WO2001011023A1 - Treatment of inflammatory or malignant disease using dnazymes - Google Patents
Treatment of inflammatory or malignant disease using dnazymes Download PDFInfo
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- WO2001011023A1 WO2001011023A1 PCT/AU2000/000932 AU0000932W WO0111023A1 WO 2001011023 A1 WO2001011023 A1 WO 2001011023A1 AU 0000932 W AU0000932 W AU 0000932W WO 0111023 A1 WO0111023 A1 WO 0111023A1
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A61P35/02—Antineoplastic agents specific for leukemia
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
Definitions
- the present invention relates to DNAzymes which are targeted against mRNA molecules encoding a subunit of the transcription factor NF- ⁇ B.
- the present invention also relates to compositions including these DNAzymes and to methods of treatment involving administration of the DNAzymes.
- Transcription factors which bind the promoter regions of genes to induce their expression at the level of mRNA synthesis, are capable of simultaneous control of many mediators of inflammation.
- a transcription factor which is necessary for the expression of a large number of mediators of inflammation is therefore a suitable target in the therapy of RA.
- the inducible transcription factor NF- ⁇ B is particularly important in the regulation of gene expression in inflammation.
- Inducers of NF- B include TNF ⁇ , IL-l ⁇ , PDGF. oxidative stress, viral products and bacterial cell wall products such as LPS.
- NF- ⁇ B can activate the transcription of cytokines (TNF ⁇ , IL-l ⁇ , IL-6, IL-8), adhesion molecules (ICAM-1, VCAM-1, E-selectin) and enzymes (iNOS. COX-2, cPLA 2 ) that form the main known contributors to the inflammatory process.
- NF- ⁇ B transcriptional activity is largely controlled by sequestration of NF- ⁇ B in the cytoplasm by a family of proteins, I ⁇ Bs. Upon stimulation of the cell I ⁇ B is degraded leading to translocation of NF- ⁇ B to the nucleus where it binds the promoter sequences of numerous genes, such as those listed above. Since NF- ⁇ B is localised in the nuclei of synovial cells in RA (Handel et al, 1995a) and the list of inducers and targets of NF- ⁇ B very closely match the profile of inflammatory mediators in RA, an important role for activated NF- ⁇ B in human RA is likely. This is supported by animal models in which NF- ⁇ B decoys and an I ⁇ B repressor effectively reduced streptococcal cell wall-induced and pristane-induced arthritis in rats (Miagkov et al, 1998).
- AP-1 Another transcription factor, may also be important in the pathogenesis of inflammatory arthritis.
- AP-1 is localised in the nuclei of fibroblast-like CDl4-negative type B synovial lining cells (Handel et al, 1995a).
- AP-1 is important for the expression of metalloproteinases, especially collagenase and stromelysin, that are likely to contribute to the erosion of bone and cartilage in RA (Brinckerhoff,1991). It should be noted that NF- ⁇ B is found predominantly in macrophages, although it is also present in a subset of fibroblasts. In contrast.
- AP-1 is found almost exclusively in synovial lining fibroblasts (Handel et al, 1995a: Kinne et al, 1994). It is proposed that a hierarchy exists, whereby NF- ⁇ B activity in macrophages (Type A synovial cells) is responsible for AP-1 activation in neighbouring fibroblasts (Type B synovial cells).
- NF- ⁇ B activity in macrophages Type A synovial cells
- Type B synovial cells Type B synovial cells
- metalloproteinase expression by synovial fibroblasts has clearly been shown to be induced by TNF ⁇ and IL-l ⁇ .
- metalloproteinase expression is AP-1 dependent, or in other words, the expression of an AP-1 dependent gene in fibroblasts is due to the effect of a cytokine. namely TNF ⁇ , which is NF- ⁇ B dependent in macrophages.
- TNF ⁇ which is NF- ⁇ B dependent in macrophages.
- the hypothesis that NF- KB activity in macrophages is of primary importance is further supported by the observation that joint erosion in RA correlates with the density of macrophages in the synovium.
- NF- ⁇ B in cancer and apoptosis NF- ⁇ B plays a role in the fundamental processes of cell proliferation and apoptosis.
- Chemotherapy and radiation in some cancer cells can induce NF- ⁇ B activity.
- Activation of NF- ⁇ B protects against apoptosis therefore leading to resistance to these therapies.
- Inhibition of NF- ⁇ B by antisense oligonucleotides or by expression of its inhibitor I- ⁇ Ba has been observed to cause tumour regression in adult T-cell leukemia (Kitajima, 1992) and human breast carcinomas (Higgins, 1993; Cai, 1997) amongst other tumours.
- NF- ⁇ B and/or AP-1 drugs have actions that directly, or indirectly, inhibit NF- ⁇ B and/or AP-1. These include glucocorticosteroids, retinoids, gold thiolates and D-penicillamine. Salicylates as well as chloroquine and the other aminoquinolines may also have indirect effects on NF- ⁇ B.
- Another transcription factor, NF-AT is indirectly inhibited by cyclosporine and tacrolimus (FK506). This list of drugs includes a significant proportion of the useful anti-rheumatic agents, highlighting the importance of transcription factor inhibition as a means of treating rheumatic diseases.
- Glucocorticosteroids The reliability and effectiveness with which glucocorticosteroids suppress inflammation has meant that they underpin the therapy of many individuals with RA and are extremely useful in crisis situation.
- Glucocorticosteroids act by binding the intracellular glucocorticoid receptor (GR), a member of the nuclear receptor class of transcription factors.
- Ligand activated GR can either form homodimers (GR- GR) to up-regulate the expression of genes possessing the GR response element (GRE) or form heterodimers with other transcription factors.
- Increased expression of GRE dependent genes may be responsible for the development of the main adverse effects of glucocorticosteroids recognized as Cushing's syndrome, although there are so many genes involved that have not been fully characterized that it is difficult to directly attribute all the unwanted metabolic effects to this mechanism.
- glucocorticosteroids are the anti-inflammatory effects.
- the metabolic effects, such as obesity, diabetes, cataracts and osteoporosis are the unwanted but unavoidable adverse effects when glucocorticosteroids are used in the treatment of inflammation.
- glucocorticosteroids are mediated in large part by inhibition of NF- ⁇ B. This is illustrated by studies on the effects of dexamethasone on synovium from the joints of osteoarthritis patients. Using electrophoretic mobility shift analyses (EMS A), DNA binding by NF- ⁇ B was induced by TNF ⁇ and inhibited by dexamethasone in human synovial tissue explants, clearly demonstrating that glucocorticosteroids are effective inhibitors of NF- B (Handel et al, 1998). There are several mechanisms by which glucocorticosteroids inhibit NF- ⁇ B activity.
- Ligand activated GR increases the expression of I ⁇ B ⁇ , an inhibitor that prevents the activation and nuclear translocation of NF- B (Scheinman et al, 1995: Auphan et al, 1995), although this mechanism does not seem to account for the glucocorticosteroid-induced repression of NF- ⁇ B activity in endothelial cells (Brostjan et al, 1996).
- Another anti-inflammatory mechanism of glucocorticosteroids involves the formation of heterodimers between GR and RelA (p65) resulting in mutual antagonism between glucocorticosteroids and NF- ⁇ B activity (Ray and Prefontaine, 1994; Caldenhoven et al, 1995).
- Gold and D-penicillamine are thiol reactive drugs. In vitro they interact with thiol groups of cysteine residues in the DNA binding domains of Jun and Fos. thus inhibiting DNA binding of AP-1 (Handel et al, 1995b; 1996). The chemical reactions of these thiol drugs are facilitated by positively charged lysine and arginine residues that flank the cysteine residues of Jun and Fos, thus accelerating the formation of gold-cysteinyl bonds and D-penicillamine-cysteine disulphides. The reaction with D-penicillamine is free radical-dependent whereas the reaction with gold is not. Both reactions are favoured under oxidative conditions of inflammation.
- the concentration of gold thiomalate required for the inhibition of AP-1 mediated transcription in cultured cells is in the low micromolar range. This concentration range is pharmacologically relevant and is below the concentration reported for the inhibition of any enzyme (Shaw, 1979). Gold thiolates also have similar inhibitory effects on NF- ⁇ B (Yang et al, 1995).
- Anti-malarial aminoquinolines Aminoquinolines, including chloroqine and hydroxychloroquine, are basic and they accumulate to very high concentrations in the acidic environment of lysosomes (Poole and Ohkuma, 1981).
- Acidic sphingomyelinase which is found in lysosomes and cannot function in the neutralized environment after aminoquinoline treatment, mediates a necessary step in a signal transduction pathway between the p55-TNF ⁇ receptor and activation of NF- ⁇ B in the nucleus (Weigmann et al, 1994; Schutze et al, 1995). Inhibition of NF-icB is therefore a likely part of the anti-arthritic action of anti-malarial drugs.
- Salicylates have been reported to inhibit NF- ⁇ B activation, in addition to their well known effects on cyclooxygenase (Kopp and Ghosh, 1994). The concentration of salicylate required for this effect is very high and the specificity for suppression of NF- KB has been called into question (Frantz and O'Neill, 1995). Of possible mechanistic relevance is the recent observation that arachidonic acid, the precursor of many pro-inflammatory lipids, is directly anti-inflammatory by stabilizing I ⁇ B, the inhibitor of NF- ⁇ B (Stuhlmeier et al, 1997).
- Cyclosporin and tacrolimus action By complexing with immunophilins cyclosporin (CsA) and tacrolimus (FK506) inhibit the activity of calcineurin, thereby blocking the nuclear translocation of nuclear factor (NF-AT).
- CsA immunophilins cyclosporin
- FK506 tacrolimus
- NF-AT nuclear translocation of nuclear factor
- the transcription factor NF-AT is important for the expression of the IL-2 gene, although the relative lack of IL-2 in rheumatoid synovium suggests that CsA action in RA may employ another mechanism.
- CsA and tacrolimus therefore have inhibitory effects on both NF-AT and NF- ⁇ B, at least in lymphocytes.
- antisense nucleic acid technology has been one of the major tools of choice to inactivate genes whose expression causes disease and is thus undesirable.
- the anti-sense approach employs a nucleic acid molecule that is complementary to, and thereby hybridizes with, an mRNA molecule encoding an undesirable gene. Such hybridization leads to the inhibition of gene expression.
- Anti-sense technology suffers from certain drawbacks.
- Anti-sense hybridization results in the formation of a DNA/target mRNA heteroduplex.
- This heteroduplex serves as a substrate for RNAse H-mediated degradation of the target mRNA component.
- the DNA anti-sense molecule serves m a passive manner, in that it merely facilitates the required cleavage by endogenous RNAse H enzyme.
- This dependence on RNAse H confers limitations on the design of anti-sense molecules regarding their chemistry and ability to form stable heteroduplexes with their target mRNAs.
- Anti- sense DNA molecules also suffer from problems associated with non-specific activity and, at higher concentrations, even toxicity.
- catalytic nucleic acid molecules As an alternative to anti-sense molecules, catalytic nucleic acid molecules have shown promise as therapeutic agents for suppressing gene expression, and are widely discussed in the literature (Haseloff (1988); Breaker (1994): Koizumi (1989): Otsuka: Kashani-Sabet (1992) : Raillard (1996): and Carmi (1996)).
- a catalytic nucleic acid molecule functions by actually cleaving its target mRNA molecule instead of merely binding to it.
- Catalytic nucleic acid molecules can only cleave a target nucleic acid sequence if that target sequence meets certain minimum requirements.
- the target sequence must be complementary to the hybridizing regions of the catalytic nucleic acid, and the target must contain a specific sequence at the site of cleavage.
- RNA molecules Catalytic RNA molecules
- ribozymes Catalytic RNA molecules
- Haseloff (1988): Symonds (1992); and Sun (1997) have been shown to be capable of cleaving both RNA (Haseloff (1988)) and DNA (Raillard (1996)) molecules.
- in vitro selection and evolution techniques has made it possible to obtain novel ribozymes against a known substrate, using either random variants of a known ribozyme or random-sequence RNA as a starting point (Pan (1992): Tsang (1994); and Breaker (1994)).
- Ribozymes are highly susceptible to enzymatic hydrolysis within the cells where they are intended to perform their function. This in turn limits their pharmaceutical applications.
- DNAzymes are single- stranded, and cleave both RNA (Breaker (1994): Santoro (1997)) and DNA
- DNAzyme A general model for the DNAzyme has been proposed, and is known as the "10-23" model.
- DNAzymes following the "10-23” model also referred to simply as “10-23 DNAzymes” have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains.
- In vitro analyses show that this type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions under physiological conditions (Santoro (1997)).
- DNAzymes show promise as therapeutic agents. However, DNAzyme success against a disease caused by the presence of a known mRNA molecule is not predictable. This unpredictability is due, in part, to two factors. First, certain mRNA secondary structures can impede a DNAzyme's ability to bind to and cleave its target mRNA. Second, the uptake of a DNAzyme by cells expressing the target mRNA may not be efficient enough to permit therapeutically meaningful results. For these reasons, merely knowing of a disease and its causative target mRNA sequence does not alone allow one to reasonably predict the therapeutic success of a DNAzyme against that target mRNA, absent an inventive step.
- the present invention provides a DNAzyme which specifically cleaves RelA(p65) mRNA, the DNAzyme comprising
- a catalytic domain which cleaves mRNA at a purine:pyrimidine cleavage site; (ii) a first binding domain contiguous with the 5' end of the catalytic domain; and (iii) a second binding domain contiguous with the 3' end of the catalytic domain, wherein the binding domains are sufficiently complementary to the two regions immediately flanking a purine:pyrimidine cleavage site within the region of RelA(p65) mRNA corresponding to nucleotides 1 to 1767 as shown in SEQ ID NO:l, such that the DNAzyme cleaves the RelA(p65) mRNA.
- the present invention provides a pharmaceutical composition comprising a DNAzyme of the first aspect and a pharmaceutically acceptable carrier.
- the present invention provides a method of inhibiting
- NF- ⁇ B activity in a cell which method comprises introducing into the cell a DNAzyme of the first aspect.
- the present invention provides a method of inhibiting NF- ⁇ B activity in a subject which method comprises administering to the subject a pharmaceutical composition of the second aspect.
- the present invention provides a method of treating an inflammatory disease in a subject which method comprises administering to the subject a therapeutically effective dose of a pharmaceutical composition of the second aspect.
- the present invention provides a method of treating atherosclerosis in a subject which method comprises administering to the subject a therapeutically effective dose of a pharmaceutical composition of the second aspect.
- the present invention provides a method of treating cancer or leukaemia in a subject which comprises administering to the subject a therapeutically effective dose of a pharmaceutical composition of the second aspect.
- Figure 1 Effects of DNAzymes ND2 on NF- ⁇ B and AP-1 dependent luciferase reporter gene in the presence of a liposome, CellFectin (Life Technologies).
- the present invention provides DNAzymes which specifically target RelA(p65) mRNA and inhibit NF- ⁇ B activity. More specifically, in a first aspect the present invention provides a
- DNAzyme which specifically cleaves RelA(p65) mRNA, the DNAzyme comprising
- a catalytic domain which cleaves mRNA at a purine:pyrimidine cleavage site; (ii) a first binding domain contiguous with the 5' end of the catalytic domain; and (iii) a second binding domain contiguous with the 3' end of the catalytic domain, wherein the binding domains are sufficiently complementary to the two regions immediately flanking a purine:pyrimidine cleavage site within the region of RelA(p65) mRNA corresponding to nucleotides 1 to 1767 as shown in SEQ ID NO:l, such that the DNAzyme cleaves the RelA(p65) mRNA.
- the binding domains are entirely complementary to the regions immediately flanking the cleavage site. It will be appreciated by those skilled in the art. however, that strict complementarity may not be required for the DNAzyme to bind to and cleave the RelA(p65) mRNA.
- DNAzyme means a DNA molecule that specifically recognises and cleaves a distinct target nucleic acid sequence, which may be either DNA or RNA.
- the catalytic domain of a DNAzyme of the present invention may be any suitable catalytic domain. Examples of suitable catalytic domains are described in Santoro and Joyce (1997) and U.S. Patent No. 5,807,718. In a preferred embodiment, the catalytic domain has the nucleotide sequence GGCTAGCTACAACGA (SEQ ID NO:2).
- the binding domain lengths can be of any permutation, and can be the same or different.
- the binding domain lengths are at least 6 nucleotides in length, and preferably both binding domains have a combined total length of at least 14 nucleotides.
- Various permutations such as 7+ 7, 8+8 and 9 + 9 are envisioned. It is well established that the greater the binding domain length, the more tightly it will bind to its complementary mRNA sequence. Accordingly, in a further preferred embodiment, each domain is nine or more nucleotides in length.
- the cleavage site corresponds to a site selected from the group consisting of:
- the cleavage site corresponds to the GT site at nucleotides 91-92.
- the DNAzyme has a sequence selected from the group consisting of: 5' GTTCGTCCAGGCTAGCTACAACGAGGCCGGGGT 3' (SEQ ID NO: 3);
- the DNAzyme has the sequence 5' GAGGGGGAAGGCTAGCTACAACGAAGTTCGTCC 3' (SEQ ID NO:4).
- the DNAzymes be as stable as possible against degradation in the intra-cellular milieu.
- One means of accomplishing this is by incorporating a 3'-3' inversion at one or more termini of the DNAzyme.
- a 3'-3' inversion (also referred to herein simply as an "inversion") means the covalent phosphate bonding between the 3' carbons of the terminal nucleotide and its adjacent nucleotide. This type of bonding is opposed to the normal phosphate bonding between the 3' and 5' carbons of adjacent nucleotides, hence the term "inversion".
- the 3'- end nucleotide residue is inverted in the binding domain contiguous with the 3' end of the catalytic domain.
- the instant DNAzymes may contain modified nucleotides or nucleotide linkages. Modified nucleotides include, for example, N3'-P5' phosphoramidate linkages, 2'-0-methyl substitutions and peptide-nucleic acid linkages. These are well known in the art.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a DNAzyme according to the first aspect and a pharmaceutically acceptable carrier.
- administering the pharmaceutical compositions of the second aspect can be effected or performed using any of the various methods and delivery systems known to those skilled in the art.
- the administering can be performed, for example, intravenously, orally, via implant, transmucosally, transdermally, topically, intramuscularly, intra-articularly, subcutaneously or extracorporeally.
- the instant pharmaceutical compositions ideally contain one or more routinely used pharmaceutically acceptable carriers. Such carriers are well known to those skilled in the art.
- the following delivery systems, which employ a number of routinely used carriers, are only representative of the many embodiments envisioned for administering the instant composition.
- Transdermal delivery systems include patches, gels, tapes and creams, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone), and adhesives and tackifiers (e.g., polyisobutylenes, silicone-based adhesives, acrylates and polybutene).
- solubilizers e.g., permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone), and adhesives and tackifiers (e.g., polyisobutylenes, silicone-based adhesives, acrylates and polybutene).
- permeation enhancers e.g., fatty acids, fatty acid esters
- Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).
- solubilizers and enhancers e.g., propylene glycol, bile salts and amino acids
- other vehicles e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid.
- Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA's).
- Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone.
- Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).
- excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.
- Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).
- suspending agents e.g., gums, zanthans, cellulosics and sugars
- humectants e.g., sorbitol
- solubilizers e.g., ethanol, water, PEG and propylene glycol
- Topical delivery systems include, for example, gels and solutions, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone).
- the pharmaceutically acceptable carrier is a liposome or a biodegradable polymer.
- liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII- tetrapalmitylspermine and dioleoyl phosphatidyl-ethanolamine (DOPE)(GIBCO BRL); (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[l-(2,3- dioleoyloxy)-N,N,N-trimethyl-ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL).
- DOPE dioleoyl phosphatidyl-ethanolamine
- nucleic acid agents described may also be achieved via one or more of the following vehicles:
- a viral-liposome complex such as Sendai virus
- the DNAzymes of the present invention are preferably administered by direct injection in to inflamed joints, either as naked DNA in solution or in liposome complexes. Asthma is preferably treated by administering DNAzyme of the present invention by aerosol. Inflammatory vascular and bowel diseases are preferably treated by intraluminal administration.
- the present invention provides a method of inhibiting NF- ⁇ B activity in a cell which method comprises introducing into the cell a DNAzyme of the first aspect.
- the present invention provides a method of inhibiting NF- ⁇ B activity in a subject which method comprises administering to the subject a pharmaceutical composition of the second aspect.
- the present invention provides a method of treating an inflammatory disease in a subject which method comprises administering to the subject a therapeutically effective dose of a pharmaceutical composition of the second aspect.
- the inflammatory disease is selected from the group consisting of inflammatory arthritis, asthma, inflammatory bowel disease, septic shock and vasculitis.
- the inflammatory arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis and seronegative arthritis.
- the present invention provides a method of treating atherosclerosis in a subject which method comprises administering to the subject a therapeutically effective dose of a pharmaceutical composition of the second aspect.
- the present invention provides a method of treating cancer or leukaemia in a subject which comprises administering to the subject a therapeutically effective dose of a pharmaceutical composition of the second aspect.
- the effective dose contains between about 0.1 mg and about 1 g of the instant DNAzyme. In another embodiment, the effective dose contains between about 1 mg and about 100 mg of the instant DNAzyme. In a further embodiment, the effective dose contains between about 10 mg and about 50 mg of the instant DNAzyme. In yet a further embodiment, the effective dose contains about 25 mg of the instant DNAzyme.
- a single therapeutically effective dose can be administered over time as a plurality of lesser doses.
- the method is performed in vivo. In another embodiment, the method is performed ex vivo.
- the word "comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
- NDl and ND2 Two DNA constructs, designated NDl and ND2, were designed based on the 10-23 catalytic motif (Santoro and Joyce, 1997) flanked by two substrate-recognition domains of 9 deoxynucleotides each. An inverted thymidine was placed at the 3-prime terminal end of the oligodeoxynucleotides. This exposes an apparent 5-prime end in order to make the constructs resistant to 3-prime exonuclease activity.
- Construct NDl is designed to cleave RelA(p65) messenger RNA at the AUG translation start site, between A80 and U81.
- Construct ND2 is designed to cleave RelA(p65) messenger RNA at the next available AU or GU site in the 3' direction, that is, cleavage between G91 and U92.
- Their respective controls, NDlc and ND2c contain randomised hybridisation arms.
- the control oligonucleotides possess the consensus 10-23 catalytic motif except for the alteration of a single base at the 5' end of the catalytic motif.
- In NDlc there is an A to C change, which is not consistent with the general purpose catalytic motif.
- ND2c there is an A to G change, which is consistent with catalytic activity (Santoro and Joyce, 1997).
- constructs are shown below, with hybridization arms underlined, inverted thymidines in parentheses (T) and the consensus 10-23 catalytic motif in bold.
- AUCUUCCCGG -3' 110 (SEQ ID NO: 39) ND2 5' GAGGGGGA AGGCTAGCTACAACGA AGTTCGTCC (T) 3'
- RNA In vitro cleavage of a synthetic RNA target by DNAzymes NDl and ND2. Oligonucleotides NDl, NDlc, ND2 and ND2c were incubated with RelA(p65) RNA (61-110) at 37°C in lOmM Mg 2+ for the indicated times. The RNA was 32 p end-labelled prior to incubation with the DNAzymes. Cleavage to a single product of the expected molecular weights was observed for NDl and ND2 (data not shown) . There is no cleavage with the control oligonucleotides NDlc and ND2c. ND2 cleaves more efficiently than NDl.
- ND2 causes a concentration dependent inhibition of NF- ⁇ B dependent gene expression. Inhibition by ND2 is significantly greater than with the controls ND2c and vehicle alone, at all concentrations. Most importantly, there is no inhibition of AP-1 dependent gene expression by either ND2 or ND2c, indicating specificity of ND2 for the transcription factor NF- ⁇ B when compared to another inducible transcription factor.
- HeLa cells stably transfected with the NF- ⁇ B dependent luciferase reporter gene were treated with ND2/CellFectin, ND2c/CellFectin and CellFectin alone, and induced with interleukin-l ⁇ (IL-l ⁇ , 10 ng/ml).
- IL-l ⁇ interleukin-l ⁇
- NF- ⁇ B DNA binding was induced with interleukin-l ⁇ (IL- l ⁇ , 10 ng/ml), nuclear extracts prepared from the cells and the extracts analysed by electrophoretic mobility shift analysis (EMSA) using NF- ⁇ B and AP-1 as the probes (data not shown).
- IL- l ⁇ interleukin-l ⁇
- AP-1 electrophoretic mobility shift analysis
- the human RelA mRNA sequence contains 126 RU dinucleotide sites which are potentially cleavable by the 10-23 DNAzyme. As only a portion of these sites were likely to be cleaved efficiently by DNAzymes under native conditions, a multiplex cleavage assay was emplyed to identify efficient cleavage sites. From the 126 possible sites about 30 were excluded from the cleavage assay as their sequences failed through computational analysis to reach minimum thermodynamic standards.
- the * , * *, *** rating was used to designate DNAzyme activity down to 500nM, 50nM and 5nM concentration respectively.
- the **** rating was used to indicate very strong activity throughout the tested concentration ranges.
- the **. rating was used to indicate DNAzymes which displayed only very week activity at the 5nM range relative to the higher concentrations tested. DNAzymes with ** rating and below were excluded from this list.
- ND2 specifically inhibits NF- ⁇ B dependent transcription in a concentration dependent manner. ND2 does not inhibit an unrelated inducible transcription factor, namely AP-1 and the control oligonucleotide ND2c does not inhibit NF- ⁇ B dependent transcription.
- ND2 specifically inhibits inducible binding of the NF- ⁇ B protein dimer p50/p65 in its DNA response element.
- a liposome reagent enhances inhibition of NF- ⁇ B dependent transcription in cell culture. It is recognised that the type of liposome will vary with cell type. It will be appreciated, however, that treatment of animal or human arthritic joints with DNAzymes in the absence of a liposomal reagent is possible.
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Priority Applications (5)
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JP2001515810A JP2003506078A (en) | 1999-08-04 | 2000-08-04 | Treatment of inflammatory or malignant diseases using DNAzymes |
EP00949004A EP1206527A1 (en) | 1999-08-04 | 2000-08-04 | Treatment of inflammatory or malignant disease using dnazymes |
AU62541/00A AU6254100A (en) | 1999-08-04 | 2000-08-04 | Treatment of inflammatory or malignant disease using dnazymes |
IL14794600A IL147946A0 (en) | 1999-08-04 | 2000-08-04 | Treatment of inflammatory or malignant disease using dnazymes |
CA002380678A CA2380678A1 (en) | 1999-08-04 | 2000-08-04 | Treatment of inflammatory or malignant disease using dnazymes |
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AUPQ2014A AUPQ201499A0 (en) | 1999-08-04 | 1999-08-04 | Treatment of inflammatory and malignant diseases |
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Cited By (13)
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EP1470257A2 (en) * | 2002-02-20 | 2004-10-27 | Ribozyme Pharmaceuticals, Inc. | RNA INTERFERENCE MEDIATED INHIBITION OF NF-KAPPA B/REL-A GENE EXPRESSION USING SHORT INTERFEREING NUCLEIC ACID (siNA) |
WO2005033314A2 (en) * | 2003-10-02 | 2005-04-14 | Philipps-Universität Marburg | Method for the production of a cell and/or tissue and/or disease phase specific medicament |
CN103243098A (en) * | 2013-04-28 | 2013-08-14 | 付玉荣 | RelA cut and TLR7 active sequence modified locked nucleic acid deoxyribozyme for targeted therapy of tuberculosis and application thereof |
CN103555731A (en) * | 2013-11-08 | 2014-02-05 | 湖南莱拓福生物科技有限公司 | Ser536 phosphorylated gene of NF-kB RelA/p65 and use thereof |
US9181551B2 (en) | 2002-02-20 | 2015-11-10 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA) |
US9260471B2 (en) | 2010-10-29 | 2016-02-16 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA) |
US9657294B2 (en) | 2002-02-20 | 2017-05-23 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA) |
WO2017087679A3 (en) * | 2015-11-17 | 2017-07-20 | Yale University | Bacterial proteases targeting the nf-kb transcription factor family |
US9994853B2 (en) | 2001-05-18 | 2018-06-12 | Sirna Therapeutics, Inc. | Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference |
US10487325B2 (en) | 2015-05-15 | 2019-11-26 | Sterna Biologicals Gmbh & Co. Kg | GATA-3 inhibitors for use in the treatment of Th2-driven asthma |
US10508277B2 (en) | 2004-05-24 | 2019-12-17 | Sirna Therapeutics, Inc. | Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference |
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Also Published As
Publication number | Publication date |
---|---|
AUPQ201499A0 (en) | 1999-08-26 |
CN1382211A (en) | 2002-11-27 |
JP2003506078A (en) | 2003-02-18 |
IL147946A0 (en) | 2002-08-14 |
CA2380678A1 (en) | 2001-02-15 |
ZA200200834B (en) | 2003-03-26 |
EP1206527A1 (en) | 2002-05-22 |
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