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.
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.
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° 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° C., typically between 50°-60° C. or greater.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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═O) and carbamate (2A, R═NH2) 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=CH2); 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.
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.
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° 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.
A. Exemplary Antisense Oligonucleotides
Exemplary backbone structures for antisense oligonucleotides of the claimed subject matter include the β-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.
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═NH2 or N(CH3)2, Y═O, and Z═O.
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.
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.
B. Antibacterial Antisense Oligonucleotides
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 Tm, when hybridized with the target sequence, of at least about 50° C., typically between about 50° to 60° C., although the Tm may be higher, e.g., 65° C. The selection of bacterial targets, and bacterial mRNA target sequences and complementary targeting sequences are considered in the two sections below.
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
This section considers a number of bacterial targets, including pathogenic bacteria, and specific bacterial protein targets against which the antisense compound can be directed.
A. Bacterial Targets
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.
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.)
Exemplary sequences for enterovirulent E. coli strains include GenBank Accession Numbers AB011549, X97542, AF074613, Y11275 and AJ007716.
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.
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.
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.
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.
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.
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° 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The disease is treated with antibiotics, such as doxycycline, erythromycin, rifampin, penicillin, gentamycin, ceftriaxone, ciprofloxacin, and azithromycin.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
B. Cell Division and Cell Cycle Target Proteins
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.
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.
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).
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.
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).
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.
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.
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
C. Selection of Oligomer Target Sequences and Lengths
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.
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.
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 AhxβAla-COOH (using the standard one letter amino acid code and Ahx for 6-aminohexanoic acid and βAla 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 IC50 of 3.7 μM that is lower than the 50% inhibitory concentration (IC50) observed for ampicillin (7.7 μM) under the same culture conditions (FIG. 14).
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)n, where X is an uncharged amino acid selected from the group consisting of alanine, β-alanine, valine, leucine, isoleucine, serine, threonine, phenyalanine, and tryptophan, and n=2 or 3. In preferred embodiments, the carrier peptide has the form (RFF)n or (RFF)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 AhxβAla, where Ahx is 6-aminohexanoic acid and βAla is β-alanine, and where the linker forms part of the carrier peptide.
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 102, as measured by the reduction in bacterial cell-forming units/ml when the peptide-conjugated PMO compound is added at a concentration of 20 μM in a culture to E coli, strain W3110 for a period of 8 hours, relative to the same activity of the PMO compound alone.
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 μM 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.
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
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.
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.
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.
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.
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 μM, 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
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.
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 μg 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.
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).
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.
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.
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.
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.
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.
A. Administration Methods
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.
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.
In one preferred embodiment, the compound is a peptide-conjugated morpholino oligomer, contained in a pharmaceutically acceptable carrier, and is delivered orally.
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.
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.)
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.
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.
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.
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.
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.
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.
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.
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.
B. Treatment Monitoring Methods
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The following examples are intended to illustrate but not to limit the invention.
Materials and Methods
Phosphorodiamidate Morpholino Oligomers
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 μM membrane (HT Tuffryn®, Gelman Sciences, Inc., Ann Arbor, Mich.), and stored at 4° 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
Peptide synthesis of RFF (CPO4073, (RFF)3AhxβAla), SEQ ID NO:79 and RTR(CP04074, RTRTRFLRRTAhxβAla, 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.
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-α-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% H2O and 95% trifluoroacetic acid (TFA). For the CP04074 peptide residue, a cleavage cocktail of 81.5% TFA, 5% Thioanisole, 5% Phenol, 5% H2O, 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).
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 μmol), HBTU (14.25 μmol), and HOBt (15 μmol) in 200 μL NMP and adding DIEA (22.5 μmol). 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° 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
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° C., and 30° C., respectively.
E. coli AS19 and SM105 were grown in LB broth (supplemented with 100 μg/ml ampicillin for transformants that expressed luciferase) to OD600=0.12, centrifuged (4,000×g, 10 min, 20° C.), and resuspended in 5% mucin (type II, Sigma Chemical Co., St. Louis,)/PBS to final concentrations as follows: AS19, 1.5×108 CFU/ml; SM105, 5.7×107 CFU/ml; AS19 (pT7myc-luc), 7.2×109 CFU/ml.
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
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° C., the reactions were cooled on ice and luciferase was measured as described (Geller, Deere et al., 2003).
Mammalian Tissue Culture
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° C. in 5% CO2. After 24 hours, the cells were pooled and 1×106 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 μM 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° 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.).
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 ˜1 hour. The lavages were diluted in PBS and plated in triplicate on LB to determine CFU.
Luciferase and Western Blot
Peritoneal lavages (1.00 ml) from mice infected with AS19 (pT7myc-luc) were centrifuged (10,000×g, 2 min, 4° C.) and the supernatants discarded. The pellets were resuspended in 50 μl PBS. An aliquot of resuspended cells was mixed with an equal volume of 2× cell culture lysis reagent (Promega, Inc., Madison, Wis.) and frozen at -85° 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×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%.
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.
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-RFFRFFRFFAhxβAla-COOH 79 RTR N-RTRTRFLRRTAhxβAla-COOH 80 RFFR N-RFFRFFRFFRAhxβAla-COOH 81 KTR N-KTRTKFLKKTAhxβAla-COOH 82 KFF N-KFFKFFKFFAhxβAla-COOH 83 KFFK N-KFFKFFKFFKAhxβAla-COOH 84 (RFF)2 N-RFFRFFAhxβAla-COOH 85 (RFF)2R N-RFFRFFRAhxβAla-COOH 86 RAhx N-RAhxAhxRAhxAhxRAhxAhxβAla-COOH 87 RFF-AcpP11 N-RFFRFFRFFAhxβAla-CTTCGATAGTG-3' 88 RTR-AcpP11 N-RTRTRFLRRTAhxβAla-CTTCGATAGTG-3' 89 RFFR-AcpP11 N-RFFRFFRFFRAhxβAla-CTTCGATAGTG-3' 90 (RFF)2-AcpP11 N-RFFRFFAhxβAla-CTTCGATAGTG 91 (RFF)2R-AcpP11 N-RFFRFFRAhxβAla-CTTCGATAGTG 92 RAhx-AcpP11 N-RAhxAhxRAhxAhxRAhxAhxβAla-CTTCGATAGTG 93
Acyl Carrier Protein as an Endogenous Bacterial Gene Target
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° 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.
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.
In Vivo Antisense Antibacterial Activity
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 μg 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.
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).
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).
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) (×106) 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
Enhanced Anti-Bacterial Properties of Peptide-Conjugated PMO
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).
Four laboratory strains of E. coli (W3110, MIC2067, DH5α, SM105) were treated with 20 μM RFF-AcpP11 PPMO (SEQ ID NO:88), 20 μM 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° 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° 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, DH5α 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.
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.
E. coli W3110 was treated with a short dilution series of RFF peptide at 5 μM, 20 μM, 50 μM and 100 μM, 20 μM RFF free peptide mixed with AcpP11 PMO, 20 μM RFF-AcpP11 P-PMO, 10 μM 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 μM compared to ampicillin at the same concentration.
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 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM) or received no treatment. Determination of 50% inhibitory concentration values (IC50) 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 IC50 values of 3.7, 12.1 and 7.7 mM for RFF-AcpP11, RTR-AcpP11 and ampicillin, respectively.
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 μM 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.
Antibacterial Activity of Peptide-Conjugated PMO Targeting Burkholderia and Pseudomonas species
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 5×105 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 μmol/L. The cultures were grown aerobically at 37° 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)3RAhxβAla (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 1×104 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 8.5×107 CFU/ml. Similar results were obtained using these PMO against B. multivorans and B. gen.II.
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° 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.
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
Patent applications by Bruce L. Geller, Corvallis, OR US
Patent applications by Lucas D. Tilley, Burlington, VT US
Patent applications by Patrick L. Iversen, Corvallis, OR US
Patent applications in class Glycoprotein (carbohydrate containing)
Patent applications in all subclasses Glycoprotein (carbohydrate containing)