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  • C12N15/1137—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 against enzymes
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  • 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
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  • C12N2310/341—Gapmers, i.e. of the type ===---===
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  • C12N2320/00—Applications; Uses
  • C12N2320/30—Special therapeutic applications
  • C12N2320/33—Alteration of splicing

Definitions

• This application concerns methods, compounds and compositions for delivering nucleic acids to a cell of interest.
• oligonucleotide-based therapies face a key problem regarding the inefficient access of oligonucleotides to their sites of action in the nucleus or cytosol of tissue cells 3 4 .
• This problem has meant that large doses must be given to attain therapeutic effects thus risking drug-related toxicities, or that complex delivery systems such as cationic lipid or polymer nanoparticles must be used thus creating toxicity and biodistribution problems associated with the delivery system itself 5 . Therefore it is clear that the discovery of alternative strategies to enhance the access of oligonucleotides to their intracellular targets will have substantial value for oligonucleotide-based pharmacology and therapeutics.
• Oligonucleotides are usually internalized via endocytosis and then traffic through various membrane-bound vesicular compartments 6 7 .
• Cells employ multiple distinct endocytotic uptake mechanisms including the 'classic' clathrin pit pathway, the caveolar pathway, one or more caveolin and clathrin-independent pathways, and macropinocytosis. Initial uptake is followed by trafficking into a variety of endomembrane compartments including early/sorting endosomes, late endosomes/multi-vesicular bodies, lysosomes, and the trans-Golgi network (TGN) 8,9 .
• TGN trans-Golgi network
• oligonucleotide Most of the oligonucleotide accumulated in cells remains sequestered in endomembrane vesicles and is pharmacologically inert, but a small fraction escapes to the cytosol and nucleus to permit activity. Recently we, and others, have found that the route of uptake and pathway of intracellular trafficking can have a strong effect on the pharmacological activity of the oligonucleotide; there are productive and less productive pathways 10"13 . These observations suggest that if it were possible to influence the intracellular trafficking of oligonucleotides, and their release from endomembrane compartments, one might be able to substantially enhance their pharmacological effects and/or the physiological activity thereof.
• a first aspect of the invention is, in a method of administering an oligonucleotide of interest into a cell, the improvement comprising: concurrently administering an intracellular trafficking route inhibitor to said cell in an amount effective to increase the delivery and/or increase the activity of said oligonucleotide in said cell.
• the cell is a mammalian cell.
• the method is carried out in vitro or in vivo.
• the method is carried out by administering said oligonucleotide to a subject in need thereof, and concurrently administering said intracellular trafficking route inhibitor to said subject.
• the intracellular trafficking route inhibitor is administered after said oligonucleotide.
• the oligonucleotide is single stranded.
• the oligonucleotide is from 2, 4, 6 or 8 to 100 or 200 nucleotides in length.
• the oligonucleotide is negatively charged.
• the oligonucleotide is an antisense oligonucleotide.
• the oligonucleotide is a splice switching oligonucleotide
• the intracellular trafficking inhibitor is an intracellular retrograde transport inhibitor (retro compound).
• a further aspect of the invention is the use of an intracellular trafficking route inhibitor as described herein for carrying out a method as described herein, or for the preparation of a medicament for carrying out a method as described herein.
• a further aspect of the invention is the use of an oligonucleotide as described herein for carrying out a method as described herein, or for the preparation of a medicament for carrying out a method as described herein.
• a further aspect of the invention is a composition comprising, consisting of or consisting essentially of: (a) an oligonucleotide as described herein; and (b) an intracellular trafficking route inhibitor as described herein, in combination in (c) a pharmaceutically acceptable carrier.
• Retro-1 Enhances the Actions of SSOs.
• Retro-1 does not affect the action of oligonucleotides delivered by electroporation.
• NIH-3T3-MDR cells were incubated with 100 nM anti-MDR1 antisense oligonucleotide (AS). After removal of the AS the cells were incubated for 2 h with or without 100 ⁇ Retro-1. After removal of the Retro compound the cells were further incubated for 48 h and then assayed for cell surface expression of Pgp using a monoclonal antibody and flow cytometry. Reduced Pgp expression is indicated by a left shift of the cytometry profile. See Methods for details. Ordinate, cell counts; abcissa, log scale of fluorescence. Blue profile, untreated MDR cells; Green profile, + AS; Red profile, + AS + Retro-1.
• HeLa cells on cover-glasses were incubated with 200 nM 3'-TAMRA conjugated SSO 623 for 24 h with or without subsequent treatment with 100 ⁇ Retro-1 for 2 h. Live cells were observed by confocal microscopy as described in Methods. Blue arrows indicate typical 'empty' nuclei. Yellow arrows indicate typical nuclei containing oligonucleotide.
• FIG. 1 Lack of Toxicity of Retro-1.
• the cytotoxicity of Retro-1 was measured on A375Luc705 and Hel_aEGFP654 cells by the Alamar Blue assay as described in Methods. Ordinate: cell viability as % of untreated control; abscissa, concentration of Retro-1 .
• FIG. 6 Lack of Co-localization with Certain Endomembrane Markers.
• HeLa cells were transfected with GFP chimeras that serve as markers for several endomembrane compartments. Thereafter cells were incubated with 200 nM 3'-TAMRA conjugated SSO 623 for 24 h. Live cells were observed by confocal microscopy as described in Methods. Green image, GFP fluorescence. Red image, TAMRA fluorescence.
• FIG. 7 Retro-1 Does Not Affect Acidic Endomembrane Compartments.
• Subjects with which the present invention is concerned are primarily human subjects, but the invention may also be carried out on animal subjects, particularly mammalian subjects such as dogs, cats, livestock and horses for veterinary purposes. While subjects may be of any suitable age, the subjects are in some embodiments neonatal, infant, juvenile, adolescent, adult, or geriatric subjects.
• Treat refers to any type of treatment that imparts a benefit to a subject or patient afflicted with a condition, such as by delaying the progression of a disorder, delaying the severity of at least one symptom of a disorder, etc.
• “Pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
• Constantly as used herein means sufficiently close in time to produce a combined effect (that is, concurrently may be simultaneously, or it may be two or more events occurring sequentially in time, typically within a short time period before or after each other).
• active agents or retro compounds may be administered before, simultaneously with, or after said oligonucleotide, so long as the intended effect on the oligonucleotide activity or delivery is achieved.
• Intracellular trafficking route inhibitor includes, but is not limited to, intracellular retrograde transport inhibitors, endosome to lysosome pathway inhibitors, etc.
• Intracellular retrograde transport inhibitors refers to compounds that inhibit or block the retrograde transport route from cell plasma membrane to the endoplasmic reticulum, via the endosomes and the Golgi apparatus, including (but not limited to) compounds that inhibit the early endosome to irans-Golgi network (TGN). See, e.g., B. Stechmann et al., Cell 141 , 231-242 (April 16, 2010).
• Enhance the delivery refers to any administration of a intracellular trafficking route inhibitor effective to increase the nuclear concentration, accumulation, and/or half-life of the oligonucleotide.
• Enhance the activity refers to, for example, any administration of a intracellular trafficking route inhibitor effective to (for SSOs) increase the alteration of pre- mRNA splicing, as reflected by an increase in the desired splice variant (which can be measured by any suitable technique, such as by a reporter gene readout; amelioration or treatment of symptoms in a subject, etc.), or (for antisense oligonucleotides) reduced levels of the corresponding target mRNA and/or protein (which can be measured by any suitable technique, such by flow cytometry for protein levels, treatment or amelioration of symptoms in a subject, etc.).
• Oligonucleotide refers to polymers of deoxyribonucleotides or ribonucleotides in either single- or double-stranded form.
• the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
• PNAs peptide-nucleic acids
• the oligonucleotide may be of any suitable length, e.g., from 2, 3, 4, 5, 6, 8 or 10 nucleotides in length, up to 50, 60, 80, 100, 150 or 200 nucleotides in length, or more.
• Suitable oligonucleotides include, but are not limited to, short hairpin RNA (shRNA), microRNAs, antisense oligonucleotides (including splice switching oligonucleotides or "SSOs”), small double stranded interference RNA (siRNA)s, and ribozymes.
• Antisense oligonucleotides are known. Examples include, but are not limited to, those described in US patents Nos. 8,067,571 ; 7,910,563; 7,563,778; 7,393,951 ; 7,307,069; 6,972,171 ; 6,417,169; 6,339,071 ; 6,312,900; 6,277,832; 5,985,558; and many others.
• SSOs splice switching oligonucleotides
• SSOs are known and described in, for example, US Patents Nos. 8,067,569; 7,888,012; 7,884,194; 7,785,834; 6,727,355; 6,653,467; 6,653,466; and 5,976,879, and in US Patent Application No. 20100130591 to Sazani and Kole (May 27, 2010), the disclosures of all of which are incorporated by reference herein in their entirety. See also J. Bauman et al., Oligonucleotides 19, 1-14 (2009); P. Sazani and R. Kole, J. Clin. Invest 12, 481-486 (2003).
• Any suitable intracellular trafficking route inhibitor may be used to carry out the present invention, including but not limited to the retrograde transport inhibitors.
• Intracellular retrograde transport inhibitors that may be used in carrying out the present invention include but are not limited to those described in Gillet et al., US Patent Application Pub. No. US201 1/0201601 (August 18, 201 1 ).
• X is -NH- or -O-;
• R 1 is a cycloalkyi radical having 5 to 10 carbon atoms, having one or two rings, saturated or not, wherein one or more carbon atom may be replaced by a nitrogen, oxygen or sulfur atoms; said cycloalkyi radical being optionally substituted by one or more groups chosen amongst a halogen atom, a hydroxyl function, a nitro function and a C-
• Pharmaceutically acceptable salt of compounds of formula (I) may also be used, such as hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or hydrogen, acetate, oxalate, benzoate, succinate, fumarate, maleate, lactate, citrates, tartrate, gluconate, methanesulphonate, benzene-sulphonate and paratoluene-sulphonate.
• halogen atom is meant the chemical elements of group VII of the periodic table of elements; including fluorine, chlorine, bromine and iodine.
• the preferred halogen is bromine (Br) and fluorine (F).
• C C 3 alkyl group means a linear or branched chain of 1 to 3 carbon atoms; said chain may be for example methyl, ethyl, propyl or isopropyl.
• the retro compound is a benzodiazepine derivative of formula
• R 3 is chosen amongst a link, a hydrogen atom, a halogen atom, a C Ce alkyl group, a C C 6 alkoxy group, a C C 6 acyloxy group, an aryloxy group, a heteroaryloxy group; these groups being optionally substituted by a C C 6 alkoxy group or a heteroaryloxy group;
• R 4 is either a link or chosen amongst a hydrogen atom, a C1-C3 acyloxy group, a C C 3 alkoxy group or a phenyl group;
• R 5 is either a link or chosen amongst a hydrogen atom, a C1-C 3 alkoxy group, a C1-C 3 acyloxy group, a C C 3 alkyl group, saturated or not, optionally substituted by a phenyl group; said phenyl group being optionally substituted by one or more radicals chosen amongst: -OH , a halogen atom, a C C 3 alkyl group, a C C 3 alkoxy group, -N0 2 , -CF 3 , and
• R 4 and R 5 can not simultaneously be a link and when R 4 or R 5 is a link then A and B are linked by a double bound
• R 5 may also form with the adjacent carbon atom a cycle of 5 or 6 atoms optionally substituted by a phenyl group; optionally interrupted by a nitrogen, oxygen or sulfur atom; preferably, it is a 5-atom cycle comprising an oxygen atom; and
• R 6 represents an oxygen atom or one or two C C 3 alkyl groups.
• the C C 6 alkyl group means a linear or branched chain of 1 to 6 carbon atoms; such radical includes for example methyl, ethyl, propyl or isopropyl.
• the C C 3 alkyl group means a linear or branched chain of 1 to 3 carbon atoms.
• C C 6 alkoxy group means a -OC m H 2m+ i group, m being an integer between 1 and 6.
• C C 3 alkoxy group means a -OC m H 2m +1 group, m' being an integer between 1 and
• C C 6 acyloxy group means a -0(CO)C n H 2n+ i or -(CO)OC n H 2n+1 group, n being an integer between 1 and 6.
• C C 3 acyloxy group means a -0(CO)C n H 2n +1 or -(CO)OC n H 2n +1 group, n' being an integer between 1 and 3.
• An aryloxy group is an aryl group linked by an oxygen atom to the rest of the compound.
• a heteroaryloxy group is a heteroaryl linked by an oxygen atom to the rest of the compound.
• compounds of formula (II) are such that R 3 is a link and/or R 4 is a hydrogen atom and/or R 5 is a phenyl group and/or when A is a carbon atom, then R 4 is a phenyl group and/or when B is a carbon atom then R 5 is a phenyl group.
• Some modified internucleoside linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and arminoalkylphosphoramidat.es, thionophosphoramidates, thionoalkylphosphonat.es, thionoalklyphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
• Preferred modified internucleoside linkages or backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages.
• morpholino linkages formed in part from the sugar portion of a nucleoside
• siloxane backbones sulfide, sulfoxide and sulfone backbones
• formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
• alkene containing backbones sulfamate backbones
• sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH.sub.2 component parts.
• nucleobases are retained and are bound directly or indirectly to atoms of the amide portion of the backbone.
• Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991 , 254, 1497.
• the invention pertains to uses of the above-described active agents (including both retro compounds and oligonucleotides) for methods and treatments as described below. Accordingly, the modulators of the present invention can be incorporated into pharmaceutical compositions suitable for administration. See, e.g., US Patent No. 7,459,547.
• pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
• 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 active compound, use thereof in the compositions is contemplated.
• a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
• routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration.
• compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
• the composition must be sterile and should 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.
• isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
• the present invention provides a method of introducing an oligonucleotide of interest into a cell, comprising (a) contacting an oligonucleotide compound as described above to the cell in an amount effective to introduce said oligonucleotide into said cell, (b) concurrently with (that is, before, after, or simultaneously with) contacting a retro compound as described above to the cell in an amount effective to enhance the activity of said oligonucleotide in said cell.
• the method may be carried out in vitro or in vivo with any type of cell, particularly animal cells.
• Animal cells may be mammalian cells, such as human, monkey, cat, dog, rat, mouse, or rabbit cells.
• the methods may be utilized for any purpose in which it is desired to introduce an oligonucleotide into a cell, including but not limited to those olgioncucleotides (including polynucleotides and RNAi agents) for those purposes described in US Patents Nos. 7,682,626; 7,674,778; 7,473,419; 7,459,547; and 7,015,040, the disclosures of which are incorporated by reference herein in their entirety.
• the oligonucleotide agent silences the PDGF beta gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PDGF beta expression, e.g., testicular and lung cancers.
• the oligonucleotide agent silences the CRK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted CRK expression, e.g., colon and lung cancers.
• the oligonucleotide agent silences the GRB2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted GRB2 expression, e.g., squamous cell carcinoma.
• the oligonucleotide agent silences the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., pancreatic, colon and lung cancers, and chronic leukemia.
• a disorder characterized by unwanted RAS expression e.g., pancreatic, colon and lung cancers, and chronic leukemia.
• the oligonucleotide agent silences the Erk1/2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erk1/2 expression, e.g., lung cancer.
• the oligonucleotide agent silences the PCNA(p21 ) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PCNA expression, e.g., lung cancer.
• the oligonucleotide agent silences the MYB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MYB expression, e.g., colon cancer or chronic myelogenous leukemia.
• the oligonucleotide agent silences the c-MYC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MYC expression, e.g., Burkitt's lymphoma or neuroblastoma.
• the oligonucleotide agent silences the JUN gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JUN expression, e.g., ovarian, prostate or breast cancers.
• the oligonucleotide agent silences the BCL-2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCL-2 expression, e.g., lung or prostate cancers or Non-Hodgkin lymphoma.
• the oligonucleotide agent silences the Cyclin E gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin E expression, e.g., lung and breast cancers.
• the oligonucleotide agent silences the WNT-1 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted WNT-1 expression, e.g., basal cell carcinoma.
• the oligonucleotide agent silences the PKC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PKC expression, e.g., breast cancer.
• the oligonucleotide agent silences the NFKB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted NFKB expression, e.g., breast cancer.
• the oligonucleotide agent silences the STAT3 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted STAT3 expression, e.g., prostate cancer.
• the oligonucleotide agent silences the survivin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted survivin expression, e.g., cervical or pancreatic cancers.
• the oligonucleotide agent silences the Her2/Neu gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Her2/Neu expression, e.g., breast cancer.
• the oligonucleotide agent silences the topoisomerase I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase I expression, e.g., ovarian and colon cancers.
• the oligonucleotide agent silences mutations in the p73 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p73 expression, e.g., colorectal adenocarcinoma.
• the oligonucleotide agent silences mutations in the p27(KIP1 ) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p27(KIP1 ) expression, e.g., liver cancer.
• the oligonucleotide agent silences mutations in the PPM1 D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PPM1 D expression, e.g., breast cancer.
• the oligonucleotide agent silences mutations in the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., breast cancer.
• the oligonucleotide agent silences mutations in the caveolin I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted caveolin I expression, e.g., esophageal squamous cell carcinoma.
• the oligonucleotide agent silences mutations in the MIB I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MIB I expression, e.g., male breast carcinoma (MBC).
• a disorder characterized by unwanted MIB I expression e.g., male breast carcinoma (MBC).
• the oligonucleotide agent silences mutations in the MTAI gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MTAI expression, e.g., ovarian carcinoma.
• the oligonucleotide agent silences mutations in the M68 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted M68 expression, e.g., human adenocarcinomas of the esophagus, stomach, colon, and rectum.
• a disorder characterized by unwanted M68 expression e.g., human adenocarcinomas of the esophagus, stomach, colon, and rectum.
• the oligonucleotide agent silences mutations in tumor suppressor genes, and thus can be used as a method to promote apoptotic activity in combination with chemotherapeutics.
• the oligonucleotide agent silences mutations in the p53 tumor suppressor-gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p53 expression, e.g., gall bladder, pancreatic and lung cancers.
• a disorder characterized by unwanted p53 expression e.g., gall bladder, pancreatic and lung cancers.
• the oligonucleotide agent silences mutations in the p53 family member DN-p63, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted DN-p63 expression, e.g., squamous cell carcinoma.
• the oligonucleotide agent silences mutations in the pRb tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted pRb expression, e.g., oral squamous cell carcinoma.
• the oligonucleotide agent silences mutations in the APC1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted APC1 expression, e.g., colon cancer.
• the oligonucleotide agent silences mutations in the BRCA1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BRCA1 expression, e.g., breast cancer.
• the oligonucleotide agent silences mutations in the PTEN tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PTEN expression, e.g., hamartomas, gliomas, and prostate and endometrial cancers.
• the oligonucleotide agent silences MLL fusion genes, e.g., MLL- AF9, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MLL fusion gene expression, e.g., acute leukemias.
• MLL fusion genes e.g., MLL- AF9
• the oligonucleotide agent silences the BCR/ABL fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCR/ABL fusion gene expression, e.g., acute and chronic leukemias.
• the oligonucleotide agent silences the TLS/FUS1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TLS/FUS1 fusion gene expression, e.g., Myxoid liposarcoma.
• the oligonucleotide agent silences the PAX3/FKHR fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PAX3/FKHR fusion gene expression, e.g., Myxoid liposarcoma.
• the oligonucleotide agent silences the AML1/ETO fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted AML1/ETO fusion gene expression, e.g., acute leukemia.
• Another aspect of the invention relates to a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder that may benefit by angiogenesis inhibition e.g., cancer.
• the method comprises providing a ligand-conjugated oligonucleotide agent, wherein said oligonucleotide agent is homologous to and can silence, e.g., by cleavage, a gene which mediates angiogenesis; and administering a therapeutically effective dosage of said ligand-conjugated oligonucleotide agent to a subject, preferrably a human.
• the oligonucleotide agent silences the Flt-1 receptor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Flt-1 receptors, eg. Cancer and rheumatoid arthritis.
• the oligonucleotide agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, eg. Cancer and retinal neovascularization.
• Another aspect of the invention relates to a method of treating a subject infected with a virus or at risk for or afflicted with a disorder or disease associated with a viral infection.
• the method comprises providing a ligand-conjugated oligonucleotide agent, wherein said oligonucleotide agent is homologous to and can silence, e.g., by cleavage, a viral gene of a cellular gene which mediates viral function, e.g., entry or growth; and administering a therapeutically effective dose of said ligand-conjugated oligonucleotide agent to a subject, preferably a human subject.
• the invention provides for a method of treating patients infected by the Human Papilloma Virus (HPV) or at risk for or afflicted with a disorder mediated by HPV, e.g, cervical cancer.
• HPV Human Papilloma Virus
• HPV is linked to 95% of cervical carcinomas and thus an antiviral therapy is an attractive method to treat these cancers and other symptoms of viral infection.
• the expression of a HPV gene is reduced.
• the HPV gene is one of the group of E2, E6, or El.
• the expression of a human gene that is required for HPV replication is reduced.
• the invention also includes a method of treating patients infected by the Human Immunodeficiency Virus (HIV) or at risk for or afflicted with a disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome (AIDS).
• HIV Human Immunodeficiency Virus
• AIDS Acquired Immune Deficiency Syndrome
• the expression of a HIV gene is reduced.
• the HIV gene is CCR5, Gag, or Rev.
• the expression of a human gene that is required for HIV replication is reduced.
• the gene is CD4 or Tsg101 .
• the invention also includes a method for treating patients infected by the Hepatitis B Virus (HBV) or at risk for or afflicted with a disorder mediated by HBV, e.g., cirrhosis and heptocellular carcinoma.
• HBV Hepatitis B Virus
• the expression of a HBV gene is reduced.
• the targeted HBV gene encodes one of the group of the tail region of the HBV core protein, the pre-cregious (pre-c) region, or the cregious (c) region.
• a targeted HBV-RNA sequence is comprised of the poly(A) tail.
• the expression of a human gene that is required for HBV replication is reduced.
• the invention also provides for a method of treating patients infected by the Hepatitis A Virus (HAV), or at risk for or afflicted with a disorder mediated by HAV.
• HAV Hepatitis A Virus
• the expression of a human gene that is required for HAV replication is reduced.
• the present invention provides for a method of treating patients infected by the Hepatitis C Virus (HCV), or at risk for or afflicted with a disorder mediated by HCV, e.g., cirrhosis.
• the expression of a HCV gene is reduced.
• the expression of a human gene that is required for HCV replication is reduced.
• Methods of the invention also provide for treating patients infected by the Respiratory Syncytial Virus (RSV) or at risk for or afflicted with a disorder mediated by RSV, e.g, lower respiratory tract infection in infants and childhood asthma, pneumonia and other complications, e.g., in the elderly.
• RSV Respiratory Syncytial Virus
• the expression of a RSV gene is reduced.
• the targeted HBV gene encodes one of the group of genes N, L, or P.
• the expression of a human gene that is required for RSV replication is reduced.
• the invention also provides a method for treating patients infected by the herpes Cytomegalovirus (CMV) or at risk for or afflicted with a disorder mediated by CMV, e.g., congenital virus infections and morbidity in immunocompromised patients.
• CMV herpes Cytomegalovirus
• a disorder mediated by CMV e.g., congenital virus infections and morbidity in immunocompromised patients.
• the expression of a CMV gene is reduced.
• the expression of a human gene that is required for CMV replication is reduced.
• Methods of the invention also provide for a method of treating patients infected by the herpes Epstein Barr Virus (EBV) or at risk for or afflicted with a disorder mediated by EBV, e.g., NK T-cell lymphoma, non-Hodgkin lymphoma, and Hodgkin disease.
• EBV herpes Epstein Barr Virus
• a disorder mediated by EBV e.g., NK T-cell lymphoma, non-Hodgkin lymphoma, and Hodgkin disease.
• the expression of a EBV gene is reduced.
• the expression of a human gene that is required for EBV replication is reduced.
• Methods of the invention also provide for treating patients infected by Kaposi's Sarcoma-associated Herpes Virus (KSHV), also called human herpesvirus 8, or patients at risk for or afflicted with a disorder mediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman's disease and AIDS-associated primary effusion lymphoma.
• KSHV Kaposi's Sarcoma-associated Herpes Virus
• a disorder mediated by KSHV e.g., Kaposi's sarcoma, multicentric Castleman's disease and AIDS-associated primary effusion lymphoma.
• the expression of a KSHV gene is reduced.
• the expression of a human gene that is required for KSHV replication is reduced.
• the invention also includes a method for treating patients infected by the JCV (JCV) or a disease or disorder associated with this virus, e.g., progressive multifocal leukoencephalopathy (PML).
• JCV JC Virus
• PML progressive multifocal leukoencephalopathy
• the expression of a JCV gene is reduced.
• the expression of a human gene that is required for JCV replication is reduced.
• Methods of the invention also provide for treating patients infected by the myxovirus or at risk for or afflicted with a disorder mediated by myxovirus, e.g., influenza.
• a disorder mediated by myxovirus e.g., influenza.
• the expression of a myxovirus gene is reduced.
• the expression of a human gene that is required for myxovirus replication is reduced.
• Methods of the invention also provide for treating patients infected by the rhinovirus or at risk for of afflicted with a disorder mediated by rhinovirus, e.g., the common cold.
• a disorder mediated by rhinovirus e.g., the common cold.
• the expression of a rhinovirus gene is reduced.
• the expression of a human gene that is required for rhinovirus replication is reduced.
• Methods of the invention also provide for treating patients infected by the coronavirus or at risk for of afflicted with a disorder mediated by coronavirus, e.g., the common cold.
• a disorder mediated by coronavirus e.g., the common cold.
• the expression of a coronavirus gene is reduced.
• the expression of a human gene that is required for coronavirus replication is reduced.
• Methods of the invention also provide for treating patients infected by the flavivirus West Nile or at risk for or afflicted with a disorder mediated by West Nile Virus.
• the expression of a West Nile Virus gene is reduced.
• the West Nile Virus gene is one of the group comprising E, NS3, or NS5.
• the expression of a human gene that is required for West Nile Virus replication is reduced.
• Methods of the invention also provide for treating patients infected by the St. Louis Encephalitis flavivirus, or at risk for or afflicted with a disease or disorder associated with this virus, e.g., viral haemorrhagic fever or neurological disease.
• a disease or disorder associated with this virus e.g., viral haemorrhagic fever or neurological disease.
• the expression of a St. Louis Encephalitis gene is reduced.
• the expression of a human gene that is required for St. Louis Encephalitis virus replication is reduced.
• Methods of the invention also provide for treating patients infected by the Tick-borne encephalitis flavivirus, or at risk for or afflicted with a disorder mediated by Tick-borne encephalitis virus, e.g., viral haemorrhagic fever and neurological disease.
• a disorder mediated by Tick-borne encephalitis virus e.g., viral haemorrhagic fever and neurological disease.
• the expression of a Tick-borne encephalitis virus gene is reduced.
• the expression of a human gene that is required for Tick-borne encephalitis virus replication is reduced.
• Methods of the invention also provide for methods of treating patients infected by the Murray Valley encephalitis flavivirus, which commonly results in viral haemorrhagic fever and neurological disease.
• the expression of a Murray Valley encephalitis virus gene is reduced.
• the expression of a human gene that is required for Murray Valley encephalitis virus replication is reduced.
• the invention also includes methods for treating patients infected by the dengue flavivirus, or a disease or disorder associated with this virus, e.g., dengue haemorrhagic fever.
• a dengue virus gene is reduced.
• a human gene that is required for dengue virus replication is reduced.
• Methods of the invention also provide for treating patients infected by the Simian Virus 40 (SV40) or at risk for or afflicted with a disorder mediated by SV40, e.g., tumorigenesis.
• SV40 Simian Virus 40
• a disorder mediated by SV40 e.g., tumorigenesis.
• the expression of a SV40 gene is reduced.
• the expression of a human gene that is required for SV40 replication is reduced.
• the invention also includes methods for treating patients infected by the Human T Cell Lymphotropic Virus (HTLV), or a disease or disorder associated with this virus, e.g., leukemia and myelopathy.
• HTLV Human T Cell Lymphotropic Virus
• the expression of a HTLV gene is reduced.
• the HTLV1 gene is the Tax transcriptional activator.
• the expression of a human gene that is required for HTLV replication is reduced.
• Methods of the invention also provide for treating patients infected by the Moloney- Murine Leukemia Virus (Mo-MuLV) or at risk for or afflicted with a disorder mediated by Mo- MuLV, e.g., T-cell leukemia.
• Mo-MuLV Moloney- Murine Leukemia Virus
• the expression of a Mo-MuLV gene is reduced.
• the expression of a human gene that is required for Mo-MuLV replication is reduced.
• Methods of the invention also provide for treating patients infected by the encephalomyocarditis virus (EMCV) or at risk for or afflicted with a disorder mediated by EMCV, e.g. myocarditis.
• EMCV encephalomyocarditis virus
• myocarditis a disorder mediated by EMCV
• EMCV leads to myocarditis in mice and pigs and is capable of infecting human myocardial cells. This virus is therefore a concern for patients undergoing xenotransplantation.
• the expression of a EMCV gene is reduced.
• the expression of a human gene that is required for EMCV replication is reduced.
• the invention also includes a method for treating patients infected by the measles virus (MV) or at risk for or afflicted with a disorder mediated by MV, e.g., measles.
• MV measles virus
• the expression of a MV gene is reduced.
• the expression of a human gene that is required for MV replication is reduced.
• the invention also includes a method for treating patients infected by the Vericella zoster virus (VZV) or at risk for or afflicted with a disorder mediated by VZV, e.g. chicken pox or shingles (also called zoster).
• VZV Vericella zoster virus
• a disorder mediated by VZV e.g. chicken pox or shingles (also called zoster).
• the expression of a VZV gene is reduced.
• the expression of a human gene that is required for VZV replication is reduced.
• the invention also includes a method for treating patients infected by an adenovirus or at risk for or afflicted with a disorder mediated by an adenovirus, e.g. respiratory tract infection.
• a disorder mediated by an adenovirus e.g. respiratory tract infection.
• the expression of an adenovirus gene is reduced.
• the expression of a human gene that is required for adenovirus replication is reduced.
• the invention includes a method for treating patients infected by a yellow fever virus (YFV) or at risk for or afflicted with a disorder mediated by a YFV, e.g. respiratory tract infection.
• a YFV gene is reduced.
• the preferred gene is one of a group that includes the E, NS2A, or NS3 genes.
• the expression of a human gene that is required for YFV replication is reduced.
• Methods of the invention also provide for treating patients infected by the poliovirus or at risk for or afflicted with a disorder mediated by poliovirus, e.g., polio.
• a disorder mediated by poliovirus e.g., polio.
• the expression of a poliovirus gene is reduced.
• the expression of a human gene that is required for poliovirus replication is reduced.
• Methods of the invention also provide for treating patients infected by a poxvirus or at risk for or afflicted with a disorder mediated by a poxvirus, e.g., smallpox.
• a poxvirus gene is reduced.
• a human gene that is required for poxvirus replication is reduced.
• the invention features methods of treating a subject infected with a pathogen, e.g., a bacterial, amoebic, parasitic, or fungal pathogen.
• a pathogen e.g., a bacterial, amoebic, parasitic, or fungal pathogen.
• the method comprises providing a ligand-conjugated oligonucleotide agent, wherein said oligonucleotide is homologous to and can silence, e.g., by cleavage of a pathogen gene; and administering a therapeutically effective dose of said ligand-conjugated oligonucleotide agent to a subject, prefereably a human subject.
• the target gene can be one involved in growth, cell wall synthesis, protein synthesis, transcription, energy metabolism, e.g., the Krebs cycle, or toxin production.
• the present invention provides for a method of treating patients infected by a Plasmodium that causes malaria.
• the expression of a Plasmodium gene is reduced.
• the gene is apical membrane antigen 1 (AMA1 ).
• AMA1 apical membrane antigen 1
• the expression of a human gene that is required for Plasmodium replication is reduced.
• the invention also includes methods for treating patients infected by the Mycobacterium ulcerans, or a disease or disorder associated with this pathogen, e.g. Buruli ulcers.
• the expression of a Mycobacterium ulcerans gene is reduced.
• the expression of a human gene that is required for Mycobacterium ulcerans replication is reduced.
• the invention also includes methods for treating patients infected by the Mycobacterium tuberculosis, or a disease or disorder associated with this pathogen, e.g. tuberculosis.
• a Mycobacterium tuberculosis gene is reduced.
• a human gene that is required for Mycobacterium tuberculosis replication is reduced.
• the invention also includes methods for treating patients infected by the Mycobacterium leprae, or a disease or disorder associated with this pathogen, e.g. leprosy.
• the expression of a Mycobacterium leprae gene is reduced.
• the expression of a human gene that is required for Mycobacterium leprae replication is reduced.
• the invention also includes methods for treating patients infected by the bacteria Staphylococcus aureus, or a disease or disorder associated with this pathogen, e.g. infections of the skin and muscous membranes.
• a Staphylococcus aureus gene is reduced.
• a human gene that is required for Staphylococcus aureus replication is reduced.
• the invention also includes methods for treating patients infected by the bacteria Streptococcus pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection.
• a Streptococcus pneumoniae gene is reduced.
• a human gene that is required for Streptococcus pneumoniae replication is reduced.
• the invention also includes methods for treating patients infected by the bacteria Streptococcus pyogenes, or a disease or disorder associated with this pathogen, e.g. Strep throat or Scarlet fever.
• a Streptococcus pyogenes gene is reduced.
• a human gene that is required for Streptococcus pyogenes replication is reduced.
• the invention also includes methods for treating patients infected by the bacteria Chlamydia pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection.
• a Chlamydia pneumoniae gene is reduced.
• a human gene that is required for Chlamydia pneumoniae replication is reduced.
• the invention also includes methods for treating patients infected by the bacteria Mycoplasma pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection.
• the expression of a Mycoplasma pneumoniae gene is reduced.
• the expression of a human gene that is required for Mycoplasma pneumoniae replication is reduced.
• Another aspect of the invention relates to a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder characterized by an unwanted immune response, e.g., an inflammatory disease or disorder, or an autoimmune disease or disorder.
• the method comprises providing a ligand-conjugated oligonucleotide agent, wherein said oligonucleotide agent is homologous to and can silence, e.g., by cleavage, a gene which mediates an unwanted immune response; and administering said ligand- conjugated oligonucleotide agent to a subject, preferrably a human subject.
• the disease or disorder is an ischemia or reperfusion injury, e.g., ischemia or reperfusion injury associated with acute myocardial infarction, unstable angina, cardiopulmonary bypass, surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty, the response to a transplantated organ or tissue, e.g., transplanted cardiac or vascular tissue; or thrombolysis.
• the disease or disorder is restenosis, e.g., restenosis associated with surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty.
• the disease or disorder is Inflammatory Bowel Disease, e.g., Crohn Disease or Ulcerative Colitis.
• the disease or disorder is inflammation associated with an infection or injury.
• the disease or disorder is asthma, lupus, multiple sclerosis, diabetes, e.g., type II diabetes, arthritis, e.g., rheumatoid or psoriatic.
• the oligonucleotide agent silences an integrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.
• the oligonucleotide agent silences a selectin or co-ligand thereof, e.g., P-selectin, E-selectin (ELAM), l-selectin, P-selectin glycoprotein-1 (PSGL-1 ).
• the oligonucleotide agent silences a component of the complement system, e.g., C3, C5, C3aR, C5aR, C3 convertase, C5 convertase.
• the oligonucleotide agent silences a chemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1 I, IL-1J, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA1 1 , CCR3.
• a chemokine or receptor thereof e.g., TNFI, TNFJ, IL-1 I, IL-1J, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA1 1 , CCR3.
• the oligonucleotide agent silences GCSF, Gro1 , Gro2, Gro3, PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1 1, MIP-1J, RANTES, MCP-1 , MCP-2, MCP-3, CMBKR1 , CMBKR2, CMBKR3, CMBKR5, AIF-1 , I-309.
• PPBP Pro-Platelet Basic Protein
• Another aspect of the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with acute pain or chronic pain.
• the method comprises providing a ligand-conjugated oligonucleotide agent, wherein said ligand is an aromatic group and said oligonucleotide is homologous to and can silence, e.g., by cleavage, a gene which mediates the processing of pain; and administering a therapeutically effective dose of said ligand-conjugated oligonucleotide agent to a subject, preferrably a human subject.
• the oligonucleotide agent silences a component of an ion channel.
• the oligonucleotide agent silences a neurotransmitter receptor or ligand.
• Another aspect of the invention relates to a method of treating a subject, e.g., a human, at risk for or afflicted with a neurological disease or disorder.
• the method comprises providing a ligand-conjugated oligonucleotide agent, wherein said ligand is an aromtic group and said oligonucleotide is homologous to and can silence, e.g., by cleavage, a gene which mediates a neurological disease or disorder; and administering a therapeutically effective dose of said ligand-conjugated oligonucleotide agent the to a subject, preferrably a human.
• the disease or disorder is Alzheimer Disease or Parkinson Disease.
• the oligonucleotide agent silences an amyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 and PSEN2, or l-synuclein.
• the disease or disorder is a neurodegenerative trinucleotide repeat disorder, e.g., Huntington disease, dentatorubral pallidoluysian atrophy or a spinocerebellar ataxia, e.g., SCA1 , SCA2, SCA3 (Machado-Joseph disease), SCA7 or SCA8.
• the oligonucleotide agent silences HD, DRPLA, SCA1 , SCA2, MJD1 , CACNL1A4, SCA7, SCA8.
• the loss of heterozygosity can result in hemizygosity for sequence, e.g., genes, in the area of LOH. This can result in a significant genetic difference between normal and disease-state cells, e.g., cancer cells, and provides a useful difference between normal and disease-state cells, e.g., cancer cells. This difference can arise because a gene or other sequence is heterozygous in euploid cells but is hemizygous in cells having LOH.
• the regions of LOH will often include a gene, the loss of which promotes unwanted proliferation, e.g., a tumor suppressor gene, and other sequences including, e.g., other genes, in some cases a gene which is essential for normal function, e.g., growth.
• Methods of the invention rely, in part, on the specific cleavage or silencing of one allele of an essential gene with a ligand-conjugated oligonucleotide agent of the invention.
• the oligonucleotide agent is selected such that it targets the single allele of the essential gene found in the cells having LOH but does not silence the other allele, which is present in cells which do not show LOH.
• polymorphisms e.g., SNPs of essential genes that are affected by LOH
• SNPs of essential genes that are affected by LOH are used as a target for a disorder characterized by cells having LOH, e.g., cancer cells having LOH.
• a disorder characterized by cells having LOH e.g., cancer cells having LOH.
• one of ordinary skill in the art can identify essential genes which are in proximity to tumor suppressor genes, and which are within a LOH region which includes the tumor suppressor gene.
• the gene encoding the large subunit of human RNA polymerase II, POLR2A, a gene located in close proximity to the tumor suppressor gene p53, is such a gene. It frequently occurs within a region of LOH in cancer cells.
• genes that occur within LOH regions and are lost in many cancer cell types include the group comprising replication protein A 70-kDa subunit, replication protein A 32-kD, ribonucleotide reductase, thymidilate synthase, TATA associated factor 2H, ribosomal protein S14, eukaryotic initiation factor 5A, alanyl tRNA synthetase, cysteinyl tRNA synthetase, NaK ATPase, alpha-1 subunit, and transferrin receptor.
• another aspect of the invention relates to a method of treating a disorder characterized by LOH, e.g., cancer.
• the method comprises optionally, determining the genotype of the allele of a gene in the region of LOH and preferably determining the genotype of both alleles of the gene in a normal cell; providing a ligand-conjugated oligonucleotide agent which preferentially cleaves or silences the allele found in the LOH cells; and administering a therapeutically effective dose of said ligand-conjugated oligonucleotide agent to the subject, preferrably a human.
• the invention also includes a ligand-conjugated oligonucleotide agent disclosed herein, e.g, an oligonucleotide agent which can preferentially silence, e.g., cleave, one allele of a polymorphic gene.
• a ligand-conjugated oligonucleotide agent disclosed herein e.g, an oligonucleotide agent which can preferentially silence, e.g., cleave, one allele of a polymorphic gene.
• the invention provides a method of cleaving or silencing more than one gene with a ligand-conjugated oligonucleotide agent.
• the oligonucleotide agent is selected so that it has sufficient homology to a sequence found in more than one gene.
• the sequence AAGCTGGCCCTGGACATGGAGAT (SEQ ID NO: ) is conserved between mouse lamin B1 , lamin B2, keratin complex 2-gene 1 and lamin A C.
• an oligonucleotide agent targeted to this sequence would effectively silence the entire collection of genes.
• the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
• the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
• a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day.
• the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. Preferred dosing is one administration per day.
• Retro compounds block the retrograde trafficking pathway 16 used by many toxins by interfering with shuttling between early endosomes and the TGN.
• the first model tests effects of splice switching antisense oligonucleotides (SSOs) in cultured cells stably transfected with a cassette comprising the coding sequence of a reporter gene interrupted by an abnormal intron 11,17 . Delivery of an appropriate SSO to the cell nucleus results in corrected splicing and increased expression of the reporter.
• a second model tests the ability of classic antisense oligonucleotides to reduce the expression of a target.
• the chosen target is the MDR1 gene product P-glycoprotein (Pgp, ABCB1 ), a membrane transporter involved in cancer drug resistance 18 .
• Pgp MDR1 gene product P-glycoprotein
• a membrane transporter involved in cancer drug resistance 18 a membrane transporter involved in cancer drug resistance 18 .
• Expression of Pgp in drug resistant cells is assayed using a monoclonal anti-Pgp antibody and flow cytometry 19 .
• cells were treated with oligonucleotides in the absence of any transfection agent, the oligonucleotides were rinsed away, and the cells were then briefly treated with the Retro compound. Several hours after removal of the Retro compound the pharmacological effects of the oligonucleotides were measured.
• Retro-1 acts by alteration of the intracellular processing of the SSO.
• Retro-1 strongly enhanced the induction of EGFP by a SSO.
• the observations at the protein level were paralleled by correction of splicing at the RNA level as indicated by an RT-PCR analysis ( Figure 1d).
• the timing involved in these experiments was quite important. In preliminary experiments we observed little effect when the Retro compound was added before or during the early phases of oligonucleotide uptake, but a much stronger effect when the Retro compound was added after the oligonucleotide had accumulated in cells.
• the Retro compound strongly enhanced the pharmacological activity of a SSO, likely by modifying its intracellular processing.
• Retro compounds have been carefully studied, suggesting that these compounds act only on a limited subset of endomembranes, primarily the endosome-TGN interface 15 .
• Figure 3b there was considerable co-localization of fluorescent oligonucleotide with Rab 9, a protein that has been implicated in endosome to TGN traffic 14 .
• Retro compounds have been used to promote release of plasmids or oligonucleotides from endomembrane stores, particularly the drug chloroquine that raises the pH of acidic endomembrane compartments and causes their destabilization 22 .
• the action of the Retro compounds is completely distinct as illustrated in Figure 7.
• chloroquine treatment markedly reduced cellular accumulation of a lysosomoptropic dye, whereas a pharmacologically effective concentration of Retro-1 had little effect on the ability of acidic endomembrane compartments to accumulate dye.
• Retro-1 can substantially enhance the pharmacological effectiveness of antisense and splice switching oligonucleotides. While it may be surprising that a compound that blocks toxin action enhances oligonucleotide action, this may be a reflection of very different trafficking processes utilized by the two types of molecules. Our results suggest that the Retro agent causes partial release of oligonucleotides from endomembrane compartments where they have accumulated but are pharmacologically inert, and allows oligonucleotides to gain entry into the cytosol and thence the nucleus where they exert their actions.
• Splice switching oligonucleotide 623 (5'-GTTATTCTTTAGAATGGTGC- 3'), its 5-base mismatch (5'-GTAATTATTTATAATCGTCC-3'), as well as a 3'-TAMRA fluorophore labeled version were synthesized in our laboratory as described 26 . These oligomers are fully modified 2'-0-Me phosphorothioates.
• the anti-MDR1 antisense oligonucleotide (5'-CCATCccgacctcgcGCTCC-3') 27 and its scrambled control were synthesized by Integrated DNA Technologies (Coralville, IA).
• A375Luc705 is a human melanoma cell line containing a firefly luciferase coding sequence interrupted by an abnormal intron 26 .
• Hel_aEGFP654 is a human cell line containing an enhanced green fluorescent protein reporter interrupted by an abnormal intron (obtained from R. Kole, U. North Carolina).
• Cells were cultured in Dulbecco's minimum essential medium (DMEM) supplemented with L-glutamine and 10% fetal bovine serum (FBS) (Gibco/lnvitrogen, Carlsbad, CA, USA). In both of these cell lines correct splicing and reporter expression can be restored by delivery of the 623 SSO to the nucleus.
• DMEM Dulbecco's minimum essential medium
• FBS fetal bovine serum
• NIH-3T3- MDR is a highly drug resistant mouse fibroblast cell line that is stably transfected with a cDNA coding for the human P-glycoprotein 27 ; these cells were kindly provided by M. Gottesman, National Cancer Institute, and were maintained in DMEM plus 10% FBS.
• Luciferase Induction Assays A375Luc705 cells were incubated with 50 nM SSO 623 or control mismatch oligonucleotide in serum free OPTI-MEM I for 4 h followed by addition of FBS to 1 .5% for an additional 12 h. The oligonucleotides were removed and the cells rinsed in buffer; thereafter medium plus 1.5% FBS was added followed by various amounts of Retro-1. The Retro compound was removed after 2 h and the cells further incubated in medium plus 1.5% FBS for an additional 6 h.
• Luciferase enzyme activity was determined using a Luciferase assay kit (Promega, Madison, Wl, USA) and was performed on a FLUOstar Omega microplate reader (BMG LABTECH, Cary, NC, USA). Protein content was determined by the BCA protein assay (Pierce, Rockford, IL, USA).
• EGFP Induction Assays HeLaEGFP654 cells were incubated with 50 nM SSO 623 in serum free OPTI-MEM I for 4 h followed by addition of FBS to 1 .5% for an additional 12 h. The oligonucleotides were removed and the cells rinsed in buffer; thereafter medium + 1.5% FBS was added followed by 100 ⁇ Retro-1. The Retro compound was removed after 2 h and the cells further incubated in medium plus 1.5% FBS for an additional 24 h. In some cases cells were treated with SSO 623 and Lipofectamine 2000 as a positive control. EGFP expression was measured by flow cytometry using a LSR II cell analyzer (Becton-Dickenson, San Jose, CA, USA) with a 488 nm laser coupled with a 525/50 filter for EGFP.
• LSR II cell analyzer Becton-Dickenson, San Jose, CA, USA
• NIH-3T3-MDR cells were incubated with various concentrations of anti-MDR1 antisense oligonucleotide in DMEM-H medium plus 2% FBS for 4 h.
• the oligonucleotides were removed by rinsing and medium + 2% FBS was added followed by the addition of Retro-1 at various concentrations. After 4 h the Retro compound was removed and incubation continued in medium plus 2% FBS for 48 h.
• cells were treated with SSO 623 and Lipofectamine 2000 as a positive control.
• P- glycoprotein expression was measured essentially as previously described 28 .
• Live cell confocal microscopy was performed using an Olympus Confocal FV300 fluorescent microscope with 60x oil immersion objectives.
• cells were transfected using Lipofectamine 2000 with plasmids encoding GFP chimeras of Rab 9 or 1 1 .
• the transfection agent was removed and the cells incubated in growth medium for 24 h.
• the cells were transfected with a baculovirus expression vector for Rab 5 or for a Golgi marker ⁇ Organelle LightsTM). Thereafter cells were incubated with 3'-TAMRA conjugated SSO 623 and observed for co-localization of red and green signals.

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