WO2004001045A1 - Inhibition of nuclear receptors - Google Patents

Inhibition of nuclear receptors Download PDF

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Publication number
WO2004001045A1
WO2004001045A1 PCT/EP2003/006183 EP0306183W WO2004001045A1 WO 2004001045 A1 WO2004001045 A1 WO 2004001045A1 EP 0306183 W EP0306183 W EP 0306183W WO 2004001045 A1 WO2004001045 A1 WO 2004001045A1
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tissue
cell
sense strand
nuclear receptor
gene
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PCT/EP2003/006183
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French (fr)
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Igor Bendik
Manuel Heim
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Dsm Ip Assets B.V.
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Priority to AU2003242688A priority Critical patent/AU2003242688A1/en
Publication of WO2004001045A1 publication Critical patent/WO2004001045A1/en

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    • 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
    • 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
    • C12N15/1138—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 receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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 N.A.
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • the present invention relates to the inhibition of nuclear receptors.
  • the inactivation is achieved by introduction of an oligonucleotide sense strand and a corresponding anti- sense strand complementary to a part of the nuclear receptor messenger RNA into a mammalian cell or tissue.
  • Inhibition of nuclear receptors is desirable where over-expression of such a receptor is leading to or responsible for a disease or deficiency state.
  • the nuclear receptors belong to a large family of proteins that confer cells with responsiveness to molecules which are signalling ligands, e.g., hormones, vitamins and derivatives thereof, such as glucocorticoids, estrogens, retinoids, vitamin D compounds, and thyroid hormones. These receptors mediate their signal by activating and/or repressing gene transcription directly by modulating the RNA polymerase transcription machinery.
  • signalling ligands e.g., hormones, vitamins and derivatives thereof, such as glucocorticoids, estrogens, retinoids, vitamin D compounds, and thyroid hormones.
  • nuclear receptors Inhibition of nuclear receptors" expression in individuals with high susceptibility to certain diseases or physiological states related to receptor malfunction is of high importance for prevention and therapy.
  • the nuclear receptors are cellular key players that can redirect the cell into a detrimental developmental stage in the field of hormone dependent diseases.
  • RNA interference is a biological response of cells for the protection against nucleic acid invaders like viruses and is highly potent to fight foreign gene transcripts.
  • dsRNA that is longer than 30 base pairs triggers a non-specific pathway leading to the degradation of all mRNAs thus ceasing the protein synthesis of the cell. It has been found recently that using short interfering dsRNAs of from about 21 to about 23 nucleotides having sense and antisense strands which correspond to parts of receptor mRNAs are capable of specifically inhibiting or "silencing" targeted genes [WO 01/75164 and Elbashir et al., Nature 411, 494-8 (2001), contents of which are herewith explicitly incorporated by reference into the present specification] , viz. suppress specifically expression of endogenous and heterologous genes in different mammalian cell lines, e.g., genes coding for cytoskeletal proteins and reporter genes, such as genes for luciferases, via a mechanism which still remains to be uncovered.
  • the present invention relates to a method of inhibiting or silencing genes coding for mammalian nuclear receptors, to the use of this method in research, prevention and therapy of diseases, to the olgonucleotides used in this method and to corresponding pharmaceutical compositions.
  • the present invention relates also to a cell or tissue wherein two complementary RNA strands interfere with the expression of a mammalian nuclear receptor (so called knockdown cell or tissue) and it concerns a kit comprising reagents inhibiting the expression of a nuclear receptor gene in a mammalian cell or tissue.
  • the superfamily of nuclear receptors share a modular structure in which regions, A to F, are displayed (Evans, Science 240:889-895, 1988).
  • the nuclear receptor is characterized by a variable N-terminal domain (A/B), followed by a centrally located, highly conserved DNA- binding domain (C), a variable hinge domain (D), a conserved ligand binding domain (E) and a variable C-terminal domain (F).
  • the non-conserved N-terminal domain (A/B) is highly variable in size and sequence and modulates the transciption activation.
  • the variable C-terminal domain (F) has a similar transactivation function.
  • the F-region is also needed for ligand binding.
  • Table 1 A list of the major human nuclear receptor genes including their trivial names, accession numbers and nuclear receptors they are expressing according to NCBI Gene Bank is given in Table 1.
  • the ligand/receptor complex switches to a transcriptionally active state, binds with high affinity to a hormone response element on the chromatin DNA and activates the transcription of specific target genes (Martinez and Wahli, Nuclear Hormone Receptors, chapter 6: 125 - 153, Acad. Press, 1991). All of these nuclear receptors have in common that they regulate on the transcriptional level important key pathways in embry- onic development, in adult homeostasis or in organ physiology. Various diseases and abnormalities have already been ascribed to a disturbance in these pathways like different cancers, rickets, osteoporosis and many more.
  • ERalpha In the context of the present invention the human estrogen receptor (ER) family and the human estrogen receptor alpha (ERalpha) is of specific interest. ERalpha regulates important processes in the reproductive tissues, in the bone system, in the cardiovascular system and in the central nervous system. Futhermore, deregulated ERalpha expression is involved in pathological states such as, e.g., cancer (R. Lindsay, D.W. Dempster and V.C. Jordan, eds., Estrogens and Antiestrogens - Basic and Clinical Aspects, Lippincott- Raven, Philadelphia, 1997).
  • NR2C1 TR2 TR2-11 NM 003297 TR2 orphan receptor
  • Steroid receptor TR2-11 NR2C2 TR4.
  • COUPTFB ARP1, SVP40 Apolipoprotein Al regulatory protein
  • the present invention relates to a method of inhibiting expression of a receptor gene in a mammalian cell or tissue by introduction of a sense strand and corresponding antisense strand RNA sequence of about 21 to about 23 nucleotides complementary to a part of the receptor mRNA into such cell or tissue characterized in that the receptor mRNA is a mammalian nuclear receptor mRNA.
  • inhibitor expression comprises all degrees of inhibition of expression of a gene in a mammalian cell or tissue, especially of altered expression and particularly of over-expression, from partial to complete inactivation (down-regulation), depending on the target gene and the intracellular dose of siRNA.
  • the inhibition of the target gene expression is reflected in a loss of phenotype.
  • the degree of inhibition may be estimated by comparing the values from untreated cells or tissue with those from treated cells or tissue according to the method of the present invention.
  • the consequences of inhibition may be assayed for properties of the cell or tissue by molecular biology methods such as real-time quantitative reverse transcription polymerase chain re- action (RT-PCR), RNA solution hybridization, Northern hybridization and biochemical assays like enzyme linked immunoabsorbent assay (ELISA), Western blotting or radioim- munoassay (RIA), for example.
  • mammalian cell or tissue relates on the one hand to all mammals, including and especially to human beings, and on the other hand to all types of cells or tissues in which expression of a mammalian nuclear receptor gene takes place or can be effected.
  • the cells or tissues may be, e.g., from the vascular or extravascular, the blood or lymph system, from muscles, any organ, gland, the skin, brain or cerebrospinal fluid.
  • introduction comprises all means and methods which are known to be effective, generally and specifically, to incorporate genetic material into cells and tissues in a way that the genetic material will become effective in the desired way.
  • introduction therefore comprises the methods of transformation (uptake of naked genetic material), conjugation (exchange of genetic material from cell to cell) and transfection or transduction, using plasmids or non-lethal viruses as vectors, or using liposomal technology.
  • RNA sense and anti-sense oligonucleotide sequences which are complementary to a specific part of the targeted nuclear receptor gene can be introduced either in double-stranded (ds) form, using, e.g., plasmids or liposomes or in single- stranded (ss) form, e.g., using viruses carying the ss, viz. sense and antisense oligonucleotide sequences separately.
  • alphaviruses such as Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Equine Encephalitis virus (WEE), Sindbis virus (SIV), South African Arbovirus No.
  • EEE Eastern Equine Encephalitis virus
  • VEE Venezuelan Equine Encephalitis virus
  • Everglades virus Mucambo virus
  • Pixuna virus Western Equine Encephalitis virus
  • WEE Western Equine Encephalitis virus
  • SIV Sindbis virus
  • Semliki Forest virus Semliki Forest virus (SFV), Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Ge- tah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus.
  • SFV Semliki Forest virus
  • Middelburg virus Chikungunya virus
  • O'nyong-nyong virus Ross River virus
  • Barmah Forest virus Ge- tah virus
  • Sagiyama virus Bebaru virus
  • Mayaro virus Una virus
  • Aura virus Whataroa virus
  • Babanki virus Kyzylagach virus
  • Highlands J virus Fort Morgan virus
  • Ndumu virus Buggy Creek virus.
  • Preferred alphaviruses include SFV (Liljestrom and Garoff, Bio/Technology 9, 1356-1361 1991; A new generation of animal cell expression vectors based on the SFV replicon), SIV (Xiong et al., Science 243, 1188-1191, 1989; Sindbis virus: an efficient broad host range vector for gene expression in animal cells), and VEE (Davis et al., Virology 171, 189-204 1989; In vitro synthesis of infectious Venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant), for exam- pie. Most alphaviruses and vectors are commercially available.
  • Expression plasmids which can be used as vectors are also well-known in the art and are described as well as methods of transfection, e.g., in Colosimo et al. (Bio Techniques, 29:314-331, 2000).
  • the plasmids express the short oligonucleotide sequences either individually or in a single transcript that forms secondary structures (e.g. a hairpin structure).
  • the expression plasmids can be introduced stably or transiently to generate the down- regulation. Modifications and specific improvements of RNA expression systems mentioned above are also well known in the art (e.g. expression cassettes for short RNA tran- scription, multiple copies on the plasmid) and can be used to work the present invention. Many expression plasmids are commercially available.
  • liposomes which are excellent transfer systems.
  • liposomes are described, e.g. by Elbashir et al. (Methods, 26:199-213, 2002).
  • vector means a construct or particle which is used for the introduction of genetic material in mammalian cells or tissue.
  • the preferred liposome reagent which has been used is the commercially available Oligofectamine .
  • the reagent has been used as described by the manufacturer (Invitrogen Ltd., Basel, Switzerland.)
  • the term "complementary” refers to nucleic acid sequences that can form hydrogen bonds with specific RNA sequences by either traditional Watson-Crick or other, non-traditional types (e.g. Hoogsteen type) of base-paired interactions, thus leading to degradation of the targeted gene's mRNA.
  • complementary therefore, comprises sequences which are identical (100% homolog) to the targeted gene mRNA or analogues of such sequences which differ by one or more nucleotides, preferably by 1 -3 nucleotides, from such sequences by addition, deletion, substitution or alteration.
  • RNA sequences of about 21-23 nucleotides can be identical with or complementary to a n y part of the mRNA of the targeted nuclear re- ceptor gene. It is evident to the person skilled in the art that the specificity of the inhibition of a specific receptor will depend within certain limits on the sequence differences within a gene family that may code for a certain receptor. Normally, highest specificity will be obtained with RNA sequences complementary to parts of a mRNA which is expressed from a highly variable domain.
  • sense and antisense in connection within “strand” or “sequence” are used in the way generally understood by every person skilled in the art, viz., a sense RNA sequence is identical with a sequence transcribed and processed from the gene yielding the mRNA. The antisense strand is again complementary to a sense strand.
  • the 21 - 23 nucleotide (nt)RNA molecules can be single-stranded or double stranded (as two 21-23 nt RNAs); such molecules can be blunt ended or comprise overhanging ends (e.g., 5', 3').
  • the RNA molecule is double stranded and either blunt ended or, preferably, comprises overhanging ends (as two 21-23 nt RNAs).
  • At least one strand of the RNA molecule has a 3' overhang from about 1 to 6 nucleotides (e.g., pyrimidine nucleotides, purine nucleotides) in length. In other embodiments, the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from 2 to about 4 nucleotides in length. In one embodiment the RNA molecule is double stranded, one strand has a 3' overhang and the other strand can be blunt-ended or have an overhang. In the embodiment in which the RNA molecule is double stranded and both strands comprise an overhang, the length of the overhangs may be the same or different for each strand.
  • nucleotides e.g., pyrimidine nucleotides, purine nucleotides
  • the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from 2 to about
  • the RNA of the present invention comprises 21 nucleotide strands which are paired and which have overhangs of from about 1 to about 3, particularly about 2, nucleotides on both 3' ends of the RNA.
  • the 3' overhangs can be stabilized against degradation.
  • the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides and derivatives thereof.
  • the RNA is stabilized by pyrimidine nucleotides, such as uridine, thymidine and cytosine nucleotides and derivatives thereof.
  • the about 21 - 23 nt RNA molecules of the present invention can be prepared and purified using a number of techniques well-known to those skilled in the art, e.g., by recombinant methods or by chemical synthesis, making use of common nucleic acid protecting and coupling groups (see, e.g., Caruthers et al., 1992, Methods in Enzymology 211, 3-19 and Elbashir et al., above).
  • the introduction of the ss or ds 21 - 23 nt RNA into the transfer vectors is also effected by methods well-known in the art.
  • TOM-Protecting-GroupTM (2' - O-triisopropylsilyl- oxy-methyl protecting group) which is structurally related to the tBDMS-group, introduced by Ogilvie and Usman in the early eighties for the protection of the 2'-OH groups in the chemical synthesis of RNA.
  • the TOM-group is fully compatible with the established tBDMS chemistry and has several important advantages (e.g. coupling yields >99.5% with ⁇ 2 minutes coupling time due to less sterical hindrance and short, reliable and complete deprotection under mild conditions.
  • the invention also relates to the new nucleotide sequences used in the method of the present invention, viz. to those ss RNA sequences which are identical with sequences of about 21 to 23 nucleotides of a mammalian nuclear receptor mRNA, complementary sequences and analogues thereof which analogues differ by addition, deletion, substitution or altera- tion of one or more, preferably 1 - 3, nucleotides and to corresponding ds RNA sequences and to their uses
  • the invention also relates to transfer systems containing the above-mentioned nucleotide sequences and to their uses
  • the present invention also relates to a method of treatment of a disease caused by over- expression of a nuclear receptor gene in a mammalian cell or tissue characterized in that dsRNA of about 21 to 23 nucleotide pairs targeting the mRNA of this nuclear receptor for degradation is administered to the mammal in the form of a pharmaceutical composition comprising ss or ds RNA strands, and to these pharmaceutical compositions themselves.
  • the present invention finally relates to a kit comprising reagents inhibiting expression of a nuclear receptor gene in a mammalian cell or tissue, said kit containing at least a sufficient amount of vectors comprising a sense strand and a corresponding anti-sense strand RNA sequence of about 21 to about 23 nucleotides complementary to a part of a mammalian nuclear receptor mRNA and to a knockdown cell or tissue generated by a method of the present invention.
  • a kit may include additional reagents to carry out the in vivo or in vitro delivery of the about 21 to 23 nt molecules to the samples or subjects and may also include instructions to allow a user of the kit to practice the invention.
  • the method of examining the function of a mammalian nuclear receptor gene in a cell or tissue comprises:
  • test cell or test tissue in which mRNA of the gene is degraded used above is identical with the term “knock-down cell or tissue” which latter term is widely used in the art.
  • the ss and ds RNA molecules of the present invention can be used as pharmaceutical agents in pharmaceutical compositions to treat disease states originating from altered ex- pression of one or more mammalian nuclear receptor genes and can be administered to or introduced into a mammal/patient by any standard means.
  • compositions comprise formulations for all kinds of delivery, e.g., oral, nasal, rectal, intravenous, subcutaneous, intramuscular, or intraperitoneal admini- stration and include all kinds of pharmaceutically acceptable excipients known to be useful in such compositions.
  • excipients comprise, e.g., antioxidants, preservatives, stabilizers, especially additives which protect the active molecules from degradation by nu- cleases, dyes, flavoring agents and carriers.
  • a pharmaceutically effective dose is the dose required to prevent, inhibit the occurrence of or treat a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristic of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is to be administered.
  • the inhibition of expression of nuclear ERalpha is achieved by introduction of a short sequence-specific double stranded RNA into primary osteoblasts via degradation of ERal- pha mRNA.
  • sense strand RNA CAU CCU CUC CCA CAU CAG GCA UU, SEQ ID NO:l
  • anti-sense strand RNA UGC CUG AUG UGG GAG AGG AUG UU, SEQ ID NO:2
  • ssRNA and the asRNA (20 ⁇ M each) in annealing buffer [100 mM K acetate, 30mM HEPES-KOH (pH 7.4), 2 mM Mg acetate] were heated to 90°C for 1 minute, then allowed to cool down to room temperature. The stock solution was kept at -20°C. 5 ⁇ l of lOOnM of annealed dsRNA, ssRNA and asRNA were used in the transfection. The primary human osteoblasts were plated at passage 4 into 6-well plates. When the cells reached 70% confluency, transfection was started using the OligofectamineTM kit (Invitrogen Ltd., Basel, Switzerland) for 4 hours. The transfected cells were incubated for 24 and 48 hours before harvesting. For total RNA isolation the RNAeasy protocol (Qiagen, Basel, Switzerland) was applied.
  • the quantitative determination of the ERalpha mRNA of the present invention was per- formed using the quantitative real-time reverse transcription polymerase chain reaction (kinetic RT-PCR) approach.
  • kinetic RT-PCR quantitative real-time reverse transcription polymerase chain reaction
  • the primers forward primer 5'-GGCTGGCCCAGCTCCT-3 > , SEQ ID NO:4; reverse primer 5'- CAGATGCTCCATGCCTTTGTT-3', SEQ ID NO:5
  • the 5'-FAM and 3' TAMRA CPG labeled TaqMan Probe 5-CATCCTCTC CCACATC-AGGCACATGA-3', SEQ ID NO:6 were applied.
  • the expression of the 18S rRNA was determined using the primers (forward primer S'-CGGCTACCACATCCAAGGAA-S', SEQ ID NO:7; reverse primer 5'-GCTGGAATTACCGCGGCT-3 ⁇ SEQ ID NO:8) and a 5'NIC and 3'-TAMRA CPG labeled probe (5'-TGCTGGCACCAGACTTGCCCTC-3', SEQ ID NO:9).
  • the specific degradation of the ERalpha mRNA that was mediated by the dsRNA was monitored by calculating the arbitrary expression values normalized to 18Sr RNA using the formula
  • Table 2 demonstrates the specificity of the ERalpha receptor down-regulation 24 hours post transfection using the specific dsRNA primer pair whereas the expression of the other members of the estrogen receptor family were not changed.
  • the strength of the specificity of the mRNA degradation is apparent even after 48 hours (Table 3). Only the respective target nuclear receptor messenger RNA was degraded.
  • the selective inhibition of the ERalpha lead to an expected functional consequence as demonstrated in Table 4.
  • the enzyme alkaline phosphatase is up-regulated via estrogenic compounds (genistein, 17beta-estradiol) in osteoblasts. Accordingly, the inhibition of the ERalpha resulted to the reversal of this up-regulation as shown in Table 4. This was also reflected by the results of an alkaline phosphatase activity measurement. Inactivation of the ERalpha at the protein level could be shown in Western blot analysis using ERalpha specific antibodies. ERalpha protein has disappeared after 48 hours. Only small amounts of ERalpha were still detectable after 24 hours.
  • siRNA technique knocked down the protein expression gradually to null. After 24 hours residual ERalpha protein was still present. After 48 hours ERalpha protein expression was no longer detectable. Table 2. Specific inhibition of ERalpha mRNA in human primary cells after 24 hours.
  • the mRNAs levels of the estrogen receptors alpha, beta 1, beta 2, and beta 5 were determined after 24 hours post transfection. Human primary osteoblasts were transfected either with vehicle (annealing buffer), control ( 100 nM ERalpha ssRNA oligo) or inhibition
  • ERalpha inhibition is specific also after 48 hours.
  • mRNA levels of the ERalpha, ⁇ l, ⁇ 2, and ⁇ 5 were determined after 48 hours post transfection. Normalized arbitrary units are given. The values were calculated as in Table 2. Upper and lower limits are given in brackets. The ERalpha down-regulation did not change also after 48hours post transfection. This inhibition was specific on the tran- scriptional level.
  • siRNA (lOOnM (1.18-0.85) (1.14-0.98) (1.19-0.88) (1.39-1.13) (1.35-1.10) siRNA)

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Abstract

The present invention relates to a method of inhibiting expression of a nuclear receptor gene in a mammalian cell or tissue by introduction of a sense strand and corresponding antisense strand RNA sequence of about 21 to about 23 nucleotides complementary to a part of the receptor mRNA into such cell or tissue, to the 21 - 23 nt nucleotides themselves and corresponding transfer systems and their uses in the preparation of pharmaceutical compositions useful in the treatment of diseases caused by altered expression of the nuclear receptor gene. The invention also relates to a method of examining the function of a mammalian nuclear receptor gene in a cell or tissue.

Description

Inhibition of Nuclear Receptors
The present invention relates to the inhibition of nuclear receptors. The inactivation is achieved by introduction of an oligonucleotide sense strand and a corresponding anti- sense strand complementary to a part of the nuclear receptor messenger RNA into a mammalian cell or tissue. Inhibition of nuclear receptors is desirable where over-expression of such a receptor is leading to or responsible for a disease or deficiency state.
The nuclear receptors belong to a large family of proteins that confer cells with responsiveness to molecules which are signalling ligands, e.g., hormones, vitamins and derivatives thereof, such as glucocorticoids, estrogens, retinoids, vitamin D compounds, and thyroid hormones. These receptors mediate their signal by activating and/or repressing gene transcription directly by modulating the RNA polymerase transcription machinery.
Inhibition of nuclear receptors" expression in individuals with high susceptibility to certain diseases or physiological states related to receptor malfunction is of high importance for prevention and therapy. The nuclear receptors are cellular key players that can redirect the cell into a detrimental developmental stage in the field of hormone dependent diseases.
The classic approach to interfere the hormone deregulated pathways, cells and diseases is mainly approached by inhibiting the receptors in their activity. This is achieved by antagonizing the binding in the hormone binding cavity using inactive compounds that can bind. Such an approach involves a large screening program and the use of chemical compound libraries. Such a method is laborious and cost intensive.
The use of small single-stranded antisense phosphorothioate oligodeoxynucleotides to down-regulate the expression of genes is based on the physical and chemical properties that govern the structure of nucleic acids. The antisense approach has been used extensively in the past years, however with mixed results. The antisense theory for the ablation
Mez / 9.4.2003 of gene expression is simple and elegant, but this theory has its limits and is therefore not always applicable.
On the other hand double-stranded (ds) RNA interference (RNAi) is a biological response of cells for the protection against nucleic acid invaders like viruses and is highly potent to fight foreign gene transcripts.
Introduction of dsRNA that is longer than 30 base pairs triggers a non-specific pathway leading to the degradation of all mRNAs thus ceasing the protein synthesis of the cell. It has been found recently that using short interfering dsRNAs of from about 21 to about 23 nucleotides having sense and antisense strands which correspond to parts of receptor mRNAs are capable of specifically inhibiting or "silencing" targeted genes [WO 01/75164 and Elbashir et al., Nature 411, 494-8 (2001), contents of which are herewith explicitly incorporated by reference into the present specification] , viz. suppress specifically expression of endogenous and heterologous genes in different mammalian cell lines, e.g., genes coding for cytoskeletal proteins and reporter genes, such as genes for luciferases, via a mechanism which still remains to be uncovered.
The present invention relates to a method of inhibiting or silencing genes coding for mammalian nuclear receptors, to the use of this method in research, prevention and therapy of diseases, to the olgonucleotides used in this method and to corresponding pharmaceutical compositions.
The present invention relates also to a cell or tissue wherein two complementary RNA strands interfere with the expression of a mammalian nuclear receptor (so called knockdown cell or tissue) and it concerns a kit comprising reagents inhibiting the expression of a nuclear receptor gene in a mammalian cell or tissue.
The superfamily of nuclear receptors share a modular structure in which regions, A to F, are displayed (Evans, Science 240:889-895, 1988). The nuclear receptor is characterized by a variable N-terminal domain (A/B), followed by a centrally located, highly conserved DNA- binding domain (C), a variable hinge domain (D), a conserved ligand binding domain (E) and a variable C-terminal domain (F). The non-conserved N-terminal domain (A/B) is highly variable in size and sequence and modulates the transciption activation. The variable C-terminal domain (F) has a similar transactivation function. In addition, the F-region is also needed for ligand binding. A list of the major human nuclear receptor genes including their trivial names, accession numbers and nuclear receptors they are expressing according to NCBI Gene Bank is given in Table 1. Through differential splicing and different promoter usage a multitude of different nuclear receptor proteins can be generated. These protein variants can have different functions in the different tissues, based on their spliced sequence, ligand specificity, heteromeric partner or co-activator/ -repressor availablility. Not for all nuclear receptors a ligand is known. The nuclear receptors with known ligands receive their ligand by diffusion into the cell. The ligand is recognized by the ligand binding domain and thereby initiates an allosteric alteration. As a re- suit of this alternation the ligand/receptor complex switches to a transcriptionally active state, binds with high affinity to a hormone response element on the chromatin DNA and activates the transcription of specific target genes (Martinez and Wahli, Nuclear Hormone Receptors, chapter 6: 125 - 153, Acad. Press, 1991). All of these nuclear receptors have in common that they regulate on the transcriptional level important key pathways in embry- onic development, in adult homeostasis or in organ physiology. Various diseases and abnormalities have already been ascribed to a disturbance in these pathways like different cancers, rickets, osteoporosis and many more. It is therefore anticipated that defects like mutations in the promoter or amplifications of the gene or additional known or unknown mechanisms leading to an overexpression of the respective nuclear receptor are the un- derlying reason for a range of diseases where cellular pathways are disturbed. All nuclear receptors as listed in Table 1 are important regulators in this respect and the possibility to inactivate altered nuclear receptor expression using the small interfering RNA (siRNA) technology of the present invention generates novel methods to cure diseases.
In the context of the present invention the human estrogen receptor (ER) family and the human estrogen receptor alpha (ERalpha) is of specific interest. ERalpha regulates important processes in the reproductive tissues, in the bone system, in the cardiovascular system and in the central nervous system. Futhermore, deregulated ERalpha expression is involved in pathological states such as, e.g., cancer (R. Lindsay, D.W. Dempster and V.C. Jordan, eds., Estrogens and Antiestrogens - Basic and Clinical Aspects, Lippincott- Raven, Philadelphia, 1997).
Table 1. List of Members of the Human Nuclear Receptor Family
Table 1 List of Members of the Human Nuclear Receptor Family
Accession
Nr NR/Gene Trivial Names Number Description
01 NR0B1 DAXl. AHCH XM 010297 Nuclear receptor, adrenal hypoplasia protein
02 NR0B2 SHP NM 011850 Nuclear receptor, Small heterodimer partner
03 NR1A1 TRa, c-erbA-l, THRA XM 083987 Thyroid hormone receptor alpha
04 NR1A2 TRb, c-erbA-2, THRB XM 002986 Thyroid hormone receptor beta
05 NR1B1 RARa NM 000964 Retinoic acid receptor alpha
06 NR1B2 RARb, HAP, RRB2 XM 053323 Retinoic acid receptor beta
07 NR1B3 RARg, RARD XM 029728 Retinoic acid receptor gamma
08 NR1C1 PPARa NM 005036 Peroxisome prolieferator activated receptor alpha
09 NR1C2 PPARb, NUC1, XM 004285 Peroxisome prolieferator activated receptor beta PPARd, FAAR
10 NR1C3 PPARg XM 003059 Peroxisome prolieferator activated receptor gamma
Table 1 continued
Accession
Nr NR/Gene Trivial Names Number Description
11 NR1D1 EAR1, EAR1A, REVERBa, XM 113329 Orphan nuclear hormone receptor hRev, THRAL
12 NR1D2 EARlb, REVERBb, L31785 Orphan nuclear hormone receptor BD73, RVR, HZF2
13 NR1F1 RORa, RZRa NM 002943 RAR-related orphan receptor (ROR) alpha, Nuclear receptor R alpha 14 NR1F2 RORb, RZRb XM 005384 RAR-related orphan receptor (ROR) beta, Nuclear receptor RZ beta
15 NR1F3 RORg, RZRg, NM 005060 RAR-related orphan receptor (ROR) gamma, Nuclear receptor RORc, TOR gamma
16 NR1H2 LXRb, UNR, XM 046419 Oxysterol receptor LXR-beta, Liver X receptor beta NER1, RIP15
17 NR1H3 RLDl, LXR, LXRa NM 005693 Oxysterol receptor LXR-alpha, Liver X receptor alpha
17 NR1H4 FXR, RIP14, HRR1 NM 005123 Farnesol receptor
19 NR1I1 VDR XM 007046 Vitamin D receptor, l,25(OH)2D3 receptor
20 NR1I2 PXR, SXR, BXR, ONR1, XM 113407 Pregnan X receptor, Steroid activated receptor, Orphan nuclear PAR, PRR, SAR, PARq eptor PAR1
Table 1 continued
Accession
Nr NR/Gene Trivial Names Number Description
21 NR1I3 CAR1, CARa MB67, XM 042458 Constitutive androstane receptor, Nuclear receptor interacts w RAREs
22 NR2A1 HNF4, HNF4A, TCF14 X76930 Hepatocyte nuclear factor 4, hepatic nuclear factor 4 alpha,
23 NR2A2 HNF4G XM 005182 Hepatocyte nuclear factor 4 gamma transcription factor 14
24 NR2B1 RXRA XM 011778 Retinoic acid-like receptor alpha, Retinoic X receptor
25 NR2B2 RXRB, H-2RIIBP, XM 042579 Retinoid X receptor beta RCoR-1
26 NR2B3 RXRG XM 053680 Retinoid X receptor gamma
27 NR2C1 TR2, TR2-11 NM 003297 TR2 orphan receptor, Steroid receptor TR2-11 28 NR2C2 TR4. TAK1, XM 042906 TR4 orphan receptor HTAK1, TR2R1
29 NR2E3 PNR, RNR NM 016346 Photoreceptor-specific nuclear receptor, retina specific nuclear tor
30 NR2F1 COUP-TFI, COUPTFA, NM 005654 Chicken ovalbumin upstream promoter transcription factor, v EAR3, SVP44, COTl related ear3 31 NR2F2 COUP-TFII, NM 021005 Chicken ovalbumin upstream promoter transcription factor II
COUPTFB, ARP1, SVP40 Apolipoprotein Al regulatory protein
Table 1 continued
Accession
Nr NR/Gene Trivial Names Number Description
32 NR2F6 EAR2 NM 005234 v-erbA related ear-2
33 NR3A1 ERa, ESR1 XM 045967 Estrogen receptor alpha
34 NR3A2 ERb, ESR2 NM 001437 Estrogen receptor beta
35 NR3B1 ERR1, ERRa, XM 048286 Estrogen-related receptor alpha, Estrogen-like receptor ESRL1, ESRRA
36 NR3B2 ERR2, ERRb, ESRRB XM 041087 Estrogen-related receptor beta, Estrogen-like receptor
37 NR3B3 ERR3, ERRg, ESRRG XM 039053 Estrogen-related receptor gamma, Estrogen receptor related protein 3
38 NR3C1 GR; GRL, GCR NM 000176 Glucocorticoid receptor
39 NR3C2 MR, MLR, MCR NM 000901 Mineralocorticoid receptor
40 NR3C3 PR, PGR NM 000926 Progesterone receptor
41 NR3C4 AR, DHTR, KD, XM 010429 Androgen receptor, Dihydroxytestosterone receptor AIS, TFM 42 NR4A1 NGFIB, TR3, N10, XM 083884 Orphan nuclear receptor HMR, Early response protein NAK1
NUR77, NAK1, HMR
Table 1 continued
Accession Nr NR/Gene Trivial Names Number Description 43 NR4A2 NURRl. NOT. RNRl, NM 006186 Orphan nuclear receptor NURR1 HZF-3, TINOR
44 NR4A3 NORl. CHN. CSMF, XM 037370 Neuron-derived orphan receptor 1, Mitogen induced nuclear MINOR orphan receptor
45 NR5A1 SF1, ELP, FTZ-F1, NM 004959 Steroidogenic factor 1, fushi tarazu factor homolog 1, Steroid AD4BP FTZ1 hormone receptor AD4BP
46 NR5A2 FTF, LRH1, B1F, XM 036634 Orphan nuclear receptor NR5A2 CPF, FFLR B1F2, HB1F, HB1F-2, PHR,
47 NR6A1 GCNF, GCNF1, RTR XM 056232 Germ cell nuclear factor, Retinoid receptor-related testis recep
The present invention relates to a method of inhibiting expression of a receptor gene in a mammalian cell or tissue by introduction of a sense strand and corresponding antisense strand RNA sequence of about 21 to about 23 nucleotides complementary to a part of the receptor mRNA into such cell or tissue characterized in that the receptor mRNA is a mammalian nuclear receptor mRNA.
The term "inhibiting expression" comprises all degrees of inhibition of expression of a gene in a mammalian cell or tissue, especially of altered expression and particularly of over-expression, from partial to complete inactivation (down-regulation), depending on the target gene and the intracellular dose of siRNA.
The inhibition of the target gene expression is reflected in a loss of phenotype. The degree of inhibition may be estimated by comparing the values from untreated cells or tissue with those from treated cells or tissue according to the method of the present invention. The consequences of inhibition may be assayed for properties of the cell or tissue by molecular biology methods such as real-time quantitative reverse transcription polymerase chain re- action (RT-PCR), RNA solution hybridization, Northern hybridization and biochemical assays like enzyme linked immunoabsorbent assay (ELISA), Western blotting or radioim- munoassay (RIA), for example.
The term "mammalian cell or tissue" relates on the one hand to all mammals, including and especially to human beings, and on the other hand to all types of cells or tissues in which expression of a mammalian nuclear receptor gene takes place or can be effected. The cells or tissues may be, e.g., from the vascular or extravascular, the blood or lymph system, from muscles, any organ, gland, the skin, brain or cerebrospinal fluid.
The term "introduction" comprises all means and methods which are known to be effective, generally and specifically, to incorporate genetic material into cells and tissues in a way that the genetic material will become effective in the desired way. The term "introduction", therefore comprises the methods of transformation (uptake of naked genetic material), conjugation (exchange of genetic material from cell to cell) and transfection or transduction, using plasmids or non-lethal viruses as vectors, or using liposomal technology. The short interfering (si) RNA sense and anti-sense oligonucleotide sequences which are complementary to a specific part of the targeted nuclear receptor gene can be introduced either in double-stranded (ds) form, using, e.g., plasmids or liposomes or in single- stranded (ss) form, e.g., using viruses carying the ss, viz. sense and antisense oligonucleotide sequences separately. A well-known group of non-lethal viruses which can be used as transfer vectors or from which transfer vectors have been or can be derived are alphaviruses, such as Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Equine Encephalitis virus (WEE), Sindbis virus (SIV), South African Arbovirus No. 86, Semliki Forest virus (SFV), Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Ge- tah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. Preferred alphaviruses include SFV (Liljestrom and Garoff, Bio/Technology 9, 1356-1361 1991; A new generation of animal cell expression vectors based on the SFV replicon), SIV (Xiong et al., Science 243, 1188-1191, 1989; Sindbis virus: an efficient broad host range vector for gene expression in animal cells), and VEE (Davis et al., Virology 171, 189-204 1989; In vitro synthesis of infectious Venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant), for exam- pie. Most alphaviruses and vectors are commercially available. Infection procedures are also well-known in the art (see, e.g., Strauss, J.H. and Strauss, E.G., Microbiological Reviews 58, 491 - 562, 1994; The Alpaviruses: Gene Expression, Replication, and Evolution)
Expression plasmids which can be used as vectors are also well-known in the art and are described as well as methods of transfection, e.g., in Colosimo et al. (Bio Techniques, 29:314-331, 2000). The plasmids express the short oligonucleotide sequences either individually or in a single transcript that forms secondary structures (e.g. a hairpin structure). The expression plasmids can be introduced stably or transiently to generate the down- regulation. Modifications and specific improvements of RNA expression systems mentioned above are also well known in the art (e.g. expression cassettes for short RNA tran- scription, multiple copies on the plasmid) and can be used to work the present invention. Many expression plasmids are commercially available.
The same applies for liposomes which are excellent transfer systems. Such liposomes are described, e.g. by Elbashir et al. (Methods, 26:199-213, 2002).
The terms "vector", "transfer vector" and "transfer system" are interchangeable and all mean a construct or particle which is used for the introduction of genetic material in mammalian cells or tissue.
In connection with the present invention the preferred liposome reagent which has been used is the commercially available Oligofectamine . The reagent has been used as described by the manufacturer (Invitrogen Ltd., Basel, Switzerland.) The term "complementary" refers to nucleic acid sequences that can form hydrogen bonds with specific RNA sequences by either traditional Watson-Crick or other, non-traditional types (e.g. Hoogsteen type) of base-paired interactions, thus leading to degradation of the targeted gene's mRNA. The term complementary, therefore, comprises sequences which are identical (100% homolog) to the targeted gene mRNA or analogues of such sequences which differ by one or more nucleotides, preferably by 1 -3 nucleotides, from such sequences by addition, deletion, substitution or alteration.
In the practice of the present invention the RNA sequences of about 21-23 nucleotides can be identical with or complementary to a n y part of the mRNA of the targeted nuclear re- ceptor gene. It is evident to the person skilled in the art that the specificity of the inhibition of a specific receptor will depend within certain limits on the sequence differences within a gene family that may code for a certain receptor. Normally, highest specificity will be obtained with RNA sequences complementary to parts of a mRNA which is expressed from a highly variable domain.
The term "sense" and "antisense" in connection within "strand" or "sequence" are used in the way generally understood by every person skilled in the art, viz., a sense RNA sequence is identical with a sequence transcribed and processed from the gene yielding the mRNA. The antisense strand is again complementary to a sense strand.
The 21 - 23 nucleotide (nt)RNA molecules can be single-stranded or double stranded (as two 21-23 nt RNAs); such molecules can be blunt ended or comprise overhanging ends (e.g., 5', 3'). In a specific embodiment, the RNA molecule is double stranded and either blunt ended or, preferably, comprises overhanging ends (as two 21-23 nt RNAs).
In one embodiment, at least one strand of the RNA molecule has a 3' overhang from about 1 to 6 nucleotides (e.g., pyrimidine nucleotides, purine nucleotides) in length. In other embodiments, the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from 2 to about 4 nucleotides in length. In one embodiment the RNA molecule is double stranded, one strand has a 3' overhang and the other strand can be blunt-ended or have an overhang. In the embodiment in which the RNA molecule is double stranded and both strands comprise an overhang, the length of the overhangs may be the same or different for each strand. In a particular embodiment, the RNA of the present invention comprises 21 nucleotide strands which are paired and which have overhangs of from about 1 to about 3, particularly about 2, nucleotides on both 3' ends of the RNA. In order to further enhance the stability of the si RNA of the present invention, the 3' overhangs can be stabilized against degradation. In one embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides and derivatives thereof. In another embodiment the RNA is stabilized by pyrimidine nucleotides, such as uridine, thymidine and cytosine nucleotides and derivatives thereof. The about 21 - 23 nt RNA molecules of the present invention (single stranded sense and anti-sense molecules, including analogues and overhangs, as well as ds molecules) can be prepared and purified using a number of techniques well-known to those skilled in the art, e.g., by recombinant methods or by chemical synthesis, making use of common nucleic acid protecting and coupling groups (see, e.g., Caruthers et al., 1992, Methods in Enzymology 211, 3-19 and Elbashir et al., above). The introduction of the ss or ds 21 - 23 nt RNA into the transfer vectors is also effected by methods well-known in the art.
A recent development is the use of the TOM-Protecting-Group™ (2' - O-triisopropylsilyl- oxy-methyl protecting group) which is structurally related to the tBDMS-group, introduced by Ogilvie and Usman in the early eighties for the protection of the 2'-OH groups in the chemical synthesis of RNA. The TOM-group is fully compatible with the established tBDMS chemistry and has several important advantages (e.g. coupling yields >99.5% with < 2 minutes coupling time due to less sterical hindrance and short, reliable and complete deprotection under mild conditions.
The invention also relates to the new nucleotide sequences used in the method of the present invention, viz. to those ss RNA sequences which are identical with sequences of about 21 to 23 nucleotides of a mammalian nuclear receptor mRNA, complementary sequences and analogues thereof which analogues differ by addition, deletion, substitution or altera- tion of one or more, preferably 1 - 3, nucleotides and to corresponding ds RNA sequences and to their uses
- in the preparation of pharmaceutical compositions for the treatment of diseases caused by altered expression of a mammalian nuclear receptor gene
- in the preparation of kits of reagents inhibiting the expression of mammalian nuclear receptor genes and
- in a method of examining the function of a mammalian nuclear receptor gene.
The invention also relates to transfer systems containing the above-mentioned nucleotide sequences and to their uses
- in the preparation of pharmaceutical compositions for the treatment of diseases caused by altered expression of a mammalian nuclear receptor gene
- in the preparation of kits of reagents inhibiting the expression of mammalian nuclear receptor genes and
- in a method of examining the function of a mammalian nuclear receptor gene.
The present invention also relates to a method of treatment of a disease caused by over- expression of a nuclear receptor gene in a mammalian cell or tissue characterized in that dsRNA of about 21 to 23 nucleotide pairs targeting the mRNA of this nuclear receptor for degradation is administered to the mammal in the form of a pharmaceutical composition comprising ss or ds RNA strands, and to these pharmaceutical compositions themselves.
The present invention finally relates to a kit comprising reagents inhibiting expression of a nuclear receptor gene in a mammalian cell or tissue, said kit containing at least a sufficient amount of vectors comprising a sense strand and a corresponding anti-sense strand RNA sequence of about 21 to about 23 nucleotides complementary to a part of a mammalian nuclear receptor mRNA and to a knockdown cell or tissue generated by a method of the present invention.
A kit may include additional reagents to carry out the in vivo or in vitro delivery of the about 21 to 23 nt molecules to the samples or subjects and may also include instructions to allow a user of the kit to practice the invention.
The method of examining the function of a mammalian nuclear receptor gene in a cell or tissue comprises:
(a) introducing a sense strand and a corresponding anti-sense strand RNA sequence of about 21 to about 23 nucleotides that targets mRNA of the gene for degradation into the cell or tissue, thereby producing a test cell or test tissue,
(b) maintaining the test cell or test tissue under conditions under which degradation of mRNA of the gene occurs, thereby producing a test cell or test tissue in which mRNA of the gene is degraded,
(c) observing the phenotype of the test cell or test tissue produced in (b) and, optionally, comparing the phenotype observed to that of an appropriate control cell or control tissue, thereby providing information about the function of the gene.
The term "test cell or test tissue in which mRNA of the gene is degraded" used above is identical with the term "knock-down cell or tissue" which latter term is widely used in the art.
The ss and ds RNA molecules of the present invention can be used as pharmaceutical agents in pharmaceutical compositions to treat disease states originating from altered ex- pression of one or more mammalian nuclear receptor genes and can be administered to or introduced into a mammal/patient by any standard means.
The pharmaceutical compositions comprise formulations for all kinds of delivery, e.g., oral, nasal, rectal, intravenous, subcutaneous, intramuscular, or intraperitoneal admini- stration and include all kinds of pharmaceutically acceptable excipients known to be useful in such compositions. Such excipients comprise, e.g., antioxidants, preservatives, stabilizers, especially additives which protect the active molecules from degradation by nu- cleases, dyes, flavoring agents and carriers.
A pharmaceutically effective dose is the dose required to prevent, inhibit the occurrence of or treat a disease state. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristic of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is to be administered.
Example
The inhibition of expression of nuclear ERalpha is achieved by introduction of a short sequence-specific double stranded RNA into primary osteoblasts via degradation of ERal- pha mRNA.
Based on the sequence of the human mERalpha gene two complementary oligonucletides, (viz. sense strand RNA: CAU CCU CUC CCA CAU CAG GCA UU, SEQ ID NO:l, and anti-sense strand RNA: UGC CUG AUG UGG GAG AGG AUG UU, SEQ ID NO:2) including two uridine overhangs were selected, synthesized and HPLC/dialysis-purified in accordance within known methods (Mycrosynth, Switzerland). The ssRNA and the asRNA (20μM each) in annealing buffer [100 mM K acetate, 30mM HEPES-KOH (pH 7.4), 2 mM Mg acetate] were heated to 90°C for 1 minute, then allowed to cool down to room temperature. The stock solution was kept at -20°C. 5μl of lOOnM of annealed dsRNA, ssRNA and asRNA were used in the transfection. The primary human osteoblasts were plated at passage 4 into 6-well plates. When the cells reached 70% confluency, transfection was started using the Oligofectamine™ kit (Invitrogen Ltd., Basel, Switzerland) for 4 hours. The transfected cells were incubated for 24 and 48 hours before harvesting. For total RNA isolation the RNAeasy protocol (Qiagen, Basel, Switzerland) was applied.
The quantitative determination of the ERalpha mRNA of the present invention was per- formed using the quantitative real-time reverse transcription polymerase chain reaction (kinetic RT-PCR) approach. Using the ABI sequence detection system 7700 from Applied Biosystems, Rotkreuz, Switzerland, using TaqMAN™ technology, the primers (forward primer 5'-GGCTGGCCCAGCTCCT-3>, SEQ ID NO:4; reverse primer 5'- CAGATGCTCCATGCCTTTGTT-3', SEQ ID NO:5) and the 5'-FAM and 3' TAMRA CPG labeled TaqMan Probe (5-CATCCTCTC CCACATC-AGGCACATGA-3', SEQ ID NO:6) were applied. As an internal control the expression of the 18S rRNA was determined using the primers (forward primer S'-CGGCTACCACATCCAAGGAA-S', SEQ ID NO:7; reverse primer 5'-GCTGGAATTACCGCGGCT-3\ SEQ ID NO:8) and a 5'NIC and 3'-TAMRA CPG labeled probe (5'-TGCTGGCACCAGACTTGCCCTC-3', SEQ ID NO:9). The specific degradation of the ERalpha mRNA that was mediated by the dsRNA was monitored by calculating the arbitrary expression values normalized to 18Sr RNA using the formula
2-[Ct(18S RNA)-Ct(ERalpha)] -| -8.
Table 2 demonstrates the specificity of the ERalpha receptor down-regulation 24 hours post transfection using the specific dsRNA primer pair whereas the expression of the other members of the estrogen receptor family were not changed. The strength of the specificity of the mRNA degradation is apparent even after 48 hours (Table 3). Only the respective target nuclear receptor messenger RNA was degraded.
The selective inhibition of the ERalpha lead to an expected functional consequence as demonstrated in Table 4. The enzyme alkaline phosphatase is up-regulated via estrogenic compounds (genistein, 17beta-estradiol) in osteoblasts. Accordingly, the inhibition of the ERalpha resulted to the reversal of this up-regulation as shown in Table 4. This was also reflected by the results of an alkaline phosphatase activity measurement. Inactivation of the ERalpha at the protein level could be shown in Western blot analysis using ERalpha specific antibodies. ERalpha protein has disappeared after 48 hours. Only small amounts of ERalpha were still detectable after 24 hours.
"Knock-out" of ERalpha protein in human OB cells using siRNA.
Cells were lysed and total protein (20μg/lane as determined by the BCA protein assay reagent kit, Pierce Rockford, IL, USA) was separated by SDS-polyacrylamide gel electropho- resis, transferred to Immobilon P membrane (Millipore, Bedford, MA, USA) and probed with an 1:500 diluted antibody against human ERalpha (D-12) from Santa Cruz, CA, USA. The control antibody recognized the actin protein (Sigma, Buchs, CH) in a 1:2000 dilution, which was used to normalize the samples. Detection was monitored by chemilumi- nescence using the ECL+ ™ reagent from Amersham Pharmacia Biotech, Buckinghamshire, UK. The siRNA technique knocked down the protein expression gradually to null. After 24 hours residual ERalpha protein was still present. After 48 hours ERalpha protein expression was no longer detectable. Table 2. Specific inhibition of ERalpha mRNA in human primary cells after 24 hours.
ERalpha ERβl ERβ2 ERβ5
(x lO"8) (x lO'8) (x lO"8) (x lO"8)
Vehicle 60.0 3.8 17.2 195
(78.6-53.1 ) (4.79-2.59) (2.02-1.32) (247-151)
Control 58.3 3.33 13.8 180.4
(lOOnM ssRNA) (80.6-42.2) (4.66-2.38) (1.72- 1.1) (239-136)
Inhibition 0.38 4.58 13.1 132.5
(lOOnM siRNA) (0.42-0.30) (5.29 3.96) (19.3- 9.5) (165-106 x)
The mRNAs levels of the estrogen receptors alpha, beta 1, beta 2, and beta 5 were determined after 24 hours post transfection. Human primary osteoblasts were transfected either with vehicle (annealing buffer), control ( 100 nM ERalpha ssRNA oligo) or inhibition
( lOOnM siRNA) for ERalpha. The mRNA was quantitated using kinetic multiplex RT-PCR (TaqMan). The multiplex real-time quantitative RT-PCR values are defined using the formula 2 exp-(Ct,nuciear receptor- , ISS ΓRNA). where the is the cycle threshold (Ct) of either the nuclear receptor (ERalpha, ERβl, 2, 5) or the 18S rRNA internal control. The upper limit is Calculated USing the formula 2 eXp-((Ct,nuclear receptor- SDCt,nuclear receptor)" (Ct, 18S rRNA
+SDCt,i8SrRNA)) x 10~8, where SD is the standard deviation. The lower limit is calculated
USing the formula 2 exp-( (Ct,nuclear receptor + SDα,nuclear receptor)" ( , l8S rRNA - SDCt,18SrRNA))-
Only ERalpha was regulated, whereas ERβl, β2, and β5 were not altered in their expression. ERβ3 and ERβ4 expression was not detectable neither in the non-inhibited control nor in the inhibited samples. Table 3. ERalpha inhibition is specific also after 48 hours.
ERalpha ERβl ERβ2 ERβ5
(x lO'8) (x lO"8) (x lO-8) (x lO"8)
Vehicle 52.8 3.2 16.4 225.3
(64.1 -47.3) (3.99-2.54) (21.8-11.7) (263-183)
Control 47.4 2.5 18.2 157.5
(lOOnM ssRNA) (81.1-27.6) (3.27-1.95) (32.8-10.1 ) (239-103)
Inhibition 0.23 2.2 13.5 215.6
(lOOnM siRNA) (0.4- 0.12) (3.16-1.66) (21.2-8.6) (361 -128)
The mRNA levels of the ERalpha, βl, β2, and β5 were determined after 48 hours post transfection. Normalized arbitrary units are given. The values were calculated as in Table 2. Upper and lower limits are given in brackets. The ERalpha down-regulation did not change also after 48hours post transfection. This inhibition was specific on the tran- scriptional level.
Table 4. Inducible alkaline phosphatase mRNA expression is down-regulated after ERalpha inhibition.
Pre-treat- Vehicle lOnM 500nM lOOOnM lOnM 17β- ment (DMSO) Genistein Genistein Genistein Estradiol
(48h)
Control l (per def.) 1.18 2.06 2.17 2.30
(lOOnM (1.07-0.93) (1.23-1.14) (2.37-1.79) (2.28-1.80) (2.45-2.16) ssRNA)
Inhibition l (per def.) 1.06 1.02 1.29 1.23
(lOOnM (1.18-0.85) (1.14-0.98) (1.19-0.88) (1.39-1.13) (1.35-1.10) siRNA)
Genistein and the natural estrogen induce alkaline phosphatase (ALP). Expression was determined after 24h treatment with the estrogenic compounds genistein and 17β-estra- diol. Primary human osteoblast cells have been pre-treated with either ssRNA (hERalpha) or siRNA (hERalpha). All determinations were done in triplicate in a TaqMan multiplex RT-PCR assay. Values are given as fold changes from vehicle control +/- upper and lower limits.

Claims

Claims
1. A method of inhibiting expression of a receptor gene in a mammalian cell or tissue by introduction of a sense strand and corresponding antisense strand RNA sequence of about 21 to about 23 nucleotides complementary to a part of the receptor mRNA into such cell or tissue characterized in that the receptor mRNA is a mammalian nuclear receptor mRNA.
2. A method as claimed in claim 1 wherein the two RNA sequences are introduced by infection of the cell or tissue with a viral vector.
3. A method as claimed in claim 1 wherein the two RNA sequences are introduced by transfection of the cell or tissue with an expression plasmid.
4. A method as claimed in claim 1 wherein the two RNA sequences are introduced by transfection of the cell or tissue using liposomal technology.
5. A method as claimed in any one of claims 1 - 3 wherein the nuclear receptor is the estrogen receptor.
6. A method as claimed in claim 4 wherein the sense strand RNA sequence is represented by SEQ ID No. 1 and the anti-sense strand RNA sequence is represented by SEQ ID No. 2.
7. A method as claimed in any one of claims 1 - 4 wherein the sense strand and anti-sense strand RNA sequences are analogues of the sequences represented by SEQ ID Nos. 1 and 2 which analogues differ by addition, deletion, substitution or alteration of one or more, preferably 1 - 3, nucleotides.
8. A method as claimed in any one of claims 1 - 6 to be used for the down-regulation of over-expression of a nuclear receptor gene.
9. A method of treatment of a disease caused by over-expression of a nuclear receptor gene in a mammalian cell or tissue characterized in that dsRNA of about 21 to 23 nucleotide pairs targetting the mRNA of this nuclear receptor for degradation is administered to the mammal in the form of ss or ds strands.
10. A knockdown cell or tissue generated by a method as claimed in any one of claims 1-6.
11. The use of a sense strand together within a corresponding anti-sense strand RNA sequence of about 21 to about 23 nucleotides complementary to a part of a mammalian nu- clear receptor mRNA for the preparation of a pharmaceutical composition useful in the treatment of diseases caused by altered expression of the nuclear receptor gene.
12. The use of a transfer system containing a sense strand and a corresponding anti-sense strand RNA sequence of about 21 to about 23 nucleotides complementary to a part of a mammalian nuclear receptor mRNA for the preparation of a pharmaceutical composition useful in the treatment of diseases caused by altered expression of the nuclear receptor gene.
13. A pharmaceutical composition comprising a sense strand and a corresponding anti- sense strand RNA sequence of about 21 to about 23 nucleotides complementary to a part of a mammalian nuclear receptor mRNA and optionally pharmaceutically acceptable excipients.
14. A method of examining the function of a mammalian nuclear receptor gene in a cell or tissue comprising:
(a) introducing a sense strand and a corresponding anti-sense strand RNA sequence of about 21 to about 23 nucleotides that targets mRNA of the gene for degradation into the cell or tissue, thereby producing a test cell or test tissue,
(b) maintaining the test cell or test tissue under conditions under which degradation of mRNA of the gene occurs, thereby producing a test cell or test tissue in which mRNA of the gene is degraded,
(c) observing the phenotype of the test cell or test tissue produced in (b) and, optionally, comparing the phenotype observed to that of an appropriate control cell or control tissue, thereby providing information about the function of the gene.
15. A kit comprising reagents inhibiting expression of a nuclear receptor gene in a mammalian cell or tissue, said kit containing at least a sufficient amount of vectors comprising a sense strand and a corresponding anti-sense strand RNA sequence of about 21 to about 23 nucleotides complementary to a part of a mammalian nuclear receptor mRNA.
16. A nucleotide sequence which is identical with a sequence of about 21 to 23 nucleotides of a mammalian nuclear receptor mRNA, complementary sequences and analogues thereof which analogues differ by addition, deletion, substitution or alteration of one or more, preferably 1 - 3, nucleotides.
17. A nucleotide sequence represented by anyone of SEQ ID Nos. 1 to 3 or by analoguous sequences thereof which analogues differ by addition, deletion, substitution or alteration of one or more, preferably 1 - 3, nucleotides per strand.
18. The ss nucleotide sequence represented by SEQ ID No. 1
19. The ss nucleotide sequence represented by SEQ ID No. 2
20. The ds nucleotide sequence represented by SEQ ID No. 3
21. A transfer system containing a sense strand and/or a corresponding anti-sense strand RNA as claimed in anyone of claims 16-20.
22. The invention substantially as described hereinbefore especially with reference to the foregoing Example.
PCT/EP2003/006183 2002-06-20 2003-06-12 Inhibition of nuclear receptors WO2004001045A1 (en)

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Citations (2)

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