Classifications

• • 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

Definitions

• the present invention is directed to antisense oligonucleotides, specifi ⁇ cally to antisense oligonucleotides blocking the synthesis of the human progesterone receptor (hPR).
• hPR human progesterone receptor
• antiprogestativa in -.general ac in the following manner.
• they When administered to normal women in the follicular phase of the menstrual cycle, they prevent ovulation by blocking progesterone action at the level of pituitary or hypothalamus: this suppresses the midcycle surge of gonadotropins and delays follicular development.
• progesterone In the luteal phase of the cycle, they act directly on the uterus to block the action of progesterone, resulting in release of prostaglandins from the endometrium and subsequent menstrual bleeding.
• they can terminate early pregnancy by facilitating luteolysis, menstruation, uterine motility, and detachment of the embryo.
• aON antisense oligonucleotides
• an object of the present invention to provide an aON comprising a nucleo tide sequence which is characterized by its ability to hybridize under stringent hybridization conditions to a part of the mRNA sequence of the human progesterone receptor and further characterized by its ability to block the synthesis of the human progesterone receptor or more specifically such an aON which is not longer than 100 nucleotides, preferentially which is 10 to 50 nucleotides long.
• a furthermore preferred aO is such an aON as mentioned above whereby the part of the mRNA sequence of the human progesterone receptor has the following nucleotide sequence:
• an aON comprising the following nucleotide sequence:
• a furthermore preferred aON is an aON as mentioned above whereby t part of said mRNA sequence of the human progesterone receptor has the nucleotide sequence which corresponds to nucleotides 641-701 of the corresponding cDNA sequence.
• NH3 is preferred to create such alkaline conditions and which aONs are if desired, accordingly modified.
• Figure 1 shows the effect of the aON of Example 1 on the level of expression of the hPR of T47D-(A) and on MCF7 cells (B).
• Spotted boxes refer t the experiment performed as described in Example 2 in the presenc of the aON and open boxes to this experiment without the aON ("control"), whereby “II” refers to such experiments made in the presence of ⁇ -estradiol and “I” to such experiments without the presence of ⁇ -estradiol.
• Data shown represent means +/- standard deviation of means indicated by a bar on top of the boxes of a*- minimum of 5 single determinations with a probability "p" ⁇ 0.05 o control (*) and "p" ⁇ 0.01 of control (**).
• Figure 2 shows the dose dependent effect of the aON of Example 1-on the level of expression of the hPR on T47D cells whereby "control" has the same significance as given for Figure 1 and the different doses are indicated as concentrations (1.5 ⁇ M up to 12 uM) of the aON used.
• Figure 3 shows the antagonistic affect of the aON of Example 1 on progesterone-induced estrogen receptor (ER) down regulation.
• Spotted boxes refer to the experiment performed as described in Example 3 in the presence of the aON, shadow boxes refer to progesterone treatment and open boxes are controls (no adition o aON nor hormone).
• the data shown represent means +/- standar deviation indicated by a bar on top of the boxes of a minimum of single dete ⁇ ninations with a probability "p" ⁇ 0.01 of control (**) a "p" ⁇ 0.05 of progesterone treatment at same time ( ⁇ ) as determine by the one-way ANOVA statistical test using the "Epistaf'-progra (available from Tracy L. Gustafson, M.D., 1705 Galtis School Road, Round Rock, Texas 78664, USA).
• aONs of the present invention can be designed on the basis of the know mRNA sequence of the hPR [Misrahi et al., Biochem. Biophys. Res. Comm. 143, 740-748 (1987)] whereby regions like splicing sites, ribosomal binding sites sites for the binding of regulatory proteins, parts of the mRNA responsible fo the formation of secondary or tertiary structure of the mRNA or polyadenylation sites, if present and specifically the site aroimd the start codo are preferred.
• General considerations with respect to the design of oligo ⁇ nucleotides and especially regarding the stringency of hybridization condition can be found in the state of the art, e.g. in Sambrook et al.
• the maximal length of such a sequence can be determined also by suc a person according to considerations known in the art regarding sufficient penetration efficiency through cell membranes [Loke et al., PNAS 86, 3474-347 (1989); Yakubov et al., PNAS 86, 6454-6458 (1989)].
• a specific sequence for an aON of the present invention has been defined according to the criteria mentioned above, such aON can be synthesized in liquid or solid phase by methods known in the art and describe for example by Narang [Tetrahedron 39, 3 (1983)], Itakura et al. [Ann. Rev. Biochem.
• such aONs can be purified by methods known in the art, e.g. by polyacrylamide gel electrophoresis under denaturing conditions or by reverse phase chromatography on silica gel columns, as described for example in Sambrook et al. (see above).
• the aONs of the present invention can be characterized by their ability of blocking the synthesis of the hPR, e.g. by blocking the translation of the mRN of the hPR, e.g. via hybridization of an aON of the present invention to the mRNA.
• Blocking of translation can be determined by any assay known in the art directed to translation of mRNA, e.g. in vitro translation of mRNA in wheat gerin extracts or reticulocyte lysates (see e.g. Sambrook et al.) or by injection into frog oocytes [see e.g. Kawasaki, E.S., Nucleic Acid Res. 1 4991 (1985)].
• mRNA has to be isolated from specific tissues known to produce high amounts of hPR by methods known in the art (see e.g. Sambroo et al.), translated in these assays and the blocking of translation measured by a reduced synthesis rate of the translation product.
• Such measurement can be performed according to any method used for the detection of a specific protein especially a method used for the detection of the binding of a ligand to a receptor, whereby enzyme immun-linked assays as e.g. specifically described in Example 2 are preferred.
• Blocking of hPR synthesis by aONs of the present invention can be determined also in cultures of cells which are known for their capability of synthesizing hPR at high concentrations, e.g. cultures of cell lines 'T47D" (ATCC No. HTB 133) or “MCF7" (ATCC No. HIN 22) and as specifically described, e.g. in Example 2. More significantly blo king of hPR synthesis can be determined by using the same cell culture assays however, under conditions wherein the hPR synthesis is induced by estrogen [see e.g. Horwitz and McGuire, J. Biol. Chem. 253, 2223 (1978); Eckert and Katzenellenbogen, Cancer Res.
• cell culture assays are preferred for the detei ination of the blocking of hPR synthesis since such assays are much closer to physiological conditions as in vitro translation assays mentioned above.
• aONs of the present invention can be characterized in in vivo models, e.g. by injection into the ventromedial hypothalamus of estrogen-primed female rats and analyzing the resulting change in sexual behaviour and thereby demonstrating a counter action of the physiological action of progesterone.
• aONs of the present invention can be modified in order to improve their desired properties [for a general review also with respect to general considerations regarding the design of antisense oligonucleotides for the purpose of blocking the synthesis of a specific protein see Uhlmann, E. and Peymann, A., Chem. Reviews 90, 543-584 (1990)].
• aONs can be chemically modified at the phosphodiesterbond and/or at the 3'-te ⁇ ninal ends in order to render them more stable with respect to DNAse breakdown or to improve their penetration- or hybridizatio properties as described for example in the European Patent Application, Publ. No. 386563.
• aONs can be covalently linked to a lipid as described for example in the international patent application with the publication number WO/9010448.
• DNA can be more efficiently transported across cell membranes and cleaved, e.g. by membrane-bo und intracellular cytoplasmic enzymes, to release the active aON.
• aONs can also be phosphorylated or linked to cholesterine or derivatives of cholesterol a the 3'end [Letsinger et aL, PNAS, 86, 6553-6556 (1989)].
• RNAse H When aONs are modified in order to render them more stable against DNAse breakdown or for better uptake into cells as mentioned above, care has to be taken that such modified aONs still hybridize well to their specific target mRNA and that ENA bo und in these complexes can be easily degraded by RNAse H.
• aONs of the present invention can be modified in order to ensure the specific delivery of the drug to progesterone receptor synthesizing tissues and, therefore, limiting the dispersion of the molecule administered.
• the aONs of the present invention may be administered in pharma ⁇ ceutically acceptable forms, especially in a form suitable for oral application. Dosage forms and dose rates may parallel those currently being used in clinic applications of known compounds of comparable structure.
• Pharmaceutical compositions may contain at least one aON of the present invention in association with pharmaceutically acceptable solid or liquid carrier materials. Any conventionally used carrier material can be utilized.
• the pharmaceutical compositions may contain other pharmaceutically active agents and be prepared by methods known in the art.
• aONs of the present invention and their modifications for the preparation of such pharmaceutical compositions and for a diagnostic or medical purposes, e.g. for the termination of a pregnancy or as an anticanc agent [see e.g. Clarke and Sutherland, Endocrine Reviews ⁇ , 266-301 (1990)] a also an object of the present invention.
• aONs of the present invention can be used when fixed on a solid support according to methods known in the art for the isolation or the oligonucleotides itself for the detection of hPR-mRNA.
• aONs of the present invention can be labeled with a signalling moiety, e.g. a radioactive isotope, an enzyme, or a fluorescent compound according to methods known in the art.
• the aON with the following nucleotide sequence was synthesized pn a DNA synthesizer (Milligen/Biosearc 7500) according to the instructions of the manufacturer:
• MCF7- or T47D-cells were cultured in RPMI "1640" medium [Pool Bioanalysis Italiana, Milano, Italy] containing 10% fetal calf serum (FCS at a density of 2-5X10 5 cells/well.
• FCS fetal calf serum
• the medium was replaced by an RPMI 1640 medium without phenol red [ICN Biomedical Ltd., Bucks, GB, Catalog No. 07312854500] and 10% dextran charcoal-stripped FCS with and without 10" 8 M ⁇ -estradiol and 20 ⁇ g of an aON as prepared in Example 1 was added.
• additional 10 ⁇ g per well of the same aON were added.
• the cells were washed with phosphate-buffered saline (PBS) harvested with trypsin, centrifuged 10 min., at 350xg at room temperature.
• the cellular pellet were resuspend in 10 mM Tris/1,5 mM EDTA/5 mM Na_Mo ⁇ 4 /0.4 M KC1, homogenated and centrifuged 1 h at lOO.OOOxg.
• the amoun of hPR was determined by the enzyme immune linked assay of Abbott (Abbot Laboratories, North Chicago, IL, USA) according to the instructions of the manufacturer and expressed as fmoles/mg of cytosolic proteins. Amount of protein was determined according to Bradford et al.
• T47D cells were plated in a Costar (Europe Ltd., Badhoeverdorp, Netherlands) 24 wells plate (300.000 cells/well). The cells were grown at semi-confluency in RPMI medium including phenol red, 10% FCS and 10 -8 M ⁇ -estradiol. The aON was added twice: the first time at the concentration shown in Figure 2 and 24 hours later at half of this concentration.
• T47D-cells were cultured in RPMI-1640 medium without phenol red and 10% dextran charcoal stripped FCS and 20 ⁇ g of an aON as prepared in Example 1 were added. At day 2 additional 10 ⁇ g of the same aON were added. At day 3 the medium was replaced and 10" 8 M progesterone and 20 ⁇ g of the same aON were added. After 8 and 16 hours the cells were killed and the receptor extracts were prepared as in Example 2. In these receptor extracts the amount of ER was determined with an enzyme immune linked assay of Abbott (s.a.) as described in Example 2.

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• 1991
  • 1991-07-30 IT ITMI912117A patent/IT1250722B/it active IP Right Grant
• 1992
  • 1992-07-29 EP EP92916147A patent/EP0605437A1/de not_active Withdrawn
  • 1992-07-29 WO PCT/EP1992/001745 patent/WO1993003053A1/en not_active Ceased
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