WO2012018594A2 - Polysaccharides d'origine végétale pour l'administration de thérapies à base d'arn - Google Patents

Polysaccharides d'origine végétale pour l'administration de thérapies à base d'arn Download PDF

Info

Publication number
WO2012018594A2
WO2012018594A2 PCT/US2011/045239 US2011045239W WO2012018594A2 WO 2012018594 A2 WO2012018594 A2 WO 2012018594A2 US 2011045239 W US2011045239 W US 2011045239W WO 2012018594 A2 WO2012018594 A2 WO 2012018594A2
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutical composition
rna
cancer
polysaccharide
cells
Prior art date
Application number
PCT/US2011/045239
Other languages
English (en)
Other versions
WO2012018594A3 (fr
Inventor
Alexzander A.A. Asea
Rajani Srinivasan
Original Assignee
Scott & White Healthcare
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Scott & White Healthcare filed Critical Scott & White Healthcare
Publication of WO2012018594A2 publication Critical patent/WO2012018594A2/fr
Publication of WO2012018594A3 publication Critical patent/WO2012018594A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the invention relates to the fields of biology and medicine. More particularly, the invention provides compositions and methods for the delivery of RNAs and other therapeutics using plant derived polysaccharide compositions.
  • Hsp25 The twenty-five kilo Dalton heat shock protein (Hsp25) belongs to the family of small HSP and is the murine homologue of human Hsp27, which was originally identified as an estrogen responsive gene in breast cancer cells (Oeteil et al, 1996). Unlike the large HSP, which function through ATP-dependent mechanisms, Hsp25/27 operates through ATP- independent mechanisms (Egeblad and Werb, 2002; Soldes et al, 1999). Importantly, elevated Hsp27 levels have been found in various tumors, including breast, prostate, gastric, uterine, ovarian, head and neck, and tumor arising from the nervous system and urinary system (Budhram-Mahadeo and Heads, 2007). In ER-a positive benign neoplasia, elevated levels of Hsp27 have been shown to promote the progression to more malignant phenotypes
  • PA28 is essential for the recognition of epitope on melanoma cells by specific CTL (Sun et al. , 2002) and may alter the quality of products generated by proteasome cleavage (Stohigan et al, 2000; Whitby et al, 2000).
  • the overexpression of the ⁇ 28 ⁇ / ⁇ subunit enhanced MHC class I-restricted presentation of two viral epitopes and purified PA28a and ⁇ subunit accelerated T cell epitope generation by the 20S proteasome in vitro Groettrup et al, 1996). It is presumed that PA28a facilitates a transfer of antigenic peptides into the endoplasmic reticulum (Groettrup et al, 1995).
  • a pharmaceutical composition comprising (a) an active therapeutic agent; (b) a purified water soluble polysaccharide comprising galactomannan or galacturonic acid; and, (c) a pharmaceutically acceptable formulation.
  • the active agent may be a chemotherapeutic agent, a nucleic acid, or a protein.
  • the nucleic acid may be a double-stranded RNA 10-130 nucleotides in length comprising one strand with a sequence that is at least 85% complementary to a mammalian heat shock protein coding sequence, such as one that is 17-30 nucleotides in length.
  • the heat shock protein may be human heat shock factor 27, and the RNA may have at least 85%, 90% or 95% sequence identity to SEQ ID NO:l or SEQ ID NO:2.
  • the RNA may be identical to SEQ ID NO:l or SEQ ID NO:2, and/or may comprises the sequence of SEQ ID NO: l or SEQ ID NO:2.
  • the RNA may comprise two separate but hybridized strands, may be blunt-ended, may be a hairpin, may have a 5' and/or 3' overhang, and may comprise one or more modified nucleotides.
  • the RNA and polysaccharides may be directly attached, such as with chemically conjugation. More than one RNA molecule may be conjugated to a polysaccharide. The ratio of RNA: polysaccharide may be from about 1 :20 to about 10: 1.
  • the nucleic acid is isolated or purified away from cellular components or from individual nucleic acid residues or truncated nucleic acids.
  • the nucleic acid is synthetic.
  • the nucleic acid is recombinant.
  • the chemotherapeutic agent may be a cancer therapeutic.
  • the active therapeutic agent may enhance the stability of microtubules.
  • the therapeutic active agent may be a taxane, docetaxel, paclitaxel, a DNA intercalator, or selected from the group consisting of doxorubicin, berberine, ethidium bromide, proflavine, daunomycin, and thalidomide.
  • the polysaccharide may comprises a galactomannan moiety, such as the following structure:
  • Rl or R2 is an OH, a sugar (such as glucose, mannose, etc), a substituted sugar, -OR (R is alkyl, substituted alkyl, alkene, substituted alkene, alkyne, or substituted alkyne and x + y is a number from 50 to 150 and the ratio of x:y is about 1 : 1 to about 1 :5.
  • x + y is a number that ranges from 85 to 100, and in more particular embodiments is a number that ranges from 90 to 95.
  • Rl may be OH and R2 may be OH.
  • the ratio of x:y may be about 1 : 1.
  • the polysaccharide may have the structure of a polysaccharide obtained from Trigonella foenum-graecum, or obtained from the seeds of fenugreek.
  • the polysaccharide may comprises a galacturonic acid moiety and/or a glucose moiety, and/or a glucopyranose moiety or a galactopyranose moiety.
  • the polysaccharide may have the structure of a polysaccharide obtained from psyllium (Plantago psyllium), okra (Hibiscus esculentus), prickly pear cactus (Opuntia Ficus -Indica) and Aloe vera (Aloe barbadensis) .
  • the pharmaceutical composition may comprise multiple different polysaccharides, such as wherein at least two of the different polysaccharides comprise a galactomannan or a galacturonic acid moiety.
  • the at least two polysaccharides can be obtained from psyllium (Plantago psyllium), okra (Hibiscus esculentus), fenugreek (Trigonella foenum-graecum), prickly pear cactus (Opuntia Ficus -Indica), or Aloe vera (Aloe barbadensis).
  • the pharmaceutical composition may be in the form of nanoparticles comprising RNA and polysaccharides.
  • the nucleic acid may be a double-stranded RNA 10-130 nucleotides in length comprising one strand with a sequence that is at least 85%
  • the heat shock protein may be human heat shock factor 27.
  • the target is set forth in SEQ ID NO: l , so the RNA is partially or fully complementary to a portion of SEQ ID NO: l .
  • the RNA is 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length (or any range derivable therein) and is or is at least 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementary, or any range derivable therein, to SEQ ID NO: 1.
  • the RNA may have or have at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.
  • the RNA may be identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 or may comprise the sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5.
  • the RNA comprises or consists essentially of the sequence of SEQ ID NO:2.
  • the RNA may comprise two separate but hybridized strands, may be blunt-ended, may be a hairpin, may have a 5' and/or 3' overhang, and may comprise one or more modified nucleotides.
  • composition consisting essentially of (a) an active therapeutic compound, such as a nucleic acid, (b) a purified water soluble polysaccharide comprising galactomannan or galacturonic acid; and, (c) a pharmaceutically acceptable formulation.
  • an active therapeutic compound such as a nucleic acid
  • a purified water soluble polysaccharide comprising galactomannan or galacturonic acid
  • a pharmaceutically acceptable formulation e.g., a pharmaceutically acceptable formulation.
  • the only active therapeutic compound is a nucleic acid molecule.
  • the composition does not contain any other active compounds (the polysaccharide will not have any detectable therapeutic activity relative to the active therapeutic compound or to the therapeutic goal to be achieved).
  • a method for treating a patient for a disease or condition comprising administering to the patient an effective amount of the pharmaceutical formulation as described above.
  • the disease or condition may be cancer, a bacterial or viral infection, inflammation, or toxin exposure.
  • the cancer may be breast cancer, lung cancer, head & neck cancer, esophageal cancer, prostate cancer, ovarian cancer, uterine cancer, cervical cancer, colon cancer, stomach cancer, liver cancer, testicular cancer, skin cancer, pancreatic cancer, rectal cancer, melanoma, glioma, neuroblastoma or sarcoma.
  • the method may further comprise treating the patient with an additional cancer therapy, wherein the additional therapy is tumor resection, chemotherapy, or radiation.
  • the patient may be treated with the additional cancer therapy prior to treatment with the
  • the method may further comprise identifying a patient in need of the pharmaceutical composition.
  • the pharmaceutical composition may be administered intravenously, intratumorally, intraperitoneally, intrathecally, intraarterially, subcutaneouslly, orally, topically, by lavage, or by direct injection.
  • a method for administering a therapeutic agent to a cell comprising contacting the cell with the pharmaceutical composition as described above.
  • the cell may be a cancer cell, such as one that overexpresses a heat shock protein, and the pharmaceutical composition inhibits expression of the heat shock protein.
  • a method for preparing a pharmaceutical composition comprising combining a reactive polysaccharide comprising galactomannan or galacturonic acid with a nucleic acid.
  • the reactive polysaccharide may be a free radical formed by contacting the polysaccharide with a composition comprising eerie ion, nitric acid, and/or potassium persulfate.
  • the nucleic acid may be an RNA molecule, such as one that is fully or partially double-stranded, and such as one that is 17-30 nucleotides in length.
  • the RNA comprise one strand with a sequence that is at least 85%, 90% or 95% complementary to a mammalian heat shock protein coding sequence.
  • Another embodiment comprises a composition comprising (1) a water-soluble polysaccharide comprising galactomannan or galacturonic acid and (2) a nucleic acid made by a method comprising (a) obtaining purified polysaccharide; (b) creating a reactive polysaccharide through free radical polymerization; and (c) contacting the reactive polysaccharide with the nucleic acid to form a polysaccharide grafted with the nucleic acid.
  • FIG. 1A-C Permanent gene silencing and expression of Hsp25shRNA in 4T1 breast adenocarcinoma cells using a lentiviral vector.
  • HIV-based lentivirus construct pLVTHM was employed to infect 4T1 cells.
  • Construct contains a 5 '-long terminal repeats (LTR), gene encoding GFP as reporter and woodchuck hepatitis virus response element (WPRE) as enhancer of gene expression, placed under the tight control of elongation factor alpha (EF-1 ⁇ ) promoter.
  • LTR 5 '-long terminal repeats
  • WPRE woodchuck hepatitis virus response element
  • the Hsp25shRNA stem loop was placed downstream of the HI promoter, and the self inactivating (SIN) element was placed downstream of the Hl- Hsp25shRNA sequence (top panel).
  • FIG. IB Sorted 4Tl-Hsp25shRNA cells were imaged using a digital inverted fluorescent microscope. Micropictograms are phase contrast (left panel) and fluorescence images (right panel) and was obtained under 40X magnification.
  • FIGS. 2A-B Effects of gene targeted Hsp25 silencing on 4T1 breast adenocarcinoma cell functions.
  • 4Tl-controlshRNA cells (filled circles) or 4T1- Hsp25shRNA cells (open circles) were seeded on day 0 in a 6-well plate at a density of 2.5 x 10 4 cells/well in DMEM supplemented with 10% FBS. At various times from days 1-4, cells were trypsinized and counted with a hemocytometer under a phase-contrast light microscope. Data represent the mean number of cells ⁇ S.D. and is the sum of four independently performed experiments. *, pO.01 vs 4Tl-controlshRNA (Student's t-test). (FIG.
  • FIGS. 3A-B Silencing Hsp25 protein expression enhances prohibitin expression.
  • FIG. 3A 4T1 -controlshRNA cells (filled bars) and 4Tl-Hsp25shRNA cells (open bars) were used to isolate total RNA and the relative prohibitin mRNA expression was measured using real-time PCR analysis. Data are the mean prohibitin mRNA expression ⁇ SD and is the sum of three independently performed experiments. *, pO.001 vs 4T1 -controlshRNA cells (Student's /-test).
  • FIG. 3B 4T1 -controlshRNA cells and 4Tl-Hsp25shRNA cells were lysed, proteins extracted and subjected to immunoblotting with anti-prohibitin Mab or ⁇ - actin.
  • the intensity of the bands were analyzed by densitometry with a video densitometer (ChemilmagerTM 5500; Alpha Innotech, San Leandro, CA) using the AAB software (American Applied Biology). Bars represent the mean prohibitin protein expression and is a representative experiment from three independently performed experiments with similar results.
  • FIGS. 4A-C Proteasome activity is increased by silencing Hsp25 protein expression.
  • FIG. 4A 4T1 -controlshRNA cells (filled bars) and 4Tl-Hsp25shRNA cells (open bars) were used to isolate total RNA and the relative PA28a mRNA expression was measured using real-time PCR analysis. Data are the mean prohibitin mRNA expression ⁇ SD and is the sum of four independently performed experiments. *, pO.001 vs 4T1 - controlshRNA cells (Student's t-test). (FIG.
  • Free AMC fluorescence was measured by using a 380/460 nm filter set in a fluorometer. Data are the mean proteasome activity (% control ⁇ SD) and is the sum of three independently performed experiments. *, pO.001 vs 4T1 -controlshRNA cells (Student's /-test).
  • FIGS. 5A-B Silencing hsp25 gene expression in 4T1 cells induces tumor regression.
  • FIG. 5B Colony formation of tumor derived from lungs of mice injected with 4T1- controlshRNA (top panel) or 4Tl-Hsp25shRNA cells, was platted at different dilution ratios (1 :20-1 :320). Plates were stained and the number of cells was counted. Data represent the mean number of colonies ⁇ SD and is a representative experiment from four independently performed experiments. *, pO.001 vs 4Tl-controlshRNA cells (Student's /-test).
  • FIGS. 6A-B Silencing hsp25 gene expression augments CD8+ T lymphocyte- dependent tumor recognition and killing.
  • FIGS. 6A-B Silencing hsp25 gene expression augments CD8+ T lymphocyte- dependent tumor recognition and killing.
  • FIGS. 6A-B Silencing hsp25 gene expression augments CD8+ T lymphocyte- dependent tumor recognition and killing.
  • FIGS. 6A-B Silencing hsp25 gene expression augments CD8+ T lymphocyte- dependent tumor recognition and killing.
  • FIGS. 6A-B Silencing hsp25 gene expression augments CD8+ T lymphocyte- dependent tumor recognition and killing.
  • FIGS. 6A-B Silencing hsp25 gene expression augments CD8+ T lymphocyte- dependent tumor recognition and killing.
  • FIGS. 6A-B Silencing hsp25 gene expression augments CD8+ T lymphocyte- dependent tumor recognition and killing.
  • FIGS. 6A-B Silencing hsp25 gene
  • CD8+ or CD8- T lymphocytes recovered from mice injected with 4Tl-Hsp25shRNA cells were added at different effector/target ratios (10: 1 and 30: 1), and cytotoxicity was measured by lactate dehydrogenase-cytotoxicity assay kit II, according to the manufacturer's instructions (BioVision). Data are the sum of three independently performed experiments. *, p ⁇ 0.001 versus 4 ⁇ -controlshRNA cells (Student's /-test). (FIG. 6B) 4Tl-Hsp25shRNA cells (10 4 ) were injected into the mammary glands of female BALB/c mice and tumor regression was measured using MaesteroT in vivo imaging system.
  • CD8+ T lymphocytes were collected and CD8+ T lymphocytes were isolated and enriched by negative selection according to manufacturer's instruction (Milteny Biotec). The lymphocytes recovered were designated CD8+ T cells. The fraction depleted of CD8+ T lymphocytes were designated CD8- T cells.
  • Adoptive transfer of 10 6 4Tl-Hsp25shRNA reactive CD8+ T cells or CD8- T cells (top panel) was performed via the tail vein on day 5 post TCI into mice injected with 4T1 - controlshRNA tumors. Data are fluorescence micropictogram of GFP-tagged tumors (green fluorescence) measured on various days post tumor cell injection (top panel). Bars represent
  • FIG. 7 Mechanism for the free radical initiation on the polysaccharide backbone.
  • Hsp25 4T1 breast adenocarcinoma cells express elevated levels of Hsp25, which is effectively suppressed in Hsp25 silenced cells (FIG. ID). This is important not only because elevated levels of Hsp27 in breast cancer gives rise to aggressive disease and poor prognosis (Budhram-Mahadeo and Heads, 2007), but also because elevated Hsp27 levels have been reported to confer tumor protection against Bortezomib-induced cell death (Chauhan et al, 2003). Bortezomib is characterized as a reversible proteasome inhibitor, with potent anticancer effects against multiple myeloma (Mitsiades et al, 2002).
  • Bortezomib was shown to effectively induce apoptotic cell death in DHL6 lymphoma cells (do not express significant Hsp27), but not DHL4 lymphoma cells (expressing high basal levels of Hsp27). Blocking the elevated Hsp27 expression in DHL4 lymphoma cells using antisense against Hsp27 restored sensitivity to Bortezomib, providing a therapeutic advantage to combining agents that
  • NK cell-mediated anti-tumor effector functions are increased against syngeneic tumors in vitro and in vivo by blockade of the Ly49C and Ly49I inhibitory receptors using the 5E6 monoclonal antibody (Koh et al, 2001).
  • Hsp27 plays an essential role in stabilizing actin filaments, a structural protein important for maintaining the integrity of cytoskeleton (Guay et al, 1997); 2) cell cycle progression and proliferation (Farooqui-Kabir et al, 2004), and 3) apoptosis via caspase-3 activation (Rocchi et al, 2005; Rocchi et al, 2006). Taken together, these data have obvious clinical applications in light of recent successful clinical trials with the proteasome inhibitors.
  • Drug delivery systems in cancer treatment aim to increase the therapeutic efficacy of the chemotherapeutic agent by modifying its bio-distribution and controlling the rate at which the agent is released from the carrier to the systemic circulation or tissues, thereby minimizing its interaction with non- pathological sites in the body.
  • polysaccharides are highly stable, safe nontoxic, hydrophilic, biodegradable, and abundantly available and have low processing costs as compared to current lipid-based and protein-based delivery systems (Liu et al, 2008).
  • hydrophilic groups in natural polysaccharides enable them to form non-covalent bond with biological tissues (mainly epithelia cells and mucous membranes) forming bioadhesion. This in turn could prolong the residence time and therefore increase the absorbance of the loaded drug (Liu et al, 2000).
  • Non-starch, linear polysaccharides are resistant to the digestive action of the gastrointestinal enzymes and retain their integrity in the upper gastrointestinal tract.
  • Matrices manufactured from these polysaccharides therefore remain intact in the stomach and the small intestine, but once they reach the colon they are degraded by the bacterial polysaccharidases. This property makes these polysaccharides exceptionally suitable for the formulation of colon-targeted drug delivery systems (Chaurasia et al, 2006; Shirwaikar et al., 2008).
  • HSPs Heat shock proteins
  • HSPs function as molecular chaperones and thus play a critical role in protein folding, intracellular trafficking of proteins, and coping with proteins denatured by heat and other stresses. Accordingly, the study of stress proteins has undergone explosive growth.
  • Heat shock proteins can also be triggered by exposure to different kinds of environmental stress conditions, such as infection, inflammation, exercise, exposure of the cell to toxins (ethanol, arsenic, trace metals and ultraviolet light, among many others), starvation, hypoxia (oxygen deprivation), nitrogen deficiency (in plants), or water deprivation. Consequently, the heat shock proteins are also referred to as stress proteins and their upregulation is sometimes described more generally as part of the stress response.
  • Some bacterial heat shock proteins are upregulated via a mechanism involving RNA thermometers such as the FourU thermometer, ROSE element and the Hsp90 cis-regulatory element.
  • heat shock proteins function as intra-cellular for other proteins. They play an important role in protein-protein interactions such as folding and assisting in the establishment of proper protein (shape) and prevention of unwanted protein aggregation. By helping to stabilize partially unfolded proteins, HSPs aid in transporting proteins across membranes within the cell.
  • Heat-shock proteins also occur under non-stressful conditions, simply "monitoring" the cell's proteins. Some examples of their role as “monitors” are that they carry old proteins to the cell's proteaseom and they help newly synthesised proteins fold properly. These activities are part of a cell's own repair system, called the “cellular stress response” or the “heat-shock response.”
  • Heat shock proteins appear to serve a significant cardiovascular role. Hsp90, hsp84, hsp70, hsp27, hsp20, and alpha-B-crystallin all have been reported as having roles in the cardiovasculature. Hsp90 binds both endothelial nitric oxide synthase and soluble guanylate cyclase which in turn are involved in vascular relaxation. A downstream kinase of the nitric oxide cell signalling pathway, protein kinase G, phosphorylates a small heat shock protein, hsp20. Hsp20 phosphorylation correlates well with smooth muscle relaxation and is , one significant phosphoprotein involved in the process. Hsp20 appears significant in development
  • Hsp20 also serves a significant role in preventing platelet aggregation, cardiac myocyte function and prevention of apoptosis after ischemic injury, and skeletal muscle function and muscle insulin response. Extracellular and membrane bound heat-shock proteins, especially Hsp70, are involved in binding and presenting them to the immune system.
  • Heat shock proteins are overexpressed in different human cancers. Cancer treatments that are in development include inhibitors of different heat shock proteins. Hsp90, Hsp70, and Hsp27 have been implicated in cancer. Inhibitors of these proteins are potential cancer treatments, and siRNAs targeted against each of these is contemplated in methods and compositions described herein. Other heat shock proteins include Hsp60, Hsp72, and Hsp 100, which may also be targeted for destruction with an siRNA.
  • Hsp27 is the target of one or more siRNA molecules.
  • Hsp27 is a major phosphoprotein during muscle contraction. Hsp27 functions in smooth muscle migration and appears to serve an integral role. Elevated Hsp27 levels have been found in various tumors, including breast, prostate, gastric, uterine, ovarian, head and neck, and tumor arising from the nervous system and urinary system (Budhram-Mahadeo and Heads, 2007). In estrogen receptor (ER)-a positive benign neoplasia, elevated levels of Hsp27 have been shown to promote the progression to more malignant phenotypes (O'Neill et al., 2004).
  • ER estrogen receptor
  • proteasome activator PA28
  • PA28 the proteasome activator
  • PA28 is essential for the recognition of epitope on melanoma cells by specific CTL (Sun et al, 2002) and may alter the quality of products generated by proteasome cleavage (Stohigan et al., 2000; Whitby et al, 2000).
  • the overexpression of the ⁇ 28 ⁇ / ⁇ subunit enhanced MHC class I-restricted presentation of two viral epitopes and purified PA28a and ⁇ subunit accelerated T cell epitope generation by the 20S proteasome in vitro (Groettrup et al, 1996).
  • PA28a facilitates a transfer of antigenic peptides into the endoplasmic reticulum (Groettrup et al., 1995). Together, these data suggest that an efficient, well functioning proteasome system is beneficial for specific CD8 + T lymphocyte recognition of tumors and ultimate cytolysis.
  • RNAi double-stranded RNAs that target HSPs such as HSP27.
  • RNAi or RNA interference, is a conserved biological response that is present in many, if not most, eukaryotic organisms. RNAi results in transcript silencing that is both systemic and heritable, permitting the consequences of altering gene expression to be examined throughout the development and life of an animal.
  • dsRNA long double-stranded RNA molecules
  • dsRNA long double-stranded RNA molecules
  • dsRNA can induce sequence-specific silencing of gene expression in primitive and multicellular organisms.
  • These long dsRNAs are processed by a ribonuclease called Dicer into 21 to 23 nucleotide (nt) guide RNA duplexes termed short interfering RNA (siRNA).
  • RISC RNA-induced silencing complex
  • the composition of RISC is not completely defined, but includes argonaute family proteins.
  • siRNA-RISC complexes inhibit gene function by two distinct pathways. Most siRNAs pair imperfectly with their targets and silence gene expression by translational repression. This RNAi mechanism appears to operate most efficiently when multiple siRNA-binding sites are present in the 3 '-untranslated region of the target mRNAs. In some other cases, siRNAs exhibit perfect sequence identity with the target mRNA and inhibit gene function by triggering mRNA degradation. The reduction in transcript level results in lowered levels of the target protein, resulting in phenotypic changes.
  • siRNA has been shown to be effective for short-term gene inhibition in certain transformed mammalian cell lines, there may be drawbacks associated with its use in primary cell cultures or for stable transcript knockdown because their suppressive effects are by definition of limited duration.
  • Short hairpin RNAs (shRNA), consisting of short duplex structures, in contrast to siRNAs have been proven as effective triggers of stable gene silencing in plants, C. elegans, and in Drosophila.
  • These synthetic forms of RNA may be expressed from pol II or pol III promoters and the hairpin structure is recognized and cleaved by Dicer to form siRNA that is subsequently taken up by RISC for silencing of the target gene.
  • SiRNAs may be designed based on the sequence of the endogenous target using any of a variety of techniques known to those of ordinary skill in the art. Moreover, compositions
  • the double-stranded RNAs of the present invention may contain non-natural bases and also may contain non-natural backbone linkages.
  • the following discussion describes a variety of non-natural nucleic acid analogs that may be used to construct iRNAs.
  • a locked nucleic acid is a modified nucleic acid.
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' and 4' carbons. The bridge "locks" the ribose in the 3'-endo structural conformation, which is often found in the A-form of DNA or RNA.
  • LNA nucleotides can be mixed with DNA or RNA bases in the oligonucleotide whenever desired. Such oligomers are commercially available.
  • the locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the thermal stability (melting temperature) of oligonucleotides (Kaur et al, 2006).
  • LNA nucleotides are used to increase the sensitivity and specificity of expression in DNA microarrays, FISH probes, real-time PCR probes and other molecular biology techniques based on oligonucleotides.
  • FISH probes for the in situ detection of miRNA, the use of LNA was as of 2005 the only efficient method.
  • a triplet of LNA nucleotides surrounding a single- base mismatch site maximizes LNA probe specificity unless the probe contains the guanine base of G-T mismatch (You et al, 2006).
  • oligonucleotide modifications can be made to produce the RNAs of the present invention.
  • a motif having entirely of 2'-0-methyl and 2'-fluoro nucleotides has shown enhanced plasma stability and increased in vitro potency (Allerson et al, 2005).
  • the incorporation of -0- ⁇ & and 2'-0-MOE does not have a notable effect on activity (Prakash et al. , 2005).
  • BH - isoelectronic borane
  • Boranophosphate siRNAs have been synthesized by enzymatic routes using T7 RNA polymerase and a boranophosphate ribonucleoside triphosphate in the transcription reaction. Boranophosphate siRNAs are more active than native siRNAs if the center of the guide strand is not modified, and they may be at least ten times more nuclease resistant than unmodified siRNAs (Hall et al., 2004; Hall et al, 2006).
  • Certain terminal conjugates have been reported to improve or direct cellular uptake.
  • nucleic acid analogs conjugated with cholesterol improve in vitro and in vivo cell permeation in liver cells (Rand et al, 2005).
  • Soutschek et al. (2004) have reported on the use of chemically-stabilized and cholesterol-conjugated siRNAs have markedly improved pharmacological properties in vitro and in vivo.
  • LNA bases may be included in a RNA backbone, but they can also be in a backbone of 2'-0-methyl RNA, 2'-methoxyethyl RNA, or 2'-fluoro RNA. These molecules may utilize either a phosphodiester or phosphorothioate backbone.
  • 2'-modified sugars such as nucleosides and nucleotides
  • 2'- substituents such as allyl, amino, azido, thio, O-allyl, O— C J
  • Oligomeric compounds provided herein may comprise one or more monomer, including a nucleoside or nucleotide, having a modified sugar moiety.
  • a nucleoside or nucleotide having a modified sugar moiety.
  • 95138361.1 _9 ⁇ _ furanosyl sugar ring of a nucleoside can be modified in a number of ways including, but not limited to, addition of a substituent group, bridging of two non-geminal ring atoms to form a bicyclic nucleic acid (BNA).
  • BNA bicyclic nucleic acid
  • Sugars can also be replaced with sugar mimetic groups among others. Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S.
  • oligomeric compounds comprise one or more monomers that is a BNA.
  • BNAs include, but are not limited to, (A) oc-L- Methyleneoxy (4'-CH 2 -0-2') BNA, (B) ⁇ -D-Methyleneoxy (4'-CH 2 -0-2') BNA, (C) Ethyleneoxy (4'-(CH 2 ) 2 -0-2') BNA, (D) Aminooxy (4'-CH 2 -0-N(R)-2') BNA and (E) Oxyamino (4'-CH 2 -N(R)-0-2') BNA.
  • each of the bridges of the BNA compounds is, independently,—
  • each of said bridges is, independently, 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-
  • each Rj is, independently, H, a protecting group or Ci-C 12 alkyl.
  • BNA's have been prepared and disclosed in the patent literature as well as in scientific literature (Singh et al, 1998; Koshkin et al, 1998; Wahlestedt et al, 2000; Kumar et al, 1998; WO 94/14226; WO 2005/021570; Singh et al, 1998.
  • Examples of issued US patents and published applications that disclose BNA s include, for example, U.S. Patents 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191 ; and U.S. Patent Publication Nos. 2004/0171570; 2004/0219565; 2004/0014959; 2003/0207841 ; 2004/01431 14; and 2003/0082807.
  • BNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring thereby forming a methyleneoxy (4'- CH 2 ⁇ 0-2') linkage to form the bicyclic sugar moiety (reviewed in Elayadi et al, 2001 ; Braasch et al, 2001 ; and Orum et al, 2001 ; see also U.S. Patents 6,268,490 and 6,670,461).
  • the linkage can be a methylene (— CH 2 — ) group bridging the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy (4'-CH 2 --0-2')
  • BNA is used for the bicyclic moiety; in the case of an ethylene group in this position, the term ethyleneoxy (4'-CH 2 CH 2 -0-2') BNA is used (Singh et al, 1998; Morita et al, 2003).
  • oc-L- methyleneoxy (4'-CH 2 — 0-2') BNA An isomer of methyleneoxy (4'-CH 2 — 0-2') BNA that has also been discussed is oc-L- methyleneoxy (4'-CH 2 — 0-2') BNA which has been shown to have superior stability against a 3'-exonuclease.
  • the oc-L-methyleneoxy (4'-CH 2 — 0-2') BNA's were incorporated into antisense gapmers and chimeras that showed potent antisense activity (Frieden et al, 2003).
  • BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
  • the oligomeric compounds comprise one or more high affinity monomers provided that the oligomeric compound does not comprise a nucleotide comprising a 2'-0(CH 2 ) n H, wherein n is one to six.
  • the oligomeric compounds including, but not limited to short antisense compounds of the present invention comprise one or more high affinity monomer provided that the oligomeric compound does not comprise a nucleotide comprising a 2'-OCH 3 or a 2'-0(CH 2 ) 2 OCH 3 .
  • the oligomeric compounds including, but not limited to short antisense compounds of the present invention comprise one or more high affinity monomer provided that the oligomeric compound does not comprise a a-L-Methyleneoxy (4'-CH 2 — 0-2') BNA. In certain embodiments, the oligomeric compounds including, but not limited to short antisense compounds of the present invention, comprise one or more high affinity monomer provided that the oligomeric compound does not comprise a ⁇ -D-Methyleneoxy (4'-CH 2 — O- 2') BNA.
  • the oligomeric compounds including, but not limited to short antisense compounds of the present invention comprise one or more high affinity monomer provided that the oligomeric compound does not comprise a a-L-Methyleneoxy (4'-CH 2 ⁇ 0-2') BNA or a ⁇ -D-Methyleneoxy (4'-CH 2 -0-2') BNA.
  • the naturally occurring base portion of a nucleoside is typically a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • a phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar.
  • those phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the phosphate groups are commonly referred to as forming the internucleotide backbone of the oligonucleotide.
  • the naturally-occurring linkage or backbone of RNA is a 3' to 5' phosphodiester linkage.
  • nucleobases or nucleobase mimetics are amenable with the compounds described herein.
  • a modified nucleobase such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U)
  • A purine nucleobase
  • G guanine
  • T pyrimidine nucleobases thymine
  • C cytosine
  • U uracil
  • nucleobase is a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, or a G-clamp.
  • nucleobase mimetic include more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art.
  • linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together, thereby forming an oligomeric compound.
  • the two main classes of linking groups are defined by the presence or absence of a phosphorus atom.
  • Non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino (— CH 2 — N(CH 3 ) ⁇ O— CH 2 ⁇ ), thiodiester (-O-C(O)-S-), thionocarbamate (-0 ⁇ C(0)(NH)-S-); siloxane (-0- Si(H) 2 -0-); and ⁇ , ⁇ '-dimethylhydrazine ( ⁇ CH 2 -N(CH 3 )-N(CH 3 )-).
  • Oligomeric compounds having non-phosphorus linking groups are referred to as oligonucleosides. Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound. In certain embodiments,
  • 95138361.1 -24- linkages having a chiral atom can be prepared a racemic mixtures, as separate enantiomers.
  • Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous- containing linkages are well known to those skilled in the art.
  • the oligomeric compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), a or ⁇ such as for sugar anomers, or as (D) or (L) such as for amino acids et al. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
  • oligomeric compounds having reactive phosphorus groups useful for forming linkages including for example phosphodiester and phosphorothioate internucleoside linkages.
  • Methods of preparation and/or purification of precursors or oligomeric compounds are not a limitation of the compositions or methods provided herein.
  • Methods for synthesis and purification of oligomeric compounds including DNA, RNA, oligonucleotides, oligonucleosides, and antisense compounds are well known to those skilled in the art.
  • oligomeric compounds comprise a plurality of monomelic subunits linked together by linking groups.
  • Nonlimiting examples of oligomeric compounds include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, and siRNAs.
  • these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops.
  • Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
  • the present invention provides chimeric oligomeric compounds.
  • chimeric oligomeric compounds are chimeric oligonucleotides.
  • the chimeric oligonucleotides comprise differently modified nucleotides.
  • chimeric oligonucleotides are mixed-backbone antisense oligonucleotides.
  • a chimeric oligomeric compound will have modified nucleosides that can be in isolated positions or grouped together in regions that will define a particular motif. Any combination of modifications and/or mimetic groups can be used.
  • chimeric oligomeric compounds typically comprise at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • an additional region of the oligomeric compound may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex.
  • RNA target Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligomeric compounds when chimeras are used, compared to for example phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. III. Polysaccharide Compositions
  • Embodiments involve a plant-based food grade polysaccharide-based drug delivery system (LPDS) derived from Trigonellafoenum graceum, commonly called fenugreek.
  • LPDS plant-based food grade polysaccharide-based drug delivery system
  • FP fenugreek polysaccharides
  • Rajani and Mishra, 2008 and Mathur and Mathur, 2005 which are hereby incorporated by reference.
  • FP is a neutral polysaccharide and inert to pH changes. It has been used as flocculent for water purification (Rajani and Mishra, 2008). This is first time FP is being used as a drug delivery system for siRNA based anti-cancer drugs.
  • This delivery system can be used as a platform technology for other cancer drugs which are in need of suitable, effective nontoxic carriers.
  • the endosperm galactomannans are reserve seed polysaccharides in fenugreek.
  • Galactomannans generally consist of a P-(l ⁇ 4)-linked D-mannose units (mannans) to which single galactose moieties are linked by a, 1 ⁇ 6 glycoside bond.
  • mannans D-mannose units
  • Galactomannans are a hemicellulose, which is described in Hoch 2007, which is hereby incorporated by reference for its discussion of hemicelluloses, which are embodiments of the invention, and of galactomannans and galactoglucomannans in particular.
  • FP contains mannose, galactose and a small amount of other sugars.
  • LPDS is a highly efficient drug delivery system which induces significantly less cellular toxicity than currently available delivery systems like lipofectamin and chitosan for siRNA based anticancer drug because plant derived polysaccharide FP is neutral and possess
  • HSP siRNA that is formulated with the polysaccharide compositions, discussed above.
  • Various aspects of therapeutic intervention are discussed below.
  • Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. Without inflammation, wounds and infections would never heal. Similarly, progressive destruction of the tissue compromises the survival of the organism, and chronic inflammation can also lead to a host of diseases, such as hay fever, atherosclerosis, rheumatoid arthritis and cancer. It is for that reason that inflammation is normally closely regulated by the body, and when it is not, it can lead to a variety of devastating conditions. The following is a non-inclusive discussion of some important inflammatory disorders.
  • Sepsis is a serious medical condition caused by infection and characterized by a whole-body inflammatory state caused.
  • the term sepsis has traditionally been used interchangeably with septicaemia and septicemia ("blood poisoning"). However, these terms are no longer considered synonymous; septicemia is considered a subset of sepsis.
  • SIRS systemic inflammatory response syndrome
  • the immunological response that causes sepsis is a systemic inflammatory response causing widespread activation of inflammation and coagulation pathways. This may progress to dysfunction of the circulatory system and, even under optimal treatment, may result in the multiple organ dysfunction syndrome and eventually death.
  • Sepsis is currently considered present if infection is highly suspected or proven and two or more of the following (SIRS) criteria are met:
  • hyperventilation > 20 breaths per minute or
  • a white blood cell count ⁇ 4000 cells/ or > 12000 cells/mm 3 ( ⁇ 4 x 10 9 or > 12 x 10 9 cells/), or greater than 10% band forms (immature white blood cells).
  • sepsis The more critical subsets of sepsis are severe sepsis (sepsis with acute organ dysfunction) and septic shock (sepsis with refractory arterial ).
  • SIRS systemic inflammatory response syndrome criteria
  • patients with SIRS and acute organ dysfunction may be termed "severe SIRS.”
  • Patients are defined as having "severe sepsis” if they have sepsis plus signs of systemic hypoperfusion; either end organ dysfunction or a serum lactate greater than 4 mmol/dL.
  • Patient are defined as having septic shock if they have sepsis plus hypotension after an appropriate fluid bolus (typically 20 ml/kg of crystaloid).
  • the criteria for diagnosing an adult with sepsis do not apply to infants under one month of age. In infants, only the presence of infection plus a "constellation" of signs and symptoms consistent with the systemic response to infection are required for diagnosis.
  • Sepsis treatment relies on antibiotics, surgical drainage of infected fluid collections, fluid replacement and appropriate support for organ dysfunction. This may include hemodialysis in kidney failure, mechanical ventilation in pulmonary dysfunction, transfusion of blood products, and drug and fluid therapy for circulatory failure. Ensuring adequate nutrition, if necessary by parenteral nutrition, is important during prolonged illness.
  • drotrecogin alfa activate protein C, one of the coagulation factors
  • a patient must have severe sepsis or septic shock with an APACHE II score of 25 or greater and a low risk of bleeding.
  • Low dose hydrocortisone treatment has shown promise for septic shock patients with relative adrenal insufficiency as defined by ACTH stimulation testing.
  • Standard treatment of infants with suspected sepsis consists of supportive care, maintaining fluid status with intravenous fluids, and the combination of a ⁇ -lactam antibiotic (such as ampicillin) with an aminoglycoside such as gentamicin.
  • a ⁇ -lactam antibiotic such as ampicillin
  • an aminoglycoside such as gentamicin
  • Blunt force trauma a type of physical trauma caused by impact or other force applied from or with a blunt object
  • penetrating trauma is a type of physical trauma in which the skin or tissues are pierced by an object.
  • Trauma can also be described as both unplanned, such as an accident, or planned, in the case of surgery. Both can be characterized by mild to severe tissue damage, blood loss and/or shock, and both may lead to subsequent infection, including sepsis.
  • the present invention provides to treatment of trauma, including both pre-treatment (in the case of a medical procedure) and treatment after trauma injury as occurred.
  • Surgery uses operative manual and instrumental techniques on a patient to investigate and/or treat a pathological condition such as disease or injury, to help improve bodily function or appearance, or sometimes for some other reason.
  • a pathological condition such as disease or injury
  • the present embodiments can address trauma resulting from surgeries, as defined further below.
  • a procedure is considered surgical when it involves cutting of a patient's tissues or closure of a previously sustained wound.
  • Other procedures that do not necessarily fall under this rubric such as angioplasty or endoscopy, may be considered surgery if they involve common surgical procedure or settings, such as use of a sterile environment, anesthesia, antiseptic conditions, typical surgical instruments, and suturing or stapling. All forms of surgery are considered invasive procedures; so-called noninvasive surgery usually refers to an excision that does not penetrate the structure being addressed
  • 95 13836 1 . 1 _3Q_ e.g., laser ablation of the cornea
  • a radiosurgical procedure e.g., irradiation of a tumor. Surgery can last from minutes to hours.
  • Surgical procedures are commonly categorized by urgency, type of procedure, body system involved, degree of invasiveness, and special instrumentation.
  • Elective surgery is done to correct a non-life-threatening condition, and is carried out at the patient's request, subject to the surgeon's and the surgical facility's availability.
  • Emergency surgery is surgery which must be done quickly to save life, limb, or functional capacity. Exploratory surgery is performed to aid or confirm a diagnosis.
  • Therapeutic surgery treats a previously diagnosed condition.
  • Amputation involves cutting off a body part, usually a limb or digit.
  • Replantation involves reattaching a severed body part.
  • Reconstructive surgery involves reconstruction of an injured, mutilated, or deformed part of the body.
  • Cosmetic surgery is done to improve the appearance of an otherwise normal structure.
  • Excision is the cutting out of an organ, tissue, or other body part from the patient.
  • Transplant surgery is the replacement of an organ or body part by insertion of another from different human (or animal) into the patient. Removing an organ or body part from a live human or animal for use in transplant is also a type of surgery.
  • organ system or structure When surgery is performed on one organ system or structure, it may be classed by the organ, organ system or tissue involved. Examples include cardiac surgery (performed on the heart), gastrointestinal surgery (performed within the digestive tract and its accessory organs), and orthopedic surgery (performed on bones and/or muscles).
  • Minimally invasive surgery involves smaller outer incision(s) to insert miniaturized instruments within a body cavity or structure, as in laparoscopic surgery or angioplasty.
  • an open surgical procedure requires a large incision to access the area of interest.
  • Laser surgery involves use of a laser for cutting tissue instead of a scalpel or similar surgical instruments.
  • Microsurgery involves the use of an operating microscope for the surgeon to see small structures.
  • Robotic surgery makes use of a surgical robot, such as Da Vinci or Zeus surgical systems, to control the instrumentation under the direction of the surgeon.
  • Traumatic Hemorrhage Traumatic Hemorrhage accounts for much of the wide ranging international impact of injury, causing a large proportion of deaths and creating great morbidity in the injured. Despite differences in pre -hospital care, the acute management of traumatic hemorrhage is similar around the world and follows well accepted published guidelines. A critically injured patient's care occurs as four, often overlapping segments: the resuscitative, operative, and critical care phases. The diagnosis and control of bleeding
  • 95138361 .1 _3 J _ should be a high priority during all of the phases of trauma care and is especially important in the patient who is in hemorrhagic shock.
  • Early attempts at hemorrhage control include direct control of visible sources of severe bleeding with direct pressure, pressure dressings, or tourniquets; stabilization of long bone and pelvic fractures; and keeping the patient warm.
  • warmed intravenous fluids, hypotensive resuscitation prior to surgical control of hemorrhage, and appropriate transfusion of blood and blood products are provided.
  • surgical control of the hemorrhage and any other injury, and additional transfusion is provide.
  • the critical care phase provides for postoperative support and tissue perfusion.
  • Acute Pancreatitis Acute pancreatitis is rapidly-onset inflammation of the pancreas.
  • Acute Respiratory Distress Syndrome Acute respiratory distress syndrome
  • ARDS also known as respiratory distress syndrome (RDS) or adult respiratory distress syndrome (in contrast with IRDS)
  • RDS respiratory distress syndrome
  • IRDS adult respiratory distress syndrome
  • ARDS is a severe lung disease caused by a variety of direct and indirect insults. It is characterized by inflammation of the lung parenchyma leading to impaired gas exchange with concomitant systemic release of inflammatory mediators causing inflammation, hypoxemia and frequently resulting in multiple organ failure. This condition is life threatening and often lethal, usually requiring mechanical ventilation and admission to an intensive care unit. A less severe form is called acute lung injury (ALI).
  • ALI acute lung injury
  • ARDS can occur within 24 to 48 hours of an injury or attack of acute illness. In such a case the patient usually presents with shortness of breath, tachypnea, and symptoms related to the underlying cause, i.e., shock. Long term illnesses can also trigger it, such as malaria. The ARDS may then occur sometime after the onset of a particularly acute case of the infection.
  • An arterial blood gas analysis and chest X-ray allow formal diagnosis by inference using the aforementioned criteria. Although severe hypoxemia is generally included, the appropriate threshold defining abnormal Pa0 2 has never been systematically studied. Any cardiogenic cause of pulmonary edema should be excluded. This can be done by placing a
  • Acute respiratory distress syndrome is usually treated with mechanical ventilation in the Intensive Care Unit. Ventilation is usually delivered through oro-tracheal intubation, or tracheostomy whenever prolonged ventilation ( > 2 weeks) is deemed inevitable.
  • the possibilities of non-invasive ventilation are limited to the very early period of the disease or, better, to prevention in individuals at risk for the development of the disease (atypical pneumonias, pulmonary contusion, major surgery patients). Treatment of the underlying cause is imperative, as it tends to maintain the ARDS picture.
  • Appropriate antibiotic therapy must be administered as soon as microbiological culture results are available. Empirical therapy may be appropriate if local microbiological surveillance is efficient.
  • Reperfusion injury refers to damage to tissue caused when blood supply returns to the tissue after a period of ischemia.
  • the absence of oxygen and nutrients from blood creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function.
  • the damage of reperfusion injury is due in part to the inflammatory response of damaged tissues.
  • White blood cells carried to the area by the newly returning blood release a host of inflammatory factors such as interleukins as well as free radicals in response to tissue damage.
  • the restored blood flow reintroduces oxygen within cells that damages cellular proteins, DNA, and the plasma membrane. Damage to the cell's membrane may in turn cause the release of more free radicals.
  • Such reactive species may also act indirectly in redox signaling to turn on apoptosis.
  • Leukocytes may also build up in small capillaries, obstructing them and leading to more ischemia.
  • hypoxanthine In prolonged ischemia (60 min or more), hypoxanthine is formed as breakdown product of ATP metabolism.
  • the enzyme xanthine dehydrogenase is converted to xanthine oxidase as a result of the higher availability of oxygen. This oxidation results in molecular oxygen being converted into highly reactive superoxide and hydroxyl radicals.
  • Xanthine oxidase also produces uric acid, which may act as both a prooxidant and as a scavenger of reactive species such as peroxinitrite.
  • Excessive nitric oxide produced during reperfusion reacts with superoxide to produce the potent reactive species peroxynitrite.
  • Such radicals and reactive oxygen species attack cell membrane lipids, proteins, and glycosaminoglycans, causing further damage. They may also initiate specific biological processes by redox signaling.
  • Cardiovascular Disease refers to the class of diseases that involve the heart or blood vessels (arteries and veins). While the term technically refers to any disease that affects the cardiovascular system, it is usually used to refer to those related to atherosclerosis (arterial disease). These conditions have similar causes, mechanisms, and treatments. Treatment of cardiovascular disease depends on the specific form of the disease in each patient, but effective treatment always includes preventive lifestyle changes discussed above. Medications, such as blood pressure reducing medications, aspirin and the statin cholesterol-lowering drugs may be helpful. In some circumstances, surgery or angioplasty may be warranted to reopen, repair, or replace damaged blood vessels.
  • biomarkers which may reflect a higher risk of cardiovascular disease include:
  • BNP B-type natriuretic peptide
  • cardiovascular disease include aneurysms, angina, arrhythmia, atherosclerosis, cardiomyopathy, cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valve disease, coronary artery disease, dilated cardiomyopathy, diastolic dysfunction, endocarditis, high blood pressure (hypertension), hypertrophic cardiomyopathy, nitral valve prolapse, myocardial infarction, and venous thromboembolism.
  • aneurysms angina, arrhythmia, atherosclerosis, cardiomyopathy, cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valve disease, coronary artery disease, dilated cardiomyopathy, diastolic dysfunction, endocarditis, high blood pressure (hypertension), hypertrophic cardiomyopathy, nitral valve prolapse, myocardial infarction, and venous thromboembolism.
  • the present invention contemplates the treatment of a variety of autoimmune and/or inflammatory disease states such as spondyloarthropathy, ankylosing spondylitis, psoriatic arthritis, reactive arthritis, enteropathic arthritis, ulcerative colitis, Crohn's disease, irritable bowel disease, inflammatory bowel disease, rheumatoid arthritis, juvenile rheumatoid arthritis, familial Mediterranean fever, amyotrophic lateral sclerosis, Sjogren's syndrome, early arthritis, viral arthritis, multiple sclerosis, or psoriasis.
  • spondyloarthropathy ankylosing spondylitis
  • psoriatic arthritis reactive arthritis
  • enteropathic arthritis ulcerative colitis
  • Crohn's disease irritable bowel disease
  • inflammatory bowel disease rheumatoid arthritis
  • juvenile rheumatoid arthritis juvenile rheumatoid arthritis
  • familial Mediterranean fever amyotrophic lateral sclerosis
  • Chemotherapy, Radiotherapy and Cytokine Therapy Toxicity are associated with toxicity, sometimes severe, in the cancer patient.
  • toxicity is caused at least in part by the extracellular actions of histones, the present invention seeks to reduce this toxicity using the pharmaceutical compositions of the present invention, thereby reducing or alleviating discomfort on the part of the patient, as well as permitting higher doses of the therapy.
  • Burns In medicine, a burn may be an injury caused by heat, cold, electricity, chemicals, friction or radiation. First-degree burns are usually limited to redness (erythema), a white plaque, and minor pain at the site of injury. These burns usually extend only into the epidermis. Second-degree burns additionally fill with clear fluid, have superficial blistering
  • Second-degree burns involve the superficial (papillary) dermis and may also involve the deep (reticular) dermis layer.
  • Third-degree burns additionally have charring of the skin, and produce hard, leather-like eschars.
  • An eschar is a scab that has separated from the unaffected part of the body. Frequently, there is also purple fluid. These types of burns are often painless, because nerve endings have been destroyed in the burned areas. Serious burns, especially if they cover large areas of the body, can cause death; any hint of burn injury to the lungs (e.g., through smoke inhalation) is a medical emergency.
  • Chemical burns are usually caused by chemical compounds, such as sodium hydroxide (lye), silver nitrate, and more serious compounds (such as sulfuric acid). Most chemicals (but not all) that can cause moderate to severe chemical burns are strong acids or bases. Nitric acid, as an oxidizer, is possibly one of the worst burn-causing chemicals.
  • Hydrofluoric acid can eat down to the bone and its burns are often not immediately evident.
  • caustic Most chemicals that can cause moderate to severe chemical bums are called caustic.
  • Electrical bums are generally symptoms of electric shock, being struck by lightning, being defibrillated or cardioverted without conductive gel, etc.
  • the internal injuries sustained may be disproportionate to the size of the "burns" seen - as these are only the entry and exit wounds of the electrical current.
  • Bums are assessed in terms of total body surface area (TBSA), which is the percentage affected by partial thickness or full thickness burns (superficial thickness burns are not counted). The rule of nines is used as a quick and useful way to estimate the affected
  • the first step in managing a person with a bum is to stop the burning process. With dry powder burns, the powder should be brushed off first. With other bums, the affected area should be rinsed with a large amount of clean water to remove foreign bodies and help stop
  • % TBSA excludes any first degree burn
  • Cancer results from the outgrowth of a clonal population of cells from tissue.
  • the development of cancer referred to as carcinogenesis
  • carcinogenesis can be modeled and characterized in a number of ways.
  • An association between the development of cancer and inflammation has long-been appreciated.
  • the inflammatory response is involved in the host defense against microbial infection, and also drives tissue repair and regeneration.
  • Considerable evidence points to a connection between inflammation and a risk of developing cancer, i.e., chronic inflammation can lead to dysplasia.
  • Organisms such as human papilloma virus (HPV), hepatitis B and C virus, HIV, and Helicobacter pylori all have been linked to cancer.
  • environmental conditions causing chronic irritation and subsequent inflammation can also predispose to cancer, including cigarette smoke, asbestos and silica.
  • virally-encoded genes can contribute to cellular transformation.
  • An example is the HPV oncoproteins E6 and E7.
  • other microbes associated with cancer do not operate in this fashion as they are not transforming.
  • certain strains of H. pylori contain factors that affect host cell signaling but do not contain oncogenes.
  • H. pylori induces MUC 1.
  • Yet another path to cellular dysregulation may result from the cell death that occurs in infection or other inflammatory insult. Lost cells must be repopulated by the expansion of other cells, sometimes undifferentiated precursor cells such as tissue stem cells. Not surprisingly, many inflammatory pathways function to mediate survival and proliferation. Thus, in attempting to mediating tissue repair, the inflammatory response may unwittingly provide excessive survival and proliferative signals to cells, thus leading to tumorigenesis.
  • the ability of the peptides and peptide analogs of the present invention to reduce inflammatory signalling pathways can be exploited in a pre-cancer or cancer risk situation to prevent or delay the onset of dysplastic growth.
  • the present invention also involves the treatment of inflammatory conditions.
  • treatment it is not necessary that all symptoms of the disease be addressed, or that any degree of "cure" be achieved. Rather, to accomplish a meaningful treatment, all that is required is that one or more symptoms of the disease or condition be ameliorated to some degree, an advantageous effect be provided in combination with another therapy, or that the disease progression be slowed.
  • compositions of the present invention comprise an effective amount of the oligonucleotide to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or medium.
  • phrases "pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, liposomes, cationic lipid formulations, microbubble nanoparticles, and the like.
  • the use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • the active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral or parenteral routes, including intracranial (including intraparenchymal and intraventricular), intrathecal, epidural, intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intratumoral, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual) administration. Compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • 1 -39- Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, lipids, nanoparticles, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, intrathecal, epidural and intracranial (including intraparenchymal and intraventricular) administration.
  • sterile aqueous media which can be employed will be known to those of skill
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • Treatment therapies would be provided in a combined amount effective to achieve a reduction in one or more disease parameter.
  • This process may involve contacting subjects with the both agents/therapies at the same time, e.g. , using a single composition or pharmacological formulation that includes both agents, or by contacting the subject with two distinct compositions or formulations, at the same time, wherein one composition includes the double-stranded RNAs of the present invention and the other includes the other agent.
  • the double-stranded RNA therapy may precede or follow the other treatment by intervals ranging from minutes to weeks.
  • Agents or factors suitable for use in a combined therapy include those described above for the various polyglutamine repeat diseases.
  • 4T1 is a highly metastatic breast cancer cell line derived from a spontaneously arising BALB/c mammary tumor purchased from ATCC (Manassas, VA). 4T1 and 293FT cells were maintained in monolayer cultures in DMEM supplemented with 10% fetal bovine serum. Cells were maintained at 37°C humidified atmosphere with 5% C0 2 .
  • mice Female BALB/c mice (6-8 weeks old) were purchased from Charles River Laboratories (Wilmington, MA) and housed under pathogen- free conditions in laminar flow isolation units in the Scott & White Hospital's vivarium under alternate dark and light cycles. Animals were maintained on food and water ad libitum. Mice were challenged s.c. in the abdominal mammary gland with 104 4T1 cells and tumor volume was measured at regular intervals using an electronic caliper. The tumor volume was
  • RNA of mouse Hsp25 lentivirus gene transfer vector A HIV derived three plasmid system was kindly provided by Dr. Trono (Department of Microbiology and Molecular Medicine, University of Geneva, Switzerland). Briefly, the plasmid pLVTHM was digested with Mlu I and Cla I and ligated to an oligonucleotide pair containing Hsp25shRNA or controlshRNA carrying Mlu I and Cla I restriction overhangs and transformed into Max Stbl2 competent cells. The positive clones were identified by digesting the control pLVTHM vector and the vector containing Hsp25shRNA inserts using Mlul and Xba 1 enzymes. Alternatively, positive clones were also identified by DNA sequencing.
  • Lentivirus production and transduction Lentivirus transfection was carried out according to the standard protocol (Naldini et al, 1996). Briefly, 4T1 cells were plated into six- well plates (3 x 104 cells/well), 1-ml concentrated high titer virus (5 x 10 8 ) was directly overlaid on cells and polybrene was added at a final concentration of 8 ⁇ g/ml. Five days later the cells were harvested and analyzed by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • RNA isolation and real-time PCR analysis Total RNA was isolated from 4T1- controlshRNA and 4Tl-Hsp25shRNA cells using Qiagen RNeasy kit. Oligo-dT primed 5 ⁇ g of total RNA was converted into cDNA according to manufacturer's protocol (SA Biosciences). Real-time PCR was performed using gene specific primers (SA Biosciences).
  • Proteasome activity assay A whole cell extract was prepared as follows briefly, cells were harvested, washed with phosphate buffered saline twice, and 10? cells were homogenized in 0.5 ml cell lysis buffer (50 mM HEPES, pH7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 2 mM ATP) for 30 min at 4°C. Cell lysates were centrifuged at 14,000 x g for 30 min and the clear supernatant were collected as whole extracts. The proteasome activity assay was performed by using 20S proteasome activity assay kit (Millipore).
  • a whole cell extract (30 ⁇ g) of 4Tl-controlshRNA and 4T1- Hsp25shRNA cells was incubated for 90 min at 37°C with fluorogenic proteasome substrate, Suc-LLVY-AMC in 100 ⁇ of the assay buffer with or without lactacystin proteasome inhibitor (25 ⁇ ).
  • the hydrolyzed AMC were quantified using 380/460nm filter set in a Fluoroskan Ascent fluorometer (Thermo Scientific).
  • Flow cytometry FACS experiments were conducted on a BD FACSAria flow cytometer (BD Biosciences) equipped with a 488 nm argon laser. Emission filter was 515- 545 for GFP.
  • GFP sorting 4Tl-controlshRNA and 4Tl-Hsp25shRNA cells were harvested and suspended in PBS buffer containing 2% FBS to a concentration of 10 cells/ml. Cells were appropriately gated by forward/size scatter and 2-3% cells gated events were collected per sample. Post-sorted cells were collected in cell culture medium containing 20% FBS and plated in 4T1 complete medium.
  • Fluorescence microscopy Standard fluorescence microscopy was performed using an Olympus CKX41 microscope. DP71 CCD camera was used to capture phase contrast and GFP fluorescence images with DP71 image acquisition interface software (Olympus).
  • Anti-CD4 (L3T4), anti-CD8 (Ly-2) and anti-NK (5E6) antibodies were purchased from BD Biosciences Pharmingen.
  • Antibody depletion was started four days before injection of 4Tl-controlshRNA and 4Tl-Hsp25shRNA cells into the abdominal mammary pads of mice.
  • Depletion of CD4, CD8 T-cell subsets and NK cells was accompanied by i.p. injection of 100 ⁇ g antibody/mice for every week. Effective depletion of cell subsets was confirmed by flow cytometric analysis of splenocytes one day before tumor challenge and maintained by the antibody injections once a week for the duration of the tumor challenge experiment. PBS was used as control.
  • CD8+ or CD8- T lymphocytes were added at different effector/target ratios (10: 1 and 30:1), and absorbance was measured for all controls and samples using a plate reader.
  • 4T1 -controlshRNA cells (10 4 ) were subcutaneously (s.c.) injected into mammary glands of BALB/c mice. Briefly, 10 4 4Tl-Hsp25shRNA cells were injected s.c. into mammary glands of BALB/c mice, and animals were monitored for tumor regression by using the Maestero 1 M in vivo animal imaging system (CRI). At the end of two weeks splenocytes were collected and CD8+ or CD8- T lymphocytes were isolated according to manufacturer's instructions (Milteny Biotec).
  • Adoptive transfer of 4T1- Hsp25shRNA reactive CD8+ or CD8- T lymphocytes (10 6 ) via the tail vein was done on day 5 following 4T1 -controlshRNA tumor cell injections. Tumor regression and development was monitored by using MaestroTM in vivo imaging system (CRI).
  • CRI MaestroTM in vivo imaging system
  • Hsp25shRNA permanently silences hsp25 gene expression.
  • the inventors used a lentivirus-based vector (pLVTHM) that expresses RNAi inducing the twenty-five kilo Dalton heat shock protein (Hsp25)shRNA (Hsp25shRNA) under the control of the HI promoter (FIG. 1).
  • This bicistronic vector was engineered to coexpress enhanced green fluorescent protein (GFP) as a reporter gene under the tight control of the elongation factor- 1 alpha (EF- la) promoter, permitting transduced/infected target cells to be tracked using in vivo imaging.
  • GFP enhanced green fluorescent protein
  • Stable silencing of hsp25 gene expression in 4T1 tumor cells was achieved by subcloning the Hsp25shRNA cassette into pLVTHM, a self-inactivating (SIN) lentiviral vector using Mlu I and Cla I restriction sites (4Tl -Hsp25shRNA hairpin loop sequence) (FIG. 1).
  • SIN self-inactivating
  • 95138361.1 _ g_ also constructed control/scrambled shRNA containing lentiviral vector which does not have sequence homology to the mouse genome (4T1 -controlshRNA hairpin loop sequence) (FIG. 1). These constructs were introduced into 293FT viral packaging cells to make lentivirus. The concentrated lentivirus preparation was used to infect target 4T1 breast adenocarcinoma cells. The resulting GFP expression was assessed 4 days post-infection by flow cytometry and further enriched for only highly expressing GFP-positive cells. The resulting sorted 4T1- Hsp25shRNA cells were 96.7% positive for GFP.
  • silencing Hsp25 protein suppresses tumor proliferation and metastasis.
  • the uncontrollable growth of tumors and their ability to metastasize and invade distant organs is a serious problem.
  • the inventors demonstrated that silencing Hsp25 protein expression in 4T1 cells (4Tl-Hsp25shRNA) significantly suppressed cell proliferation by 40% and 53% as compared to control cells (4T1 -controlshRNA) by day 3 and 4, respectively (FIG. 2A). They further demonstrated that Hsp25shRNA treatment adversely affects the directional cell migration of 4T1 cells in vitro, almost to the same extent as serum starvation, as judged by the wound healing experiment.
  • Hsp25 represses proteasome activity and tumor suppressor genes.
  • 2D SDS-PAGE combined with LC-MS/MS techniques to compare the protein profiles between controlshRNA and Hsp25shRNA stably transfected 4T1 cells. Three unique spots were selected from 4T1-
  • the inventors measured the chymotrypsin-like activity of 20S proteasome in 4T1- controlshRNA and 4Tl-Hsp25shRNA cell extracts. The inventors demonstrated that 4T1- Hsp25shRNA cells showed 50% more proteasome activity than 4Tl-controlshRNA tumor cells (FIG. 4C). Together, these results indicate that silencing of Hsp25 enhances the tumor suppressor gene prohibitin and proteasome function via PA28a.
  • Silencing Hsp25 expression induces tumor regression via enhanced specific CD8+ cytotoxic T lymphocyte (CTL) function.
  • CTL cytotoxic T lymphocyte
  • 4Tl-controlshRNA and 4Tl-Hsp25shRNA tumor cells were injected subcutaneously (s.c.) into the mammary pad of female BALB/c mice. As early as 7 days post-tumor cells injection (TCI), tumors could be visualized growing in the mammary pad of all mice. Mice injected with 4Tl-controlshRNA tumors grew progressively and were sacrificed by day 34 post-TCI, due to the tumor burden (FIG. 5A).
  • mice injected with 4Tl-Hsp25shRNA tumor cells demonstrated a steady regression of tumors after day 7 post-tumor cell inoculation with no detectable GFP signal after day 25 (FIG. 5A).
  • Efficient Hsp25 silencing >95%) could still be demonstrated in 4Tl-Hsp25shRNA tumor before they completely disappeared (day 13 post-tumor cell injection).
  • gross pathology of multiple organs, including lungs, brain, bone and liver demonstrated an absence of tumor metastasis in mice injected with 4T1-
  • mice injected with 4T1 -controlshRNA revealed micrometastasis in lung tissues.
  • mice injected with 4Tl-Hsp25shRNA had no visible micrometastasis.
  • the inventors performed colonogenisity assays on lung tissues in the presence of complete media containing 6-thioguanine. 4T1 breast adenocarcinoma cells are resistant to 6-thioguanine, however, all other contaminating cells will be destroyed. Mice injected with the 4T1 -controlshRNA cells exhibited large numbers of colonies at all dilution, reflecting robust metastasis of tumors to the lungs (FIG. 5B).
  • the inventors performed in vivo depletion of cells known to play an important role in tumor regression.
  • the inventors demonstrated that in vivo depletion of CD8+ T lymphocytes prior to injection with 4T1- controlshRNA cells, drastically increased tumor growth rate and by day 34 post-TCI the size of the tumors were approximately 10 times larger than mice injected with PBS only.
  • the in vivo depletion of CD4+ T lymphocytes did not significantly alter tumor growth rate or tumor volume in mice injected with 4T1 -controlshRNA cells.
  • the in vivo depletion of NK cells using the 5E6 monoclonal antibody induced complete tumor regression.
  • mice injected with 4Tl-Hsp25shRNA cells no tumor growth was seen in any of the mice by the end of the experiment.
  • the in vivo depletion of CD8+ T lymphocytes and NK cells, prior to injection with 4Tl-Hsp25shRNA cells resulted in tumor growth.
  • Similar depletion of CD4+ T lymphocytes initially resulted in increased tumor growth, followed by tumor regression.
  • the in vivo depletion of CD8+ T lymphocytes prior to injection with 4Tl-Hsp25shRNA cells resulted in increased tumor growth, gross pathology of lung, brain and bone did not reveal any signs of metastasis to the lungs.
  • injection of 4Tl -Hsp25shRNA cells into the breast pad of BALB/c-nu/nu mice resulted in tumor growth without metastasis.
  • CD8+ T lymphocytes but not CD8- T lymphocytes effector cells harvested from the spleen of mice injected with 4T1- Hsp25shRNA cells exhibited potent-specific lysis against 4T1 -controlshRNA tumor target cells at various effector: target ratios in vitro (FIG. 6A).
  • both CD8+ and CD8- T lymphocytes from mice injected with 4T1 -controlshRNA cells did not mediate significant lysis above base-line levels against 4T1 -controlshRNA targets.
  • 4Tl-Hsp25shRNA reactive CD8+ T lymphocytes were adoptively transferred into 4T1 -controlshRNA tumor-bearing mice.
  • the adoptive transfer of 4Tl-Hsp25shRNA reactive CD8+ T lymphocytes into 4T1 -controlshRNA tumor-bearing mice induced significant tumor regression starting by day 17 post-TCI and by day 28 there was no detectable tumor growth (FIG. 6A).
  • 4T1 -controlshRNA tumor-bearing mice adoptively transferred with CD8- T cell fraction were not protected and mice rapidly developed tumors (FIG. 6B) and metastasis.
  • Extracted polysaccharides are used to prepare polysaccharide drug (siRNA) composites using free radical grafting method.
  • the polysaccharide drug (siRNA) composites are synthesized by grafting drugs onto purified polysaccharide by free radical polymerization method in aqueous system using eerie ion, nitric acid redox or potassium persulfate, water initiator (FIG. 7).
  • Table 1 below represents effects of the variation in drug-to-polysaccharide concentration on transfection efficiency. From the results it can be seen that increase in the concentration of the polysaccharide increases the drug payload, indicated by increase in the transfection efficiency.
  • the concentration ratios will be increased from 1 ⁇ g/mL to 30 ⁇ g/mL of polysaccharide: 0.002g to O. lg of initiator: and 25 nm to 100 nm of siRNA based drug till the maximum grafting efficiency is achieved. This is because change in the ratio of polysaccharide and siRNA also effects the % grafting efficiency thus the bond strength (Rajani et al, 2002)
  • Table 2 Represents the effects of varying the concentration ratios of polysaccharide, siRNA and initiator (catalyst)
  • LPSD efficiently binds AA1907 (siRNA-based drug for triple negative breast cancer).
  • grafting was achieved by using potassium persulfate redox initiation in the inert gas atmosphere. Characterization of the polysaccharide drug conjugate using FTIR demonstrates that grafting successfully combined the drug to the polysaccharide LPDS backbone.
  • siRNA-based drug One of the major concerns as pointed out in the beginning is the large size of siRNA- based drug. In vitro experiments were performed with GFP-tagged siRNA and LPDS to study
  • the inventors demonstrated that the LPDS efficiently binds siRNA and delivers it into tumors without significant toxicity.
  • the "gold standard" lipofectamine delivery system showed superior transfection efficiency (98%) as compared to the LPDS (56%), this was accompanied by significant cell death (87%), as compared to LPDS (0%). Studies are designed to increase the efficiency of LPDS and maintain its relatively nontoxic abilities.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.

Abstract

L'invention concerne des compositions et des procédés mettant en jeu des polysaccharides et des acides nucléiques. Dans des modes de réalisation particuliers, l'acide nucléique est un siARN dirigé contre une séquence codante de protéine de choc thermique de mammifère. Dans des modes de réalisation supplémentaires, le polysaccharide comprend du galactomannane ou de l'acide galacturonique.
PCT/US2011/045239 2010-07-26 2011-07-25 Polysaccharides d'origine végétale pour l'administration de thérapies à base d'arn WO2012018594A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US36750710P 2010-07-26 2010-07-26
US61/367,507 2010-07-26
US201061424434P 2010-12-17 2010-12-17
US61/424,434 2010-12-17

Publications (2)

Publication Number Publication Date
WO2012018594A2 true WO2012018594A2 (fr) 2012-02-09
WO2012018594A3 WO2012018594A3 (fr) 2012-05-18

Family

ID=45559981

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/045239 WO2012018594A2 (fr) 2010-07-26 2011-07-25 Polysaccharides d'origine végétale pour l'administration de thérapies à base d'arn

Country Status (1)

Country Link
WO (1) WO2012018594A2 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040063654A1 (en) * 2001-11-02 2004-04-01 Davis Mark E. Methods and compositions for therapeutic use of RNA interference
US20060240092A1 (en) * 2005-04-01 2006-10-26 Kurt Breitenkamp Polymeric micelles for drug delivery
US20070213287A1 (en) * 2000-03-03 2007-09-13 Genetronics, Inc. Nucleic Acid Formulations for Gene Delivery and Methods of Use
US20090142391A1 (en) * 2007-08-14 2009-06-04 Mutz Mitchell W Conjugated RNAi Therapeutics

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070213287A1 (en) * 2000-03-03 2007-09-13 Genetronics, Inc. Nucleic Acid Formulations for Gene Delivery and Methods of Use
US20040063654A1 (en) * 2001-11-02 2004-04-01 Davis Mark E. Methods and compositions for therapeutic use of RNA interference
US20060240092A1 (en) * 2005-04-01 2006-10-26 Kurt Breitenkamp Polymeric micelles for drug delivery
US20090142391A1 (en) * 2007-08-14 2009-06-04 Mutz Mitchell W Conjugated RNAi Therapeutics

Also Published As

Publication number Publication date
WO2012018594A3 (fr) 2012-05-18

Similar Documents

Publication Publication Date Title
US20230398074A1 (en) Nucleic Acid-Based Therapy of Muscular Dystrophies
AU2015252917B2 (en) Compositions and methods for modulating PKK expression
US7858592B2 (en) Interfering RNAs against the promoter region of P53
EP1313514B1 (fr) Methodes de traitement d'une pathologie en rapport avec le gene bcl-2 au moyen d'oligomeres antisens de bcl-2
CN107075515B (zh) C/EBPα组合物和使用方法
AU2015252841B2 (en) Compositions and methods for modulating growth hormone receptor expression
ES2790574T3 (es) Modulación de expresión de prekallikrein (PKK)
JP6896420B2 (ja) 抗癌組成物
EP1874930B1 (fr) Utilisation d'une approche fondee sur un arni cible sur la galectine 1 pour traiter le cancer
CN116726203A (zh) 编码免疫调节多肽的mrna的组合及其用途
KR20160002848A (ko) 표적 세포 내에서 치료적 단백질의 표적화된 생산을 위한 시스템 및 방법
KR20240036132A (ko) 대상에게서 ｓｍｎ２ 스플라이싱을 조정하기 위한 조성물 및 방법
US20180282728A1 (en) Organic compositions to treat beta-catenin-related diseases
JP2015518710A (ja) ヘモグロビン遺伝子ファミリー発現を調節するための組成物及び方法
ES2919552T3 (es) Procedimientos de utilización de polinucleotidos codificadores de ligando ox40
ES2932304T3 (es) Composiciones y métodos para la modulación del empalme de SMN2 en un sujeto
JP2021504343A (ja) 尿素サイクル異常症の治療のためのオルニチントランスカルバミラーゼをコードするポリヌクレオチド
KR20180104692A (ko) Angpt2 및 pdgfb를 표적화하는 rna 복합체를 사용하는 혈관신생 관련 질환의 치료
TW201726920A (zh) 具高活性及減低脫靶之siRNA構造
KR102246814B1 (ko) 치료용 올리고뉴클레오타이드
WO2013056670A1 (fr) Petits arn d'interférence, leurs utilisations et procédé destiné à l'inhibition de l'expression du gène plk1
WO2018194089A1 (fr) Virus coxsackie génétiquement modifié, et composition pharmaceutique
CA2846074A1 (fr) Compositions et methodes de traitement d'un cancer metastatique
WO2005080568A1 (fr) Procedes et compositions de traitement ou de prevention de lesions ischemiques secondaires
WO2012018594A2 (fr) Polysaccharides d'origine végétale pour l'administration de thérapies à base d'arn

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11815051

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11815051

Country of ref document: EP

Kind code of ref document: A2