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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • 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
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

• This invention relates to novel pharmaceutical compositions and methods for treating malignant glioma.
• High-grade glioma or GBM is the most common primary malignant brain tumor, as well as the most devastating, accounting for 19 percent of all primary brain tumors.
• MGC malignant glial cell
• compositions and methods for the treatment of glioma comprising nucleic acid molecules effective at reducing the expression of DRR in tumor cells.
• the nucleic acid molecules of the invention include, for example, therapeutic RNAs or therapeutic oligonucleotides such as antisense oligonucleotides, antisense RNAs, or vectors which encode antisense oligonucleotides or antisense RNAs.
• the tumor cells are glioma cells, such as malignant glioma cells or glioblastoma cells.
• kits comprising the pharmaceutical compositions of the invention, and instructions for use thereof.
• the kits provided herein may further comprise a second active compound suitable for treating glioma and/or for delaying the progression thereof, for simultaneous, separate or sequential administration to a subject.
• a method for inhibiting malignant glial cell invasion in a subject in need thereof comprising providing to tumor cells an antisense molecule of the invention, or a fragment or derivative thereof, wherein the antisense molecule reduces the expression of DRR in the tumor cells.
• malignant glial cell invasion is inhibited in a subject by providing to tumor cells a DNA molecule comprising the sequence encoding SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15 or 16 or a fragment or derivative thereof, wherein the DNA encodes an antisense molecule suitable for reducing the expression of DRR in the tumor cells.
• a method for diagnosis or prognosis of glioma in a subject comprising measuring DRR expression in the glioma cells of the subject, wherein DRR expression indicates invasiveness of the cells, is provided.
• a method for visualizing invasive glioma cells in a subject comprising contacting glioma cells with a molecule which specifically binds DRR protein or mRNA and measuring DRR protein or mRNA levels in the cells, wherein cells which express DRR are invasive, is provided.
• (n 8 for each cell line); asterisk, P ⁇ 0.001 ;
• (K) shows quantification of the effect of DRR expression on cell shape showing that DRR expression promotes an elongated cell shape;
• (N) shows quantification of cell proliferation in DRR + , WT, and DRR " cells.
• Fig. 2 shows that DRR is expressed in neurons and human gliomas but not in normal glia.
• DRR immunolabeling of normal human brain at low (A and B) and high (C and D) magnification shows that DRR is found within the cortex but not in white matter (wm).
• Expression of the glial marker GFAP does not overlap with DRR (A-D).
• DRR is not expressed in the aneuronal molecular layer (ml) of the cortex (C).
• High magnification imaging shows that DRR is highly expressed in neurons (E) but not in white matter (F).
• Rat brain cultures similarly show that DRR expression overlaps with the neuronal marker MAP2 in neurons (G-l) but not with the glial marker GFAP in glia (J-L).
• FIG. 4 shows that DRR association with actin and LC2 is required for cell invasion.
• A shows 3D invasion assays of WT, DRR + , DRR APEPE and DRR AHRE in a 3D collagen matrix.
• B shows a closer view of the spheroid margins showing cell invasion. Asterisk indicates the spheroid edge in DRR APEPE cells.
• C Quantitative analysis of cell invasion after 24, 48 and 72h.
• Fig. 6 shows that DRR promotes focal adhesion disassembly.
• DRR " , DRR + (A) and DRR PEPE (B) were starved for 24h and left untreated or treated for 4h with 10 ⁇ nocodazole. The MT depolymerizer was then washed out for the indicated time. DRR expression promotes FA disassembly whereas DRR deficiency leads to more stable FAs.
• Fig. 7 shows that DRR organizes the actin and microtubular cytoskeletons.
• C A working model summarizing the role of DRR in cytoskeletal organization and invasion. We propose that with LC2, DRR acts as an actin-MT crosslinker. DRR targets MTs to FAs promoting their disassembly, cell rear retraction, and cell invasion.
• Fig. 8 shows DRR protein expression in DRR " and DRR + stable cell lines.
• A shows protein blotting showing increased DRR expression in the DRR + cell line and reduced DRR expression in the DRR " cell line in comparison to wild- type cells.
• B shows DRR + cells implanted into mouse brain showing elongated cell shape and invasion into corpus callosum (cc). Arrows indicate MGCs that have invaded the corpus callosum.
• Fig. 11 shows that DRR regulates focal adhesion dynamics and invasion in multiple glioma cell lines.
• Control U343 or U343-DRR " cells (A) and control C6 or C6-DRR " cells (B) were colabeled for actin (phalloidin) and FAs
• Fig. 13 shows a comparison of amino acid sequences within regions required for DRR-actin association across species.
• DRR-RNAi vector also contains GFP. Tumor spheroids were generated from these cells and implanted into a collagen matrix. Brightfield (upper lanes) and fluorescence images (lower lanes) were captured at 1 to 14 days post-implantation. Non-transfected tumors (A) and control GFP-transfected tumors (B) readily invade, whereas DRR-RNAi transfected tumors (C) do not. (D) shows quantification of invasion distance from spheroid edge, wherein D indicates days, GFP is green fluorescent protein and GBM is glioblastoma (high grade glioma).
• Fig. 17 shows comparison of efficacy of different DRR antisense
• Fig. 18 shows changes in the actin cytoskeletal and focal adhesions when DRR expression is reduced.
• DRR+ cells were transfected with a non- targeting control antisense (ctl Antisense), the indicated DRR antisense (Antisense G4 (SEQ ID NO: 14), Antisense G5 (SEQ ID NO: 15) or
• Antisense G6 (SEQ ID NO: 16) or left untransfected (Untransfected). At 72 hours, cells were fixed, counterstained, and analyzed by confocal microscopy to visualize vinculin (left column; green) and actin (right column; red).
• the above-mentioned oligonucleotide has a structure selected from the group consisting of:
• the alkyl group is a lower alkyl group.
• the lower alkyl group is selected from the group consisting of methyl, ethyl and propyl groups.
• the functionalized alkyl group is selected from the group consisting of methylamino, ethylamino and propylamino groups.
• antisense oligonucleotides may have a peptide nucleic acid (PNA, sometimes referred to as "protein” or “peptide” nucleic acid) backbone, in which the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone wherein nucleosidic bases are bound directly or indirectly to aza nitrogen atoms or methylene groups in the polyamide backbone (see for example, Nielsen et al., Science, 1991 ,254: 1497 and U. S. Pat. No. 5,539, 082).
• the phosphodiester bonds may be substituted with structures that are chiral and enantiomerically specific.
• modifications are 2'-0-alkyl-and 2'- halogen- substituted nucleotides.
• Some specific examples of modifications at the 2' position of sugar moieties which are useful in the present invention are OH, SH, SCH 3 , F, OCN, O (CH 2 ) n , NH 2 or O (CH 2 ) n CH 3 where n is from 1 to about 10; Ci to CIO lower alkyl, substituted lower alkyl, alkaryl or aralkyl ; CI; Br; CN ; CF 3 ; OCF 3 ; 0-, S-, or N-alkyl ; O-, S-, or N- alkenyl ;'SOCH 3 S0 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl ; heterocycloalkaryl; aminoalkylamino ; polyalkylamino; substituted silyl ; an RNA cleaving group; a reporter group
• the antisense oligonucleotide has the formula:
• the oligonucleotide of the invention is an aptamer comprising one or more internucleotide linkages selected from the group consisting of: a) phosphodiester; b) phosphotriester; c) phosphorothioate;d) methylphosphonate; e) boranophosphate; and f) any combination of (a) to (e) .
• the aptamer may have any number of arabinonucleotides at any location in the aptamer, for example:
• the aptamer is fully substituted with arabinonucleotides.
• arabinonucleotides For example: 5' -AAAAAAAAAAAAAAA-3'.
• antisense oligonucleotides such as those described in WO/2003/037909 may be used in the methods and compositions of the invention.
• such oligonucleotides have the structure: [R -XJ a -R 2 ]a wherein a is greater than or equal to 1 ; wherein each of R 1 and R 2 are independently at least one nucleotide; and wherein X is an acyclic linker.
• the oligonucleotide comprises at least one modified deoxyribonucleotide, i.e. either R 1 , R 2 or both may comprise at least one modified deoxyribonucleotide.
• the modified deoxyribonucleotide is selected from the group consisting of ANA, PS-ANA, PS-DNA, RNA-DNA and DNA-RNA chimeras, PS- [RNA-DNA] and PS-[DNA- RNA] chimeras, PS- [ANA-DNA] and PS-[DNA-ANA] chimeras, RNA, PS- RNA, PDE- or PS-RNA analogues, locked nucleic acids (LNA) , phosphorodiamidate morpholino nucleic acids, N3'-P5' phosphoramidate DNA, cyclohexene nucleic acid, alpha-L-LNA, boranophosphate DNA, methylphosphonate DNA, and combinations thereof.
• the ANA is FANA (e.g. PDE- or PS-FANA) .
• the above-mentioned PDE- or PS-RNA analogues are selected from the group consisting of 2' -modified RNA wherein the 2'- substituent is selected from the group consisting of alkyl, alkoxy, alkylalkoxy, F and combinations thereof.
• the acyclic linker is selected from the group consisting of an acyclic nucleoside and a non-nucleotidic linker.
• the acyclic nucleoside is selected from the group consisting of purine and pyrimidine seconucleosides.
• the purine seconucleoside is selected from the group consisting of secoadenosine and secoguanosine.
• AO 1 is an oligonucleotide chain, which in embodiments is selected from the group consisting of ANA (e.g. FANA), DNA, PS-DNA, 5' -RNA-DNA- 3' chimeras, as well as other RNase H-competent oligonucleotides, for example arabinonucleic acids (2' -OH substituted ANA) (Damha, M.J. et al . J. Am . Chem . Soc . 1998, 120, 12976), cyclohexene nucleic acids (Wang J. et al . J. Am . Chem . Soc.
• the internucleotide linkages of the AON1 and AON2 include but are not necessarily limited to phosphodiester, phosphotriester, phosphorothioate, methylphosphonate, and/or phosphoramidate (5'N-3'P and 5'P-3'N) groups.
• the substituent directly attached to the C2'-atom of the arabinose sugar in ANA-X-ANA chimera constructs includes but is not limited to fluorine, hydroxyl, amino, azido, alkyl (e.g.
• alkoxy groups e.g., 2'-OMe, 2'-OEt, 2'-OPr, 2'-0Bu, 2'-OCH 2 CH 2 OMe, etc.
• each of R 1 and R 2 noted above independently may comprise at least two nucleotides connected via an internucleotide linkage, wherein said internucleotide linkage is selected from the group consisting of phosphodiester, phosphotriester, phosphorothioate, methylphosphonate, phosphoramidate (5'N-3'P and 5'P-3'N) groups and combinations thereof.
• each of R 1 and R 2 noted above independently comprise ANA.
• the above-noted ANA comprises a 2 ' - substituent selected from the group consisting of fluorine, hydroxyl, amino, azido, alkyl (e.g.
• the therapeutic molecule e.g. therapeutic RNA or therapeutic oligonucleotide
• the therapeutic molecule is an oligonucleotide which is complementary to or specifically hybridizes with a fragment or portion of the DRR mRNA.
• reducing the expression of a target gene refers to the ability of the present therapeutic molecule or therapeutic oligonucleotide, e.g. antisense, or other therapeutic molecules (e.g., aptamer), to block expression of the target gene in a specific and post- transcriptional manner.
• fragment and “derivative” also refer to nucleic acids that may differ from the original nucleic acid in that one or more nucleotides of the original sequence are substituted by other nucleotides and/or (chemically) modified by methods available to the skilled person, provided that the function of the resulting molecule is not abolished or inhibited.
• the "fragment” and “derivative” may typically show at least 80%, e.g., at least 85%, at least 90%, at least 95% or even at least 99% sequence identity to the original nucleic acid. Sequence identity between two nucleotide sequences can be calculated by aligning the said sequences and determining the number of positions in the alignment at which the two sequences contain the same nucleic acid base vs. the total number of positions in the alignment.
• the invention relates to the use of an RNA sequence containing the sequence of SEQ ID NO: 1 , 2, 5, 6, 7, 8, 9, 10, 14, 15 or 16, a fragment or derivative thereof, to prepare an antisense molecule.
• the therapeutic molecules are prepared from ribonucleic acids of the present invention as defined above, are delivered into target cells, preferably human GBM cells.
• Expression vectors capable of giving rise to transcripts which form therapeutic nucleic acids as defined herein, can for instance be cloning vectors, binary vectors or integrating vectors.
• the invention thus also relates to a vector comprising any of the DNA sequences described herein.
• the expression vector is preferably a eukaryotic expression vector, or a retroviral vector, a plasmid, bacteriophage, or any other vector typically used in the biotechnology field. Such vectors are known to the person skilled in the art.
• the DNA nucleic acid can be operatively linked to regulatory elements which direct the synthesis of mRNA in eukaryotic cells.
• the invention relates to a pharmaceutical composition for the inhibition of cancer invasion, preferably glioma, and more preferably glioblastoma, comprising a therapeutic molecule and/or expression vector according to the invention, and a pharmaceutically acceptable carrier.
• the invention relates to a pharmaceutical composition for the inhibition of malignant glial cell (MGC) invasion.
• MMC malignant glial cell
• the pharmaceutical composition according to the invention may further comprise at least one additional cancer therapeutic, as discussed above.
• Suitable administration forms are, for example, percutaneous or topical administration, for example in the form of ointments, tinctures, sprays or transdermal therapeutic systems, or the inhalative administration in the form of nasal sprays or aerosol mixtures, or, for example, microcapsules, implants or wafers.
• the pharmaceutical preparations can also contain additives, of which many are known in the art, for example fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
• additives of which many are known in the art, for example fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings, aromatizers, thickeners, diluents, buffer substances, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, coating agents or antioxidants.
• the present inventors contemplate that the therapeutic benefits of knocking-down and thus significantly reducing DRR expression in tumor cells may be mediated by inhibiting the invasion of the tumor into the brain, for example by inhibiting malignant glial cell (MGC) invasion, so as to reduce or delay the cancer invasion into adjacent healthy tissues (e.g., the brain in the case of glioma), based on the inventors' novel and unexpected findings that DRR is highly expressed in the invasive component of malignant gliomas and drives MGC invasion in both in vivo and in vitro invasion assays.
• MMC malignant glial cell
• the invention further provides methods for down- regulating DRR expression, for example decreasing DRR expression by more than 50%, by more than 70%, or by more than 90%.
• DRR expression is decreased or reduced by about 50%, about 60%, about 70%, about 80%, or about 90%.
• the invention relates to a method for inhibiting or reducing the migration or invasiveness of tumor cells, preferably cells of glioma such as glioblastoma, comprising administering a therapeutic molecule, a vector or a composition of the invention to a subject in need thereof.
• the invention further provides a method for enhancing the efficacy of cancer therapies for the treatment of cancer, in particular glioma (preferably glioblastoma), selected from the group comprising resection, chemotherapy, radiation therapy, immunotherapy, and/or gene therapy, comprising administering a therapeutic molecule, a vector or a composition as defined herein, and simultaneously, separately or sequentially administrating said cancer therapy.
• glioma preferably glioblastoma
• enhancing the efficacy of a cancer therapy refers to an improvement of conventional cancer treatments and includes reduction of the amount of the anti-cancer composition which is applied during the conventional cancer treatment, e.g.
• DRR as a biomarker for invasive brain cancer cells. Detection of elevated DRR expression can be used to identify invasive tumor cells and for diagnosis and/or prognosis of a tumor, based on DRR expression. Accordingly methods for diagnosis and prognosis of malignant glioma are provided, along with use of DRR as a biomarker for invasiveness.
• the kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
• a label can be provided on the container to indicate that the composition is used for a specific application.
• Directions and or other information can also be included on an insert which is included with the kit.
• the present invention relates to the use of an anti-DRR therapeutic approach to treat malignant gliomas
• the present therapeutic approach is based on the use of anti-DRR molecules relating to antisense-, viral-vector-, or any other related approaches aiming to knock-down DRR expression in human tumor cells.
• the technical feasibility of the present approach is further illustrated by means of the following non-limiting examples.
• DRR " cell lines were generated using short hairpin RNAs (Paddison et al., 2002) and retroviral transduction. The distal C-terminal sequence
• Neurobasal medium supplemented with B-27, N-2, l-glutamine (500 ⁇ ) and penicillin/streptomycin (100 units/ml) (Invitrogen).
• oligonucleotides Purification of crude oligonucleotides was done either by preparative denaturing polyacrylamide gel electrophoresis (PAGE) using 24% acrylamide gels, or by reverse phase HPLC on a Waters 1525 HPLC using a Varian Pursuit 5 reverse phase C18 column with a stationary phase of lOOmmol triethylammonium acetate in water with 5% ACN, and a mobile phase of HPLC-grade acetonitrile. Gel bands were extracted overnight in DEPC- treated autoclaved Millipore water, and lyophilized to dryness. All purified oligonucleotides were desalted with Nap-25 Sephadex columns from GE Healthcare. Sequences were verified by analytical denaturing PAGE and/or ESI-LCMS.
• DRR DRR is Expressed in Neurons and Human Gliomas but not in Normal Glia
• the rate constant for GFP-paxillin incorporation into FAs was (6.2 ⁇ 0.9) x 10 "3 min "1 and the rate constant for GFP-paxillin disassembly was (8.6 ⁇ 0.7) x 10 "3 min "1 .
• FAs were not dynamic in WT control cells. We were unable to detect FAs that formed or disassembled within the 170 minute imaging interval (Fig 5D). These data strongly support a mechanism whereby DRR drives cell invasion by enhancing FA dynamics.
• FA disassembly requires polymerized microtubules (MTs) (Kaverina et al., J. Cell Biol. 142:181-190,1998; Kaverina et al., J. Cell Biol. 146:1033-1044, 1999; Krylyshkina et al., J. Cell Biol.
• MTs microtubules
• gliomas Human high grade gliomas were surgically resected and immediately placed in culture. Two weeks later they were transfected with a control GFP vector or DRR-RNAi (SEQ ID NO: 1) (vector also contains GFP). Tumor spheroids were generated from these cells and implanted into a collagen matrix. Brightfield (upper lanes) and fluorescence images (lower lanes) were captured at 1 to 14 days post-implantation (Fig. 16). Non-transfected tumors (Fig. 16A) and control GFP-transfected tumors (Fig. 16B) readily invade, whereas DRR-RNAi transfected tumors (Fig. 16C) do not. Fig. 16D shows quantification of invasion distance from spheroid edge.
• DRR is an important regulator of glioma invasion and a target for therapeutic treatment of glioma.
• DRR is a useful biomarker to delineate invasive regions and grade malignant gliomas.

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