US20050020526A1 - Oligodeoxynucleotide intervention for prevention and treatment of sepsis - Google Patents

Oligodeoxynucleotide intervention for prevention and treatment of sepsis Download PDF

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US20050020526A1
US20050020526A1 US10/818,158 US81815804A US2005020526A1 US 20050020526 A1 US20050020526 A1 US 20050020526A1 US 81815804 A US81815804 A US 81815804A US 2005020526 A1 US2005020526 A1 US 2005020526A1
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sepsis
odn
bacterial
treatment
causative agent
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Yin Chen
Xin Tan
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CytoGenix Inc
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Priority claimed from US10/453,410 external-priority patent/US20040248101A1/en
Priority claimed from US10/743,956 external-priority patent/US20050136393A1/en
Application filed by CytoGenix Inc filed Critical CytoGenix Inc
Priority to US10/818,158 priority Critical patent/US20050020526A1/en
Priority to US10/574,254 priority patent/US8889397B2/en
Priority to AU2004245978A priority patent/AU2004245978A1/en
Priority to CA002528131A priority patent/CA2528131A1/en
Priority to EP04754036A priority patent/EP1646719A2/en
Priority to PCT/US2004/017331 priority patent/WO2004108840A2/en
Priority to JP2006515087A priority patent/JP2006526416A/en
Priority to KR1020057023173A priority patent/KR20060065580A/en
Priority to BRPI0410951-1A priority patent/BRPI0410951A/en
Assigned to CYTOGENIX, INC. reassignment CYTOGENIX, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHEN, YIN, TAN, XIN XING
Publication of US20050020526A1 publication Critical patent/US20050020526A1/en
Priority to IL172324A priority patent/IL172324A0/en
Priority to US14/524,744 priority patent/US20150094236A1/en
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Definitions

  • Severe sepsis and septic shock are life threatening complication of infections and the most common cause of death in intensive care units (Angus et al., 2001, Crit. Care Med., 29:1303-1310).
  • Recent US and European surveys have estimated that severe sepsis accounts for 2-11% of all admissions to hospital or intensive care units (Martin et al., 2003, New Engl. J. Med., 348, 1546-1554). The incidence of this condition is rising due to the aging of the population and increasing numbers of immuno-compromised and critically ill patients.
  • Sepsis comprises a complex clinical syndrome that results from the body's response to infection caused by bacterial and/or fungal pathogens invading the body (Cohen, 2002, Nature, 420, 885-891).
  • a potent, complex, immunologic cascade ensures a prompt, protective response to microorganism invasion in humans.
  • a deficient immunologic defense may allow infection to become established.
  • an excessive or poorly regulated response may harm the host through maladaptive release of indigenously generated inflammatory compounds.
  • Gram negative bacilli mainly Escherichia coli, Klebsiella species, and Pseudomonas aeruginosa
  • Gram positive cocci mainly staphylococci and streptococci
  • LPS lipopolysaccharides
  • Gram positive bacteria are usually responsible for infections of skin and soft tissue, infections associated with intravascular devices, primary bloodstream infections, or respiratory infections. It is generally thought that the distinct cell wall substances of gram-positive bacteria and fungi trigger a similar cascade of events as gram negative bacteria, although the structures involved are not generally as well studied as gram-negative endotoxin. Gram positive bacteria can cause sepsis by at least two mechanisms: by producing exotoxins that act as superantigens and by components of their cell walls stimulating immune cells (Calandra, 2001, J. Chemother., 13:173-180).
  • Superantigens are molecules that bind to MHC class II molecules of antigen presenting cells and to V ⁇ chains of T cell receptors. In doing so, they activate large numbers of T cells to produce massive amounts of proinflammatory cytokines. Staphylococcal enterotoxins, toxic shock syndrome toxin-1, and streptococcal pyrogenic exotoxins are examples of bacterial superantigens.
  • TLR2 Toll-like receptor 2
  • the endothelial damage causes further exacerbation of inflammation, resulting in neutrophil activation, neutrophil-endothelial cell adhesion, and further elaboration of inflammatory cytokines (Esmon, 1998, Immunologist, 6, 84-89). These inflammatory processes further contribute to vascular endothelial dysfunction. Concurrently, the endothelial cells release Tissue Factor (TF), triggering the extrinsic coagulation cascade and accelerating the production of thrombin (Carvalho & Freeman, J. Crit. Illness, 9, 51-75; Esmon, 1998, Immunologist, 6, 84-89). This uncontrolled cascade of inflammation and coagulation fuels the progression of sepsis, resulting in hypoxia, widespread ischemia, organ dysfunction, and ultimately death for a large number of patients.
  • TF Tissue Factor
  • adjunctive treatments that is, other than antibiotics and supportive care
  • severe sepsis and septic shock have been tested in clinical trials.
  • These include neutralization of microbial toxins such as LPS, non-specific anti-inflammatory and immunosuppressive drugs, neutralization of pro-inflammatory cytokines, and correction of abnormalities in coagulation.
  • the results have been mixed (Vincent et al., 2002, Clin. Infect. Dis., 34:1084-1093), and it does not appear that any one treatment addresses all sepsis conditions and/or causative agents.
  • the lipo-polysaccharide endotoxin found in the cell wall of gram-negative bacteria plays a key role in initiating the humoral cascades observed in septic shock.
  • Several anti-endotoxin antibody products have been developed and have undergone human trials (Ziegler et al., 1982, N. Engl. J. Med., 307, 1225-1230; Angus et al., 2000, JAMA, 283, 1723-1730; McCloskey et al., 1994, Ann. Intern. Med., 121, 1-5).
  • none of the anti-endotoxin strategies have been shown to be of benefit in large clinical trials.
  • TNF tumor necrosis factor
  • IL-1 inter-leukin-1
  • Coagulation abnormalities are common in patients with sepsis and microvascular thrombosis.
  • the ensuing tissue damage may have an important role in the pathophysiology of organ dysfunction.
  • Treatment with activated protein C a protein with anti-thrombotic, pro-fibrinolytic, and anti-inflammatory effects, reduces mortality from severe sepsis (Bernard et al., 2001, N. Engl. J. Med., 344: 699-709). So far as is known, in the current market, it is the only drug to treat sepsis. However, treatment with this drug results in relatively modest improvements in patient mortality, and at the price of a slight increase in bleeding events (Id.).
  • Antibiotics must be broad spectrum and cover gram-positive, gram-negative, and anaerobic bacteria because all classes of these organisms produce identical clinical presentations. Antibiotics must be administered parenterally in doses adequate to achieve bactericidal serum levels. Many studies have found that clinical improvement correlates with the achievement of serum bactericidal levels rather than the number of antibiotics administered.
  • ODNs oligodeoxynucleotides
  • Oligonucleotide-mediated intervention (OMI) technology provides a powerful set of tools to alter the activity of any gene of known sequence at the genomic level, including triplex forming oligonucleotides for targeted gene expression, at the messenger RNA (mRNA) level using antisense, competitively inhibitory and DNA enzyme oligos and at the protein level using ssDNA as aptamers (Chen, 2002, Expert Opin. Biol. Ther.
  • Antisense, DNA enzyme, triplex, competitive inhibition and aptamer technologies provide an efficient alternative to more difficult methods such as creating gene knockout in cells and organisms.
  • Antisense oligonucleotides block gene expression by Watson-Crick base pairing between an ODN and its target mRNA thereby preventing translation of that MRNA by Ribosomes.
  • ODNs Antisense oligonucleotides
  • Antisense ODNs have been used to inhibit gene expression in eukaryotic cells and have been used to validate gene targets, and there is one antisense ODN-based product in the market and a number of others in advanced clinical trials (Uhlman, 2001, Expert Opinion on Biological Therapy, 1:319-328).
  • PNA peptide nucleic acid
  • An additional object of the present invention is to provide ODNs, and their sequences, that will knock down (silence) the bacterial, fungal, and host genes above.
  • An additional object of the present invention is to provide the said ODNs, and their sequences, as therapeutic anti-sepsis agents.
  • An additional object of the present invention is to identify delivery means of the said transfecting therapeutic ODN into target bacterial or fungal cells.
  • An additional object of the present invention is to provide a method of the treatment of bacterial or fungal sepsis using the said ODNs.
  • An additional object of the present invention is to provide plasmid constructions that are used to knock down the bacterial, fungal, and host genes above.
  • An additional object of the present invention is to provide the plasmid constructions that are used as therapeutic anti-sepsis agents.
  • An additional object of the present invention is to identify delivery means of the transfecting therapeutic ODN into target bacterial or fungal cells.
  • Still another object of the present invention is to provide a method of treatment of sepsis in an animal patient comprising the steps of contacting the causative agent of sepsis with an ODN comprising a sequence targeted to a specific gene of the causative agent for altering the expression of the specific gene to inhibit growth of the causative agent, kill the causative agent, or inhibit the synthesis or secretion of toxin by the causative agent.
  • An additional object of the present invention is to provide a method of the treatment of bacterial or fungal sepsis using a therapeutic ODN.
  • the present invention relates to a new strategy for combating sepsis.
  • the present invention comprises a list of genes critical to bacterial and fungal viability, a list of gene associated with the exaggerated innate immune response in sepsis, a list of ODNs (and their DNA sequences) effective in knocking down the said genes, a list of the said ODNs (and their DNA sequences) as therapeutic anti-sepsis agents, a method of using the said ODNs to treat bacterial sepsis, a list of plasmid constructions effective in knocking down said genes, a list of said plasmid constructions as therapeutic anti-sepsis agents, a delivery method of said plasmid constructions into bacterial cells, a delivery method of said plasmid construction into fungal cells, a delivery method of said plasmid constructions into host cells, a method of using said plasmid constructions to treat sepsis.
  • the present invention comprises methods for producing ODNs in bacterial or fungal cells in vivo so that, when said ODNs reach and knock down their target genes, and thereby kill bacterial or fungal cells or inhibit their growth, the bacterial or fungal accumulation in the bloodstream would be held constant or diminished and the sepsis syndrome would be reduced or eliminated.
  • the present invention comprises a method for producing ODNs in host cells in vivo so that, when the said ODNs silence their target genes, the host exaggerated innate immune response would be abrogated and thereby, the sepsis syndrome would be relieved.
  • the present invention comprises a method for delivering ODN-expressing plasmid constructions into bacterial or fungal cells in vivo so that, when the ODNs are produced intracellularly and knock down their target genes, and thereby kill bacterial or fungal cells, the bacterial or fungal accumulation in the bloodstream would be diminished and thereby, the sepsis syndrome would be relieved.
  • the present invention comprises a method for delivering the ODN-expressing plasmid constructions into host cells in vivo so that, when the ODNs are produced intracellularly and knock down their target genes, the host exaggerated innate immune response would be abrogated and thereby, the sepsis syndrome would be relieved.
  • FIG. 1 shows survival of infected mice with or without ODN treatment.
  • This log-phase preparation of bacteria was diluted in PBS, and 3 ⁇ 10e8 CFU of bacteria was i.p. injected to induce mouse sepsis.
  • the ODN was either mixed with bacteria in vitro and then was injected to mice, or mice were pretreated by ODN before infection. Serum was gathered and proinflammatory cytokines (IL-6, TNF, IL-1) and bacterial load tested, and mouse behavior monitored at various time points after injection.
  • proinflammatory cytokines IL-6, TNF, IL-1
  • FIG. 2 shows changes in mouse proinflammatory cytokine IL-6. Serum was collected at 4 hr and 24 hr after bacterial infection, and IL-6 concentration was measured using commercial kit.
  • FIG. 3 shows bacterial growth inhibition by ODN.
  • ODN Immediately after diluting the O/N cultures 1/50, ODN was added to final concentration of 4 uM, 40 ⁇ M or 400 ⁇ M, with addition of equal volume water as a negative control, and incubated with shaking at 30° C. After 2, 4 or 6 h, the growth was measured viable cell count, which was done by diluting the cultures and plating them on LB plates with streptomycin.
  • FIG. 4 shows bacterial growth inhibition by ODN expression plasmid AS830103.
  • the competent cells XL10-gold(kan) were transformed with the ODN expression plasmid or the plasmid without ODN insert, and plated onto LB media with chloramphenicol and incubated at 37° C. O/N.
  • the present invention relates to a new strategy for combating sepsis caused by bacterial and fungal pathogens, wherein selected ODNs and the expression plasmid used to produce them were used as therapeutic anti-sepsis agents.
  • sepsis that can be treated in accordance with the present invention include, but are not limited to, those caused by infections in the lung, abdomen, bloodstream, skin, soft tissue, caused by infections associated with intravascular devices, or caused by respiratory infections.
  • a target gene can be found among those involved in one of the major process complexes: cell division, cell wall synthesis, protein synthesis (translation), nucleic acid synthesis, fatty acid metabolism, and gene regulation.
  • examples of bacterial target genes that can be knocked down in accordance with the present invention include, but are not limited to FtsZ, MurB, acpP, 16s rRNA, PBPs, DNAA, DNAC, pcrA, rpoB, rpoA, rpoC, rpsC, rpsD, rpsF, rpsi, rpsJ, rpsM, rpsR, FabK, FabH, rplB, rplC, rplJ, rplK, rplM, rplN, rplO, rplP, rplR, rplT, rplV, rplX, rpmA, rpmL, valS, serS, proS, cysS, alaS, pheS, sporC, tsf, tufA,
  • the target genes critical to fungal viability can be found among those involved in one of the major process complexes: cell division, cell wall synthesis, protein synthesis (translation), nucleic acid synthesis, fatty acid metabolism, and gene regulation. Therefore, examples of fungal target genes that can be knocked down in accordance with the present invention include, but are not limited to, ERG1, ERG2, ERG3, ERG4, FRG5, ERG6, ERG7, ERGI11, ERG24, ERG25, ERGX, ERGY, CHS1, CHS2, CHS3, CWP1, CWP2, KRE1, KRE2, KRE5, KRE11, TIP1, GFA1.
  • ODN technology can down-regulate the over-expression of the host genes associated with the exaggerates innate immune response in sepsis, so that appropriate host response to an infection remains.
  • host target genes that can be knocked down in accordance with the present invention include, but are not limited to, tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), interleukin-15 (IL15), nitric oxide synthase (NOS), high mobility group 1 protein (HMG-1), migration inhibitory factor (MIF), Kinins, platelet-activating factor receptor antagonist (PAFra), soluble phospholipase A2 (sPLA2).
  • TNF tumor necrosis factor
  • IL-1 interleukin-1
  • IL-6 interleukin-6
  • IL-12 interleukin-12
  • IL15 interleukin-15
  • NOS nitric oxide synthase
  • NOS nitric oxide synthas
  • TNF is considered to be one of the most important inflammatory mediators in the sepsis cascade.
  • TNF is significantly elevated during sepsis (Casey et al., 1993, Ann. Intern. Med. 119:771-778; van der Poll and Lowry, 1995, Shock, 1-12), and levels of TNF have been associated with severity of sepsis and clinical outcome (Calandra et al., 1990, J. Infect. Dis. 161:982-987; Cannon et al., 1990, J. Infect. Dis., 161:79-84).
  • most of the deleterious effects of sepsis can be mimicked by the administration of TNF (Okusawa et al., 1988, J. Clin. Invest., 81:1162-1172; Natanson et al., 1989, J. Exp. Med., 169:823-832).
  • ODN therapeutics examples include, but are not limited to, CYXO080103, wherein, its sequence is 5′(CTT TCA ACA GTT TTG ATG ACC TTT GCT GAC CAT ACA ATT GCG ATA TCG TGG GGA GTG AGA G)3′, and its potential targets are btuE (GenBank ID: NP — 416225.1), CaiB (GenBank ID: NP — 414580.1), ydgD (GenBank ID: NP — 418152.1), ygcQ (GenBank ID: NP — 417249.2), ftsH (GenBank ID: NP — 417645.1), ppiB (GenBank ID: NP — 415058.1), yihl (GenBank ID: NP — 418308.1), zntA (GenBank ID: NP — 417926.1), yicI (GenBank ID: NP — 417926.1), yicI
  • LNA locked nucleic acids
  • PNA peptide nucleic acids
  • PMO phosphorothioates
  • Examples of means to deliver the said ODN and the said ODN expression plasmids into bacterial or fungal cells for treatment of sepsis include, but are not limited to, cationic polymers such as PEI, EPEI, and porphyrins; peptides such as Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys, Xaa Xaa Xaa Lys Lys Arg Arg Xaa Xaa Xaa Xaa Xaa Thr Trp Xaa Glu Thr Trp Trp Trp Xaa Xaa Xa, Lys Xaa Xa Trp Trp Trp Glu Thr Trp Trp Trp Xaa Xaa Ser Gln Pro Lys Lys Xaa Arg Lys Xaa, Tyr Gly Phe Lys Lys Xaa Arg Arg Pro Trp Thr Trp Trp Trp Glu Thr Trp Trp Xa
  • Examples of means to deliver the said ODN and the said ODN expression plasmids into host cells for treatment of sepsis, in accordance with the present invention include, but are not limited to, direct injection of naked DNA; cationic polymers such as PEI, EPEI, and porphyrins; peptides such as Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys, Xaa Xaa Xaa Lys Lys Arg Arg Xaa Xaa Xaa Xaa Xaa Thr Trp Xaa Glu Thr Trp Trp Trp Xaa Xaa Xa, Lys Xaa Xa Trp Trp Trp Glu Thr Trp Trp Trp Xaa Xaa Ser Gln Pro Lys Lys Xaa Arg Lys Xaa, Tyr Gly Phe Lys Lys Xaa Arg Arg Pro Trp Thr Trp Trp Trp
  • Examples of means to deliver the said ODN expression plasmids into host cells for treatment of sepsis include, but are not limited to, viral vectors such as retroviruses and adenoviruses; direct injection of naked DNA; cationic liposome such as DOTAP and DOTMAcationic polymers such as PEI and EPEI; peptides such as Xaa Xaa Xaa Lys Lys Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Thr Trp Xaa Glu Thr Trp Trp Trp Xaa Xaa Xa, Lys Xaa Xa Trp Trp Glu Thr Trp Trp Trp Xaa Xaa Ser Gln Pro Lys Lys Xaa Arg Lys Xaa, Tyr Gly Phe Lys Lys Xaa Arg Arg Pro Trp Thr Trp Trp Trp Glu Thr Trp X
  • E. coli SM101 a temperature-sensitive UDP-N-acetylglucosamine acyltransferase mutant that lose all detectable acyltransferase activity, and its wild-type K12, were i.p. injected as described below to induce sepsis in mouse.
  • SM101 has a defect in lipid A biosynthesis that causes the outer membrane to be permeable to high-molecular-weight substances. The lipid A content of SM101 is reduced 2-3-fold compared with the wild-type.
  • SM101 were grown in LB medium at 37° C.
  • SM101 Log-phase cultures of SM101 were grown to an optical density at 600 nm of 1.1 (equivalent to 5 ⁇ 108 CFU/ml), followed by centrifugation and resuspension in sterile phosphate-buffered saline (PBS) at 4° C.
  • PBS sterile phosphate-buffered saline
  • Serum was gathered and pro-inflammatory cytokines (IL-6, TNF, IL-1) and bacterial load tested, and mouse behavior monitored at various time points after injection. All mice bled at every 24 hours. At 6 hours, mice showed evidence of infection (lethargy, warm to the touch, scruffy).
  • mice As shown in FIG. 1 , around 60% mice died within 48 hours after infection. As shown in FIG. 2, 3 of 5 mice showed significant decrease in serum IL-6 concentration.
  • Table 1 shows mouse bacterial load in blood after infection. Serum sample was collected at 24 hrs after infection, for cell growth assay by measuring viable cell count. Viable cell count was done by diluting the cultures and plating them on LB plates. The plates were then incubated overnight at 37° C. and the number of colonies was enumerated by visual inspection.
  • ODN The inhibition of bacterial growth by ODN was evaluated by examining the effect of DNA dose on the ability of ODN to inhibit SM101 growth.
  • an ODN having the sequence CTC ATA CTC T was added to the 1/50 diluted O/N SM101 cell cultures, at final concentration of 40 ⁇ M or 400 ⁇ M, with addition of equal volume water as a negative control, and incubated with shaking at 30° C. After 2, 4 or 6 h, the growth was measured by either the optical density at 600 nm (OD600) or viable cell count, which was done by diluting the cultures and plating them in triplicate on LB plates with streptomycin. As shown in FIG. 3 , upon addition of ODN, cell growth was inhibited by 86-96%.
  • the ODN expression plasmid As080103 having the sequence CYGXO80103 listed above, and plasmid pssxGb without ODN insert as negative control, were transformed into E. coli XL10-gold(kan).
  • the resulting cell cultures were plated on LB media with chloramphenicol and incubated at 37° C. O/N.
  • no XL10-gold(kan) carrying ODN expression plasmid grew on the LB media.
  • mice Six-week-old mice Balb/c (in groups of five) were used for infection experiments. A serial dilution of SM101 was injected intraperitoneally (i.p.) into mice in 400- ⁇ l aliquots. The animals were observed for 100 h. Mice inoculated with bacteria were scored for their state of health on a scale of 5 to 0, based on progressive disease states reflected by several clinical signs.
  • a normal and unremarkable condition was scored as 5; slight illness, defined as lethargy and ruffled fur, was scored as 4; moderate illness, defined as severe lethargy, ruffled fur, and hunched back, was scored as 3; severe illness, with the above signs plus exudative accumulation around partially closed eyes, was scored as 2; a moribund state was scored as 1; and death was scored as 0. While the experiments were not conducted in a double-blind manner, all animals were evaluated by two or more independent observers. The signs of sepsis will be also detected by relative change in both cytokines/chemokines and clearance/persistence of organisms from peritoneal cavity and spleen.
  • the efficacy of ODN therapy was evaluated in two separate experiments using the above-described SM101 bacteremia mouse model. The first examined the effect of DNA dose on the ability of ODN to rescue mice from SM101 bacteremia. The second studied the effect on the outcome of delaying treatment for various periods.
  • the dose-ranging study five groups of mice (five mice in each) were challenged by i.p. injection of the LD70 of SM101. Each of these groups was treated with a single injection of the ODN CTC ATA CTC T, administered i.p. immediately after the bacterial challenge at 4 nmol, 40 nmol, 400 nmol and 0 nmol.
  • a fifth group was not challenged with bacteria, receiving only the injection of ODN (at the highest dose). The state of the health of these animals was monitored for one week.
  • FIG. 1 shows survival of infected mice with or without ODN treatment.
  • a standard DNA packaging reaction mixture (25 ⁇ l) contains 0.5 mg the said plasmid DNA, 2 ⁇ 1010 phage equivalent(peg) of prohead, 20 pmol of gp 18 and 3 pmol of gp 19 in complete pac buffer.
  • the reaction mixture was incubated at 30° C. for 30 min for DNA packaging and the reaction was terminated by the addition of 1 ⁇ l of 2 mg/ml of DNase I. After incubation at 30° C. for 20 min, the filled heads are converted to infectious particles by incubation with a head acceptor extract containing tail and tail fiber proteins.
  • Proheads are prepared through a sucrose gradient centrifuge of lysates of bacterial cells infected with bacteriophage T3, as described by Nakasu et al. (Nakasu et al., 1983, Virology, 127, 124-133). gp 18 and gp 19 proteins are purified as decribed by Hamada et al (Hamada et al., 1986, Virology, 151, 110-118). The head acceptor extracts are isolated from the lysates of bacterial cells infected by bacteriophage T3, and purified through ammonium sulfate precipitation. The resulting infectious particles contain the said ODN expression plasmid and are used to deliver the ODN expression plasmid to the target bacterial cells.
  • mice TABLE 1 Mouse bacterial load after infection Septicemia: Mice Bled at 24 hours Post Infection # Mice Bled CFU/ml *Control 6 0, 0, 3.6 ⁇ 10 3 , 1.6 ⁇ 10 3 , 4.6 ⁇ 10 3 , >10 5 PNA [10 uM] 4 0, 0, 0, 0 IP, PNA [100 uM] 4 0, 0, 0, 0 *Septicemic mice succumbed to infection prior to 46 h.

Abstract

A method for producing ODNs in bacterial or fungal cells in vivo for treatment of sepsis so that, when the ODNs reach and knock down their target genes, and thereby kill bacterial or fungal cells or inhibit their growth, the bacterial or fungal accumulation in the bloodstream is held constant or diminished and the sepsis syndrome is reduced or eliminated. The invention also contemplates of certain ODNs for use in treatment of sepsis.

Description

    CROSS REFERENCE TO RELATED APPLICATION
  • The present application is a continuation-in-part of co-pending application Ser. No. 10/453,410, filed Jun. 3, 2003 and IDENTIFICATION OF NOVEL ANTIBACTERIAL AGENTS BY SCREENING THE SINGLE-STRANDED DNA EXPRESSION LIBRARY, and co-pending application Ser. No. 10/743,956, filed Dec. 23, 2003 and entitled OLIGODOXYNUCLEOTIDE (ODN) LIBRARIES, THEIR USE IN SCREENING FOR ANTIBACTERIAL AGENTS, AND CATALYTIC ODN SEQUENCE FOR USE AS AN ANTIBACTERIAL AGENT. Both of the applications listed in this paragraph are hereby incorporated into the specification of the present application in their entirety by this specific reference thereto.
    • BACKGROUND OF THE INVENTION
  • Severe sepsis and septic shock are life threatening complication of infections and the most common cause of death in intensive care units (Angus et al., 2001, Crit. Care Med., 29:1303-1310). Recent US and European surveys have estimated that severe sepsis accounts for 2-11% of all admissions to hospital or intensive care units (Martin et al., 2003, New Engl. J. Med., 348, 1546-1554). The incidence of this condition is rising due to the aging of the population and increasing numbers of immuno-compromised and critically ill patients.
  • Sepsis comprises a complex clinical syndrome that results from the body's response to infection caused by bacterial and/or fungal pathogens invading the body (Cohen, 2002, Nature, 420, 885-891). Normally, a potent, complex, immunologic cascade ensures a prompt, protective response to microorganism invasion in humans. However, a deficient immunologic defense may allow infection to become established. Further, an excessive or poorly regulated response may harm the host through maladaptive release of indigenously generated inflammatory compounds. Additionally, diabetic individuals or others who suffer from lymphedema, especially in the feet and legs, are at risk of infection from exogenous opportunistic pathogens normally present on the skin due to the growth potential afforded by the edematous medium or the lack of circulation. Such infections may result in severe cellulitis or related sequelae that can lead to sepsis.
  • Gram negative bacilli (mainly Escherichia coli, Klebsiella species, and Pseudomonas aeruginosa) and Gram positive cocci (mainly staphylococci and streptococci) are the most common microbial pathogens isolated from patients with severe sepsis and septic shock. (Bochud, 2001, Intensive Care Med., 27 (Suppl 1): S33-S48). Although infections by Gram negative bacteria were predominant in the 1960s and early 1970s, Gram positive infections have increased in the past two decades and now account for about half of cases of severe sepsis. Fungi, mostly Candida, account for about 5% of all cases of severe sepsis, but these fungal infections are also increasing in many countries.
  • Gram negative infections usually occur in the lung, abdomen, bloodstream, or urinary tract. Endotoxins in the form of lipopolysaccharides (LPS), are an important component of the outer membrane of Gram negative bacteria, and have a pivotal role in inducing Gram negative sepsis (Alexander & Rietschel, 2001, J. Endotoxin Res., 7:167-202). LPS binding protein in host cells binds to LPS in the bacteria and transfers it to CD14 (Ulevitch & Tobias, 1999, Curr. Opin. Immunol., 11:19-22), a protein anchored in the outer leaflet of the plasma membrane. A series of remarkable investigations have recently led to the identification of a Toll-like receptor 4 (TLR4) as the co-receptor for LPS (Ulevitch & Tobias, 1999, Curr. Opin. Immunol., 11:19-22).
  • Gram positive bacteria are usually responsible for infections of skin and soft tissue, infections associated with intravascular devices, primary bloodstream infections, or respiratory infections. It is generally thought that the distinct cell wall substances of gram-positive bacteria and fungi trigger a similar cascade of events as gram negative bacteria, although the structures involved are not generally as well studied as gram-negative endotoxin. Gram positive bacteria can cause sepsis by at least two mechanisms: by producing exotoxins that act as superantigens and by components of their cell walls stimulating immune cells (Calandra, 2001, J. Chemother., 13:173-180).
  • Superantigens are molecules that bind to MHC class II molecules of antigen presenting cells and to Vβ chains of T cell receptors. In doing so, they activate large numbers of T cells to produce massive amounts of proinflammatory cytokines. Staphylococcal enterotoxins, toxic shock syndrome toxin-1, and streptococcal pyrogenic exotoxins are examples of bacterial superantigens.
  • Gram positive bacteria without exotoxins can also induce shock, probably by stimulating innate immune responses through similar mechanisms to those in Gram negative sepsis. Indeed, Toll-like receptor 2 (TLR2) has been shown to mediate cellular responses to heat killed Gram positive bacteria and their cell wall structures (peptidoglycan, lipoproteins, lipoteichoic acid, and phenol soluble modulin) (Takeuchi et al., 1999, Immunity, 11:443-451).
  • Following the initial host-microbial interaction, there is widespread activation of the innate immune response, releasing the classic pro-inflammatory cytokines IL-1, IL-6 TNF-α, and many other cytokines including IL-12, IL-15, and IL18. This, in turn, activates a second level of inflammatory cascades including cytokines, lipid mediators and reactive oxygen species. These early immune responses have direct damaging actions on the vascular endothelium (Wheeler et al, 1999, N. Engl. J. Med., 340:207-214). The endothelial damage causes further exacerbation of inflammation, resulting in neutrophil activation, neutrophil-endothelial cell adhesion, and further elaboration of inflammatory cytokines (Esmon, 1998, Immunologist, 6, 84-89). These inflammatory processes further contribute to vascular endothelial dysfunction. Concurrently, the endothelial cells release Tissue Factor (TF), triggering the extrinsic coagulation cascade and accelerating the production of thrombin (Carvalho & Freeman, J. Crit. Illness, 9, 51-75; Esmon, 1998, Immunologist, 6, 84-89). This uncontrolled cascade of inflammation and coagulation fuels the progression of sepsis, resulting in hypoxia, widespread ischemia, organ dysfunction, and ultimately death for a large number of patients.
  • Numerous adjunctive treatments (that is, other than antibiotics and supportive care) for severe sepsis and septic shock have been tested in clinical trials. These include neutralization of microbial toxins such as LPS, non-specific anti-inflammatory and immunosuppressive drugs, neutralization of pro-inflammatory cytokines, and correction of abnormalities in coagulation. The results have been mixed (Vincent et al., 2002, Clin. Infect. Dis., 34:1084-1093), and it does not appear that any one treatment addresses all sepsis conditions and/or causative agents.
  • As recited above, the lipo-polysaccharide endotoxin found in the cell wall of gram-negative bacteria plays a key role in initiating the humoral cascades observed in septic shock. Several anti-endotoxin antibody products have been developed and have undergone human trials (Ziegler et al., 1982, N. Engl. J. Med., 307, 1225-1230; Angus et al., 2000, JAMA, 283, 1723-1730; McCloskey et al., 1994, Ann. Intern. Med., 121, 1-5). However, despite some encouraging results from early studies, none of the anti-endotoxin strategies have been shown to be of benefit in large clinical trials.
  • Serum levels of tumor necrosis factor (TNF) and inter-leukin-1 (IL-1) are elevated in patients with septic shock. Both produce hemodynamic effects that duplicate those found in sepsis, and studies indicate that both mediators play key roles in sepsis and septic shock such that TNF may be a central mediator in sepsis. Similar to anti-endotoxin antibodies, antibodies to TNF or IL-1 are hypothesized to be useful in septic shock. However, anti-TNF or anti-IL-1 antibodies have yet to be demonstrated any beneficial effect in sepsis or septic shock (Abraham et al., 1998, Lancet, 351, 929-933).
  • While theoretical and experimental animal evidence exists supporting the use of large doses of corticosteroids in severe sepsis and septic shock, all randomized human studies found that corticosteroids do not prevent the development of septic shock, reverse the shock state, or improve mortality. Lefering & Neugebauer, 1995, Crit. Care Med., 23, 1294-1303; Cronin, 1995, Crit. Care Med., 23, 1430-1439.
  • Coagulation abnormalities, especially disseminated intravascular coagulation, are common in patients with sepsis and microvascular thrombosis. The ensuing tissue damage may have an important role in the pathophysiology of organ dysfunction. Treatment with activated protein C, a protein with anti-thrombotic, pro-fibrinolytic, and anti-inflammatory effects, reduces mortality from severe sepsis (Bernard et al., 2001, N. Engl. J. Med., 344: 699-709). So far as is known, in the current market, it is the only drug to treat sepsis. However, treatment with this drug results in relatively modest improvements in patient mortality, and at the price of a slight increase in bleeding events (Id.).
  • Prophylactic administration of antibiotics is still the main treatment of choice for sepsis in hospital (Cunha, 1995, Med. Clin. North Am., 79, 551-558). Antibiotics must be broad spectrum and cover gram-positive, gram-negative, and anaerobic bacteria because all classes of these organisms produce identical clinical presentations. Antibiotics must be administered parenterally in doses adequate to achieve bactericidal serum levels. Many studies have found that clinical improvement correlates with the achievement of serum bactericidal levels rather than the number of antibiotics administered.
  • Unfortunately, numerous classes of antibiotics have become less effective as a result of the rapid emergence of antibiotic resistance by many common bacterial pathogens such as S. aureus, Streptococcus pneumoniae and Enterococcus faecalis (Nicolaou & Boddy, 2001, Scientific American, p.56-61). Methicillin-resistant S. aureus (MRSA), penicillin-resistant S. pneumococcus and vancomycin-resistant E. faecalis (VRE) are now common pathogens that are difficult to treat effectively (Pfaller, et al., 1998, Antimicrobial Agents and Chemotherapy, 42:1762-1770; Jones, et al., 1999, Microbiol. Infect. Dis., 33:101-112). Probably more alarming is the emergence of multi-drug resistance pathogens (Swartz, 1994, Proc Natl. Acad. Sci. USA, 91:2420-2427; Baquero, 1997, J. Antimicrobial Chemotherapy, 39:1-6). Opportunistic fungal pathogens resistant to antifungal agents have also been increasingly documented in recent years and their frequency will likely continue to increase (Rex, 1997, Clin. Infect. Dis., 24:235-247). Candida spp., Cryptococcus neoformans, and Aspergillus spp. are among the leading fungi responsible for the invasive infections, and antifungal resistance has been described with each of these fungi.
  • Until recently, the principal approach of the pharmaceutical industry to this growing problem has been to seek incremental improvements in existing drugs (Piddock, 1998, Curr. Opin. Microbiol., 1, 502-508). Although these approaches make a significant contribution to fighting against bacterial infections, difficulty remains in meeting the increasing needs of the medical community. Thus, there is an urgent need for new discovery strategies to discover and develop new classes of antibiotics.
  • Recent advances in DNA sequencing technology have made it possible to elucidate the entire genome sequences of pathogenic bacteria and fungi. Such sequence information provides the necessary information to identify potential gene targets, and therefore enable construction of oligodeoxynucleotides (ODNs) for anti-bacterial or anti-fungal use. Oligonucleotide-mediated intervention (OMI) technology provides a powerful set of tools to alter the activity of any gene of known sequence at the genomic level, including triplex forming oligonucleotides for targeted gene expression, at the messenger RNA (mRNA) level using antisense, competitively inhibitory and DNA enzyme oligos and at the protein level using ssDNA as aptamers (Chen, 2002, Expert Opin. Biol. Ther. 2(7) 735-740). This technology has shown potential for developing highly specific and efficacious antibacterial agents (Harth et al., 2000, Proc. Natl. Acad. Sci. U.S.A, 97, 418-423; Good & Nielsen, 1998, Nature Biotech., 16, 355-358; Gasparro et al., 1991, Antisense Research and Development, 1, 117-140). Two patents suggesting a role for AS-ODN molecules in the regulation of bacterial growth have been issued (McDevitt et al., 2002, J. Appl. Microbiol. Symp. Suppl., 92, 28s-34s).
  • Antisense, DNA enzyme, triplex, competitive inhibition and aptamer technologies provide an efficient alternative to more difficult methods such as creating gene knockout in cells and organisms. Antisense oligonucleotides (ODNs) block gene expression by Watson-Crick base pairing between an ODN and its target mRNA thereby preventing translation of that MRNA by Ribosomes. (Crooke, 1999, Biochim. Biophys. Acta 1489:31-44). Antisense ODNs have been used to inhibit gene expression in eukaryotic cells and have been used to validate gene targets, and there is one antisense ODN-based product in the market and a number of others in advanced clinical trials (Uhlman, 2001, Expert Opinion on Biological Therapy, 1:319-328). However, antisense technology is not used extensively in prokaryotic systems. Prokaryotic cells (the cells involved in sepsis) have themselves developed endogenous antisense mechanisms for gene regulation (Simons & Kleckner, 1988, Annu. Rev. Genet., 22:567-600). Earlier results indicated that gene expression in bacteria may be accessible to inhibition by modified ODNs (Jayayaraman et al., 1981, Proc. Natl. Acad. Sci. USA, 78:1537-1541; Gasparro et al., 1991, Antisense Res Dev., 1:117-140) and that peptide nucleic acid (PNA) can inhibit gene expression in bacteria (Good & Nielsen, 1998, Nature Biotech., 16:355-358). PNA, a DNA mimic in which the nucleotide bases are attached to a pseudo-peptide backbone, hybridizes with complementary DNA, RNA, or PNA oligomers through Watson-Crick base pairing and helix formation.
  • Techniques using a screening library such as is described in co-pending U.S. pat. application Ser. No. 10/453,410, assigned to the same Assignee as the present application, have enabled both identification of genes critical to bacterial viability and the ODNs effective in silencing those critical genes, thus inhibiting bacterial growth/replication or killing the bacteria. The application of these ODNs and their expression plasmids to control bacterial and/or fungal pathogens causing sepsis is one focus of the present invention. On the other hand, sepsis represents an excessive innate immune response to microbial products, and many processes in the complex pathophysiology of sepsis are simultaneously over-activated. The complexity of this response affords ample targets for ODN therapy. For that reason, the design and application of ODNs and their expression plasmids to down-regulate the over-activated processes is another focus of the present invention. It is, therefore, an object of the present invention to identify the genes necessary for bacterial and fungal viability, and the host genes associated with the exaggerated innate immune response in sepsis.
  • An additional object of the present invention is to provide ODNs, and their sequences, that will knock down (silence) the bacterial, fungal, and host genes above.
  • An additional object of the present invention is to provide the said ODNs, and their sequences, as therapeutic anti-sepsis agents.
  • An additional object of the present invention is to identify delivery means of the said transfecting therapeutic ODN into target bacterial or fungal cells.
  • An additional object of the present invention is to provide a method of the treatment of bacterial or fungal sepsis using the said ODNs.
  • An additional object of the present invention is to provide plasmid constructions that are used to knock down the bacterial, fungal, and host genes above.
  • An additional object of the present invention is to provide the plasmid constructions that are used as therapeutic anti-sepsis agents.
  • An additional object of the present invention is to identify delivery means of the transfecting therapeutic ODN into target bacterial or fungal cells.
  • Still another object of the present invention is to provide a method of treatment of sepsis in an animal patient comprising the steps of contacting the causative agent of sepsis with an ODN comprising a sequence targeted to a specific gene of the causative agent for altering the expression of the specific gene to inhibit growth of the causative agent, kill the causative agent, or inhibit the synthesis or secretion of toxin by the causative agent.
  • An additional object of the present invention is to provide a method of the treatment of bacterial or fungal sepsis using a therapeutic ODN.
    • SUMMARY OF THE INVENTION
  • The present invention relates to a new strategy for combating sepsis. The present invention comprises a list of genes critical to bacterial and fungal viability, a list of gene associated with the exaggerated innate immune response in sepsis, a list of ODNs (and their DNA sequences) effective in knocking down the said genes, a list of the said ODNs (and their DNA sequences) as therapeutic anti-sepsis agents, a method of using the said ODNs to treat bacterial sepsis, a list of plasmid constructions effective in knocking down said genes, a list of said plasmid constructions as therapeutic anti-sepsis agents, a delivery method of said plasmid constructions into bacterial cells, a delivery method of said plasmid construction into fungal cells, a delivery method of said plasmid constructions into host cells, a method of using said plasmid constructions to treat sepsis.
  • The present invention comprises methods for producing ODNs in bacterial or fungal cells in vivo so that, when said ODNs reach and knock down their target genes, and thereby kill bacterial or fungal cells or inhibit their growth, the bacterial or fungal accumulation in the bloodstream would be held constant or diminished and the sepsis syndrome would be reduced or eliminated.
  • The present invention comprises a method for producing ODNs in host cells in vivo so that, when the said ODNs silence their target genes, the host exaggerated innate immune response would be abrogated and thereby, the sepsis syndrome would be relieved.
  • The present invention comprises a method for delivering ODN-expressing plasmid constructions into bacterial or fungal cells in vivo so that, when the ODNs are produced intracellularly and knock down their target genes, and thereby kill bacterial or fungal cells, the bacterial or fungal accumulation in the bloodstream would be diminished and thereby, the sepsis syndrome would be relieved.
  • The present invention comprises a method for delivering the ODN-expressing plasmid constructions into host cells in vivo so that, when the ODNs are produced intracellularly and knock down their target genes, the host exaggerated innate immune response would be abrogated and thereby, the sepsis syndrome would be relieved.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows survival of infected mice with or without ODN treatment. This log-phase preparation of bacteria was diluted in PBS, and 3×10e8 CFU of bacteria was i.p. injected to induce mouse sepsis. To treat mice with ODN, the ODN was either mixed with bacteria in vitro and then was injected to mice, or mice were pretreated by ODN before infection. Serum was gathered and proinflammatory cytokines (IL-6, TNF, IL-1) and bacterial load tested, and mouse behavior monitored at various time points after injection.
  • FIG. 2 shows changes in mouse proinflammatory cytokine IL-6. Serum was collected at 4 hr and 24 hr after bacterial infection, and IL-6 concentration was measured using commercial kit.
  • FIG. 3 shows bacterial growth inhibition by ODN. Immediately after diluting the O/N cultures 1/50, ODN was added to final concentration of 4 uM, 40 μM or 400 μM, with addition of equal volume water as a negative control, and incubated with shaking at 30° C. After 2, 4 or 6 h, the growth was measured viable cell count, which was done by diluting the cultures and plating them on LB plates with streptomycin.
  • FIG. 4 shows bacterial growth inhibition by ODN expression plasmid AS830103. The competent cells XL10-gold(kan) were transformed with the ODN expression plasmid or the plasmid without ODN insert, and plated onto LB media with chloramphenicol and incubated at 37° C. O/N.
    • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • The present invention relates to a new strategy for combating sepsis caused by bacterial and fungal pathogens, wherein selected ODNs and the expression plasmid used to produce them were used as therapeutic anti-sepsis agents.
  • Examples of sepsis that can be treated in accordance with the present invention include, but are not limited to, those caused by infections in the lung, abdomen, bloodstream, skin, soft tissue, caused by infections associated with intravascular devices, or caused by respiratory infections.
  • Examples of microorganisms that can be treated in accordance with the present invention include, but are not limited to, Gram-negative bacteria such as Bacteroides, Fusobacterium, Escherichia, Klebsiella, Salmonella, Shigella, Proteus, Pseudomonas, Vibrio, Legionella, Haemophilus, Bordetella, Brucella, Campylobacter, Neisseria, Branhamella; Gram-positive bacteria such as Streptococcus, Staphylococcus, Peptococcus, Bacillus, Listeria, Clostridium, Propionebacteria; organisms that stain poorly or not at all with Gram's stain such as Mycobacteria, Treponema, Leptospira, Borrelia, Mycoplasma, Clamydia, Rickettsia and Coxiella; and Fungi such as Candida, Aspergillosis, Blastomycosis, Coccidioidomycosis, Cryptococcosis, Histoplasmosis, Paracoccidiomycosis, Sporotrichosis, Zygomycosis.
  • Examples of bacterial target genes that can be knocked down in accordance with the present invention include, but are not limited to, those identified from library screening and those chosen based upon knowledge about bacterial physiology. A target gene can be found among those involved in one of the major process complexes: cell division, cell wall synthesis, protein synthesis (translation), nucleic acid synthesis, fatty acid metabolism, and gene regulation. Therefore, examples of bacterial target genes that can be knocked down in accordance with the present invention include, but are not limited to FtsZ, MurB, acpP, 16s rRNA, PBPs, DNAA, DNAC, pcrA, rpoB, rpoA, rpoC, rpsC, rpsD, rpsF, rpsi, rpsJ, rpsM, rpsR, FabK, FabH, rplB, rplC, rplJ, rplK, rplM, rplN, rplO, rplP, rplR, rplT, rplV, rplX, rpmA, rpmL, valS, serS, proS, cysS, alaS, pheS, sporC, tsf, tufA, fus, secA, secV, pyrc.
  • The target genes critical to fungal viability can be found among those involved in one of the major process complexes: cell division, cell wall synthesis, protein synthesis (translation), nucleic acid synthesis, fatty acid metabolism, and gene regulation. Therefore, examples of fungal target genes that can be knocked down in accordance with the present invention include, but are not limited to, ERG1, ERG2, ERG3, ERG4, FRG5, ERG6, ERG7, ERGI11, ERG24, ERG25, ERGX, ERGY, CHS1, CHS2, CHS3, CWP1, CWP2, KRE1, KRE2, KRE5, KRE11, TIP1, GFA1.
  • ODN technology can down-regulate the over-expression of the host genes associated with the exaggerates innate immune response in sepsis, so that appropriate host response to an infection remains. Examples of host target genes that can be knocked down in accordance with the present invention include, but are not limited to, tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), interleukin-15 (IL15), nitric oxide synthase (NOS), high mobility group 1 protein (HMG-1), migration inhibitory factor (MIF), Kinins, platelet-activating factor receptor antagonist (PAFra), soluble phospholipase A2 (sPLA2). In particular, TNF is considered to be one of the most important inflammatory mediators in the sepsis cascade. TNF is significantly elevated during sepsis (Casey et al., 1993, Ann. Intern. Med. 119:771-778; van der Poll and Lowry, 1995, Shock, 1-12), and levels of TNF have been associated with severity of sepsis and clinical outcome (Calandra et al., 1990, J. Infect. Dis. 161:982-987; Cannon et al., 1990, J. Infect. Dis., 161:79-84). Moreover, most of the deleterious effects of sepsis can be mimicked by the administration of TNF (Okusawa et al., 1988, J. Clin. Invest., 81:1162-1172; Natanson et al., 1989, J. Exp. Med., 169:823-832).
  • Examples of the ODN therapeutics that are used to treat sepsis in accordance with the present invention include, but are not limited to, CYXO080103, wherein, its sequence is 5′(CTT TCA ACA GTT TTG ATG ACC TTT GCT GAC CAT ACA ATT GCG ATA TCG TGG GGA GTG AGA G)3′, and its potential targets are btuE (GenBank ID: NP—416225.1), CaiB (GenBank ID: NP—414580.1), ydgD (GenBank ID: NP—418152.1), ygcQ (GenBank ID: NP—417249.2), ftsH (GenBank ID: NP—417645.1), ppiB (GenBank ID: NP—415058.1), yihl (GenBank ID: NP—418308.1), zntA (GenBank ID: NP—417926.1), yicI (GenBank ID: NP—418116.1), fhuA(GenBank ID: NP—414692.1), rpID (GenBank ID: NP—417778.1), ilvB (GenBank ID: NP—418127.1), lepB (GenBank ID: NP—417063.1), aroK (GenBank ID: NP—417849.1), mfd (GenBank ID: NP—415632.1), ripA (GenBank ID: NP—415166.1), accA (GenBank ID: NP—414727.1), pgpA (GenBank ID: NP—414952.1); CYGXacpP, wherein, its sequence is 5′(CTC ATA CTC T)3′ in PNA form, and its target is the bacterial essential fatty acid biosynthesis gene acpP (DenBank ID: NP—309499); CYGXFtsZDZ. wherein, its sequence is 5′(GTT TCG AAG GCT AGC TAC AAC GAT CAT CCA G)3′, and its target is the bacterial essential cell division gene FtsZ (GenBank ID: NP—308126).
  • Examples of the DNA therapeutics that are used to treat sepsis in accordance with the present invention include, but are not limited to, regular ODN and its expression plasmid, its modification forms such as locked nucleic acids (LNA), peptide nucleic acids (PNA), phosphorothioates, or phosphorothioates morpholino oligomer (PMO).
  • Examples of means to deliver the said ODN and the said ODN expression plasmids into bacterial or fungal cells for treatment of sepsis, in accordance with the present invention include, but are not limited to, cationic polymers such as PEI, EPEI, and porphyrins; peptides such as Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys, Xaa Xaa Xaa Lys Lys Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Thr Trp Xaa Glu Thr Trp Trp Xaa Xaa Xaa, Lys Xaa Xaa Trp Trp Glu Thr Trp Trp Xaa Xaa Ser Gln Pro Lys Lys Xaa Arg Lys Xaa, Tyr Gly Phe Lys Lys Xaa Arg Arg Pro Trp Thr Trp Trp Glu Thr Trp Trp Thr Glu Xaa, wherein any Xaa can be any amino acid. The said ODN expression plasmids can also be delivered into bacterial cells by packaging the said plasmids into infectious particles using phage extracts, as detailed below in Example 6.
  • Examples of means to deliver the said ODN and the said ODN expression plasmids into host cells for treatment of sepsis, in accordance with the present invention include, but are not limited to, direct injection of naked DNA; cationic polymers such as PEI, EPEI, and porphyrins; peptides such as Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys, Xaa Xaa Xaa Lys Lys Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Thr Trp Xaa Glu Thr Trp Trp Xaa Xaa Xaa, Lys Xaa Xaa Trp Trp Glu Thr Trp Trp Xaa Xaa Ser Gln Pro Lys Lys Xaa Arg Lys Xaa, Tyr Gly Phe Lys Lys Xaa Arg Arg Pro Trp Thr Trp Trp Glu Thr Trp Trp Thr Glu Xaa, wherein any Xaa can be any amino acid. Examples of means to deliver the said ODN expression plasmids into host cells for treatment of sepsis, in accordance with the present invention include, but are not limited to, viral vectors such as retroviruses and adenoviruses; direct injection of naked DNA; cationic liposome such as DOTAP and DOTMAcationic polymers such as PEI and EPEI; peptides such as Xaa Xaa Xaa Lys Lys Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Thr Trp Xaa Glu Thr Trp Trp Xaa Xaa Xaa, Lys Xaa Xaa Trp Trp Glu Thr Trp Trp Xaa Xaa Ser Gln Pro Lys Lys Xaa Arg Lys Xaa, Tyr Gly Phe Lys Lys Xaa Arg Arg Pro Trp Thr Trp Trp Glu Thr Trp Trp Thr Glu Xaa, wherein any Xaa can be any amino acid.
  • The present invention can be better understood by reference to the following actual examples demonstrating the operational capability of the invention. However. these examples are but one embodiment of the present invention, presented for the purpose of exemplifying the invention in accordance with the requirements of the Patent Statute. These examples therefore do not represent the full scope of the invention; reference is made to the claims that are appended hereto for a determination of the scope of the present invention.
    • EXAMPLE 1 Development of the Mouse Sepsis Model
  • E. coli SM101, a temperature-sensitive UDP-N-acetylglucosamine acyltransferase mutant that lose all detectable acyltransferase activity, and its wild-type K12, were i.p. injected as described below to induce sepsis in mouse. SM101 has a defect in lipid A biosynthesis that causes the outer membrane to be permeable to high-molecular-weight substances. The lipid A content of SM101 is reduced 2-3-fold compared with the wild-type. To prepare the bacteria for mouse infection, SM101 were grown in LB medium at 37° C. Log-phase cultures of SM101 were grown to an optical density at 600 nm of 1.1 (equivalent to 5×108 CFU/ml), followed by centrifugation and resuspension in sterile phosphate-buffered saline (PBS) at 4° C. This log-phase preparation of bacteria was serially diluted in PBS, and 3×10e8 CFU of bacteria was i.p. injected to induce mouse sepsis. Serum was gathered and pro-inflammatory cytokines (IL-6, TNF, IL-1) and bacterial load tested, and mouse behavior monitored at various time points after injection. All mice bled at every 24 hours. At 6 hours, mice showed evidence of infection (lethargy, warm to the touch, scruffy). As shown in FIG. 1, around 60% mice died within 48 hours after infection. As shown in FIG. 2, 3 of 5 mice showed significant decrease in serum IL-6 concentration. Table 1 shows mouse bacterial load in blood after infection. Serum sample was collected at 24 hrs after infection, for cell growth assay by measuring viable cell count. Viable cell count was done by diluting the cultures and plating them on LB plates. The plates were then incubated overnight at 37° C. and the number of colonies was enumerated by visual inspection.
    • EXAMPLE 2 Inhibition of Bacterial Growth by ODN
  • The inhibition of bacterial growth by ODN was evaluated by examining the effect of DNA dose on the ability of ODN to inhibit SM101 growth. In this study, an ODN having the sequence CTC ATA CTC T was added to the 1/50 diluted O/N SM101 cell cultures, at final concentration of 40 μM or 400 μM, with addition of equal volume water as a negative control, and incubated with shaking at 30° C. After 2, 4 or 6 h, the growth was measured by either the optical density at 600 nm (OD600) or viable cell count, which was done by diluting the cultures and plating them in triplicate on LB plates with streptomycin. As shown in FIG. 3, upon addition of ODN, cell growth was inhibited by 86-96%.
    • EXAMPLE 3 Inhibition of Bacterial Growth by ODN Expression Plasmid
  • In this study, the ODN expression plasmid As080103, having the sequence CYGXO80103 listed above, and plasmid pssxGb without ODN insert as negative control, were transformed into E. coli XL10-gold(kan). The resulting cell cultures were plated on LB media with chloramphenicol and incubated at 37° C. O/N. As shown in FIG. 4, no XL10-gold(kan) carrying ODN expression plasmid grew on the LB media.
    • EXAMPLE 4 Establishing Lethal Dose (LD70 ) in the Mouse Model
  • Six-week-old mice Balb/c (in groups of five) were used for infection experiments. A serial dilution of SM101 was injected intraperitoneally (i.p.) into mice in 400-μl aliquots. The animals were observed for 100 h. Mice inoculated with bacteria were scored for their state of health on a scale of 5 to 0, based on progressive disease states reflected by several clinical signs. A normal and unremarkable condition was scored as 5; slight illness, defined as lethargy and ruffled fur, was scored as 4; moderate illness, defined as severe lethargy, ruffled fur, and hunched back, was scored as 3; severe illness, with the above signs plus exudative accumulation around partially closed eyes, was scored as 2; a moribund state was scored as 1; and death was scored as 0. While the experiments were not conducted in a double-blind manner, all animals were evaluated by two or more independent observers. The signs of sepsis will be also detected by relative change in both cytokines/chemokines and clearance/persistence of organisms from peritoneal cavity and spleen. These experiments established that 109 CFU of strain SM101 is the LD70 for 6-week-old mice Balb/c, with ˜60% mice that received the LD70 dose dying within 48 h. This LD70 was used in all the DNA therapy experiments described in this study. A similar approach was used to establish the LD70 for E.coli strain K-12.
    • EXAMPLE 5 Treatment of Sepsis using ODN
  • The efficacy of ODN therapy was evaluated in two separate experiments using the above-described SM101 bacteremia mouse model. The first examined the effect of DNA dose on the ability of ODN to rescue mice from SM101 bacteremia. The second studied the effect on the outcome of delaying treatment for various periods. In the dose-ranging study, five groups of mice (five mice in each) were challenged by i.p. injection of the LD70 of SM101. Each of these groups was treated with a single injection of the ODN CTC ATA CTC T, administered i.p. immediately after the bacterial challenge at 4 nmol, 40 nmol, 400 nmol and 0 nmol. As an additional control, a fifth group (two mice) was not challenged with bacteria, receiving only the injection of ODN (at the highest dose). The state of the health of these animals was monitored for one week. FIG. 1 shows survival of infected mice with or without ODN treatment.
    • EXAMPLE 6 Delivering Plasmid to Target Bacterial Cells by Bacteriophage T3 Extracts
  • A standard DNA packaging reaction mixture (25 μl) contains 0.5 mg the said plasmid DNA, 2×1010 phage equivalent(peg) of prohead, 20 pmol of gp 18 and 3 pmol of gp 19 in complete pac buffer. The reaction mixture was incubated at 30° C. for 30 min for DNA packaging and the reaction was terminated by the addition of 1 μl of 2 mg/ml of DNase I. After incubation at 30° C. for 20 min, the filled heads are converted to infectious particles by incubation with a head acceptor extract containing tail and tail fiber proteins. Proheads are prepared through a sucrose gradient centrifuge of lysates of bacterial cells infected with bacteriophage T3, as described by Nakasu et al. (Nakasu et al., 1983, Virology, 127, 124-133). gp 18 and gp 19 proteins are purified as decribed by Hamada et al (Hamada et al., 1986, Virology, 151, 110-118). The head acceptor extracts are isolated from the lysates of bacterial cells infected by bacteriophage T3, and purified through ammonium sulfate precipitation. The resulting infectious particles contain the said ODN expression plasmid and are used to deliver the ODN expression plasmid to the target bacterial cells.
  • Although described with reference to the figures and specific examples set out herein, those skilled in the art will recognize that certain changes can be made to the specific elements of the invention that are described herein without changing the manner in which those elements function to achieve their intended results. All such changes and modifications which do not depart from the spirit of the present invention are intended to fall within the scope of the following non-limiting claims.
    TABLE 1
    Mouse bacterial load after infection
    Septicemia: Mice Bled at 24 hours Post Infection
    # Mice
    Bled CFU/ml
    *Control 6 0, 0, 3.6 × 103, 1.6 × 103, 4.6 × 103, >105
    PNA [10 uM] 4 0, 0, 0, 0
    IP, PNA [100 uM] 4 0, 0, 0, 0

    *Septicemic mice succumbed to infection prior to 46 h.

Claims (4)

1. The ODN expression plasmid As080103.
2. A method of treatment of sepsis in an animal patient comprising the steps of contacting the causative agent of sepsis with an ODN comprising a sequence targeted to a specific gene of the causative agent for altering the expression of the specific gene to inhibit growth of the causative agent, kill the causative agent, or inhibit the synthesis or secretion of toxin by the causative agent.
3. A DNA, RNA, or PNA oligonucleotide for treatment of sepsis having one or more of the following nucleotide sequences:
5′(CTT TCA ACA GTT TTG ATG ACC TTT (SEQ. ID No. 1) GCT GAC CAT ACA ATT GCG ATA TCG TGG GGA GTG AGA G)3′; 5′(CTC ATA CTC T)3′; (SEQ. ID No. 2) or 5′(GTT TCG AAG GCT AGC TAC AAC GAT (SEQ. ID No. 3) CAT CCA G)3′.
4. A cell having one or more of the sequences of claim 4 transformed therein.
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