WO1994017086A1 - Regulation de genes par ciblage d'une helice triple intramoleculaire potentielle - Google Patents

Regulation de genes par ciblage d'une helice triple intramoleculaire potentielle Download PDF

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WO1994017086A1
WO1994017086A1 PCT/US1994/000348 US9400348W WO9417086A1 WO 1994017086 A1 WO1994017086 A1 WO 1994017086A1 US 9400348 W US9400348 W US 9400348W WO 9417086 A1 WO9417086 A1 WO 9417086A1
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oligonucleotide
seq
strand
composition
nucleotides
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PCT/US1994/000348
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Kyonggeun Yoon
Meiqing Lu
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Apollon, Inc.
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Definitions

  • the present invention is related to
  • the oligonucleotides of the present invention are substantially complementary to a single stranded purine strand. Certain preferred embodiments do not form a substantially stable intermolecular triple helix with the target gene at physiological pH in vitro .
  • oligonucleotides having a circular or stem-loop functioning structure that may form both Watson- Crick and Hoogsteen bonds with the putative single-stranded target DNA.
  • the invention also provides methods for
  • compositions for decreasing transcription of a target gene preparing a composition for decreasing transcription of a target gene, methods for decreasing transcription of a target gene, and methods for treating a vertebrate suspecting of having a disease involving the expression of the target gene.
  • TFOs triplex-forming oligonucleotides
  • TFOs are designed to inhibit transcription by binding to double-stranded DNA in a sequence-specific manner to compete with and/or displace transcriptional factors that interact with the targeted cis-acting enhancer sequence.
  • 91/06626 provide examples that demonstrate the basic design of triplex-forming oligonucleotides.
  • Two recognized motifs for the formation of a triple helix are known as the "CT” motif and the “GT” motif.
  • CT CT
  • GT GT
  • the "CT” motif involves the use of a polypyrimidine oligomer as the triplex-forming
  • the TFO is oriented in a parallel direction to the purine-rich strand of the duplex.
  • the "GT" motif provides for the use of a G for every GC base pair and a T for every AT base pair. In this case, the TFO is oriented in an anti-parallel direction to the purine-rich strand of the duplex.
  • the present invention involves the targeting of a putative single-stranded portion of a DNA sequence that is believed to result in vivo from the formation of an intramolecular triple helix within the target DNA.
  • regions can be identified by their nuclease sensitivity and involvement in transcription in vitro , and by their substantial mirror symmetry, which is purine-rich on one strand.
  • this structure has never been
  • the present invention involves oligonucleotides targeted to particular regions of DNA that are believed to have a non-B form.
  • the present invention provides compositions for decreasing transcription of a target gene having a putative single-stranded region of DNA in vivo that is significantly involved in transcription and which region includes at least about 10 nucleotides, at least about 65% of which are purines on one strand, having substantially mirror symmetry in at least two palindromic arms having at least about 5 nucleotides each, the arms being either contiguous, or having about 1 to about 50 nucleotides between them.
  • Certain compositions of the present invention comprise an oligonucleotide, or derivative thereof,
  • compositions comprise substantially complementary circular
  • oligonucleotides are oligonucleotides having a stem-loop functioning structure.
  • the invention involves the preparation of the oligonucleotides.
  • the present invention involves the use of the oligonucleotides described above for the inhibition of transcription of genes having polypurine-rich sequences as described above.
  • the oligonucleotides may be employed in the treatment of diseases involving such genes.
  • the present invention therefore additionally provides methods for decreasing transcription of such genes, methods for preparing compositions for decreasing transcription, and methods for treating a vertebrate suspected of having a disease involving the expression of such gene.
  • diseases include, for example, cancer, viral and autoimmune diseases, and diseases requiring transplantation.
  • FIG. 1 illustrates the putative structure of H- DNA in vivo , illustrating the conformation of the helices.
  • H-DNA consists of an intramolecular DNA triplex region wherein pyrimidine-rich strand donates the third strand involved in Hoogsteen hydrogen bonding with the duplex. This formation causes the complementary region of the purine-rich strand to be single stranded. The two halves of the
  • pyrimidine-rich strand involved in the intramolecular triplex are antiparallel.
  • the pyrimidine-rich strand is shown as a black strand.
  • FIG. 2 shows the putative H-DNA structure of the c-myc substrate representing the upstream region of the gene.
  • the tandem H-DNA triplex model for the nuclease sensitive element (NSE) structure is shown.
  • An H-DNA structure is manifested using the Hoogsteen base pairing scheme. Watson and Crick base pairs are represented by dashes, T:AT
  • Figure 3 demonstrates the thymidine uptake of HL60 cells incubated with various oligonucleotides as described in Example 1.
  • Figure 4 displays the thymidine uptake of K562 cells incubated with various oligonucleotides as described in Example 1.
  • Figure 5 represents the steady state levels of c- myc and ⁇ -actin mRNAs measured by reverse-PCR, as detected by ethidium bromide staining.
  • HL60 cells were incubated with oligonucleotides KY10016 (SEQ ID NO: 2) and KY10027 (SEQ ID NO: 31), as described in Example 3.
  • Total RNA was isolated from HL60 cells, diluted in a sequential manner and subjected to reverse-PCR.
  • Lanes 1-4 represent the reverse-PCR product of HL60 cells incubated with KY10016. The number of cells in the reaction varies from 1000, 100, 10 and 1, respectively (Lanes 1 to 4).
  • Lanes 5-8 represent the reverse-PCR product of HL60 cells incubated with KY10027. The number of cells in the reaction varies from 1000, 100, 10, and 1, respectively (Lanes 5 to 8).
  • Lane 9 indicates the PCR product of
  • Lanes 10 and 11 indicate the PCR product of HL60 cells in the presence of c- myc primers only and ⁇ -actin primers only.
  • the size of the PCR products of the c-myc and ⁇ -actin mRNAs are 326 bp and 218 bp, respectively.
  • Figure 6 illustrates the band shift analysis of triplex formation. Binding of various oligonucleotides to the duplex target (Table II) was assayed as described in Example 1. In Lanes 1-4 and 9-11, the duplex targets at a constant concentration (5x10 M) were mixed with various concentrations of oligonucleotides in the reaction mixture containing 0.15 M NaCl, 10 mM MgCl 2 and 5 mM Tris-HCl at pH 7.0.
  • Lanes 5-8 and 12-15 the duplex targets at a constant concentration (5x10 -8 M) were mixed with various concentrations of oligonucleotides in the reaction mixture containing 10 mM Tris-HCl, 5 mM MgCl 2 , 1 mM spermine and 10% sucrose. Lanes 1 to 4 and Lanes 5-8 contain oligonucleotide
  • Lanes 9 to 11 contain
  • oligonucleotide KY10016 at various concentrations, 2x10 -8 , 2x10 -7 and 2x10 -6 M, respectively.
  • Lanes 12 to 15 contain oligonucleotide KY10016 at various concentrations, 2x10 -9 ,
  • Figure 7 depicts the inhibition of cell growth in
  • Figure 8 depicts the inhibition of cell growth in HuH7 cells by the oligonucleotides of the invention as described in Example 2.
  • Figure 9 is a schematic of the formation of H-DNA when two arms comprising inverted repeats are present, and the 3 arm is involved in the Hoogsteen base pairing.
  • Figure 10 is a schematic of the formation of H-DNA when two arms comprising inverted repeats are present and the 5' arm is involved in the Hoogsteen base pairing.
  • H-DNA can be characterized, for example, by
  • nuclease hypersensitivity sensitivity to single-stranded- specific nucleases and strong purine/pyrimidine strand asymmetry.
  • Kolluri et al. Nucleic Acids Research 1992, 20, 111-116.
  • Homopurine-homopyrimidine sequences are frequently found in upstream regulatory regions, in which they are suspected to play a function in gene expression.
  • a high degree of supercoiling or low pH i.e., less than 7.
  • H-DNA a triple helix by forming Hoogsteen base pairs consisting of T-A-T and C + -G-C triplets.
  • the structure is termed "H-DNA" due to the presence of a flexible hinge between the duplexes as well as the
  • the pyrimidine-rich strand donates the third strand, leaving the complementary region of the purine-rich strand single- stranded, as shown in Figures 9 and 10.
  • either arm of the purine strand can be involved in the Hoogsteen base pairing in the formation of the intramolecular triplex.
  • the arms are designated "S1" and “S2", respectively, in the Figures.
  • the complementary arms on the second strand are designated "S1'" and "S2'",
  • the region complementary to L which forms part of the single stranded region in both instances is designated "L".
  • the single-stranded region will also include either S2 or S1.
  • Watson Crick bonds are depicted as solid lines.
  • Hoogsteen bonds are depicted as dash lines.
  • the heavy arrows indicate the arms as inverted repeats on one strand. The inventors are not aware of a preferred
  • the promoter region Since the promoter region is targeted, it is not necessary that the purine-rich strand be the strand normally transcribed in the gene of interest, as shown in the examples which follow.
  • H-DNA may be widespread in genomic DNA since TC-AG repeats long enough to hybridize with (dT-dC) 18 are frequently found in human DNA. These regions are found in areas important for transcription, replication, and recombination.
  • the formation of H-DNA in vivo could influence DNA-protein interaction and could absorb negative supercoiling generated by transcription. H-DNA structures may be verified, for example, using reagents that provide information about DNA
  • oligonucleotides are designed to target the putative single-stranded region of the H-DNA in an antisense manner with high stability and
  • the oligonucleotides of the invention may bind to a putative transcriptional factor that interacts with H-DNA. It is believed that a family of transcriptional factors may be implicated since the
  • oligonucleotide of the present invention may be able to decrease transcription of several genes implicated in, for example, cancer.
  • NSE c-myc nuclease sensitive element
  • transcriptional factors (Kolluri et al., Nucleic Acid Res . , 1992, 20, 111-116; Postel et al. Science 1993 261, 478-480), be involved in transcriptional regulation, and form H-DNA in vitro .
  • a model has been postulated whereby two tandem triplexes are involved in the H-DNA structure, and the pyrimidine-rich strand participates in both triple helices, as illustrated in Figure 2.
  • TFOs TFO with high affinity for c-myc is reported in Postel et al., Proc . Nat 'l . Acad . Sci . USA , 1991, 88, 8227-8231 and Cooney, etal., Science , 1988, 241, 456-459.
  • a "G"-rich oligonucleotide the PU1 oligonucleotide (SEQ ID NO: 30) was shown to form a triplex with substrate DNA comprising the nucleotides -153 through -117 from the P1 promoter. Reduction in cytoplasmic c-myc P1 mRNA levels in Hela cells using PU1 were reported.
  • the present invention involves targeting the putative single-stranded region of the H-DNA in an antisense manner.
  • "C"-rich oligonucleotides directed against c-myc including the PY1 sequence disclosed as ineffective in Postel, provided a surprisingly superior result in demonstrating inhibition of c-myc transcription as compared to a conventional
  • Oligonucleotides of the present invention can be prepared for any homopurine-homopyrimidine sequences found in regulatory regions that are potentially capable of forming H-DNA.
  • the present invention thus provides therapeutic oligonucleotides for transcriptional regulation of many genes.
  • Other genes having homopurine and homopyrimidine regulatory sequences include Erb-B2, K-ras, EGF receptor, IL2 receptor alpha, collagen type I, IL2, beta-actin,
  • glucocorticoid receptor HIV ppt
  • HIV LTR HIV LTR
  • HSV I HBV ppt.
  • regulatory sequences are often present upstream of the transcription initiation site, but they may also be within an intron or in the 3' untranslated sequences, for example.
  • the present invention specifically provides compositions for decreasing transcription of target genes such as those listed above, which potentially have a
  • Nuclease sensitivity is defined as at least about 20% greater than average sensitivity to nucleases such as DNase I and S1, and preferably at least about 50% greater than average. Average sensitivity may be measured, for example, by subjecting isolated nuclei to DNase digestion and probing with a radiolabeled DNA fragment in an area of a gene that is not actively transcribed. See, for example, Postel et al., Proc . Natl . Acad . Sci . USA 1991, 88, 8227-8231.
  • Nuclease sensitivity reflects an alteration of chromatin that results in the disruption of normal nucleosome structure. See, for example, Larsen and Wientraub, Cell 1982, 29, 609-622. This may be directly related to gene transcription because nucleosomes restrict transcriptional activity. Indeed, there appears to be a correlation between the hypersensitivity and transcriptional activity. For example, the DNase hypersensitive site of the c-myc upstream region disappears in HL60 cells coincident with the cessation of c-myc transcriptional initiation. This region of the c- myc gene has been shown to be required for a high level of c- myc gene expression and to bind several nuclear factors.
  • the present invention thus provides the advantage of binding to a region capable of forming a non-B DNA structure, which may be present only during active transcription.
  • An area of the gene that is significantly involved in transcription is defined as a region that has been found, using molecular genetics, to be important in the initiation and/or enhancement of transcription, and/or a region where transcriptional factors bind or interact.
  • An example of a test that may be performed to determine whether a region is transcriptionally important is a nuclear run-on assay.
  • an enhancer mapping assay is a CAT assay where the CAT gene is coupled with the region suspected to be involved in transcription.
  • CAT assay where the CAT gene is coupled with the region suspected to be involved in transcription.
  • at least about a 50% reduction in CAT expression in the absence of the DNA element denotes significant involvement in transcription. More preferred embodiments of the invention entail DNA regions whose absence result in at least about a 75%
  • the target region is a putative single- stranded region formed due to an intramolecular triple helix created by substantial mirror symmetry within a purine-rich or a pyrimidine-rich segment.
  • Substantial mirror symmetry is defined as a sequence having at least 2 arms constituting inverted repeats. The inverted repeats need not be a perfect match so long as there is substantial symmetry, preferably with at least about 75% of the bases within the arms
  • the arms may be contiguous or non-contiguous and are each preferably at least about 5 nucleotides long, and more preferably about 5 to about 30 nucleotides long. Those sequences that are non-contiguous preferably have about 1 to about 50 nucleotides between the two arms.
  • Such intramolecular triple helices are stabilized in vitro by a pH lower than 7 and negative supercoiling.
  • the length of the purine-pyrimidine tract involved also
  • the putative single-stranded target region is from about 8 to about 30 nucleotides long, and comprises at least about 65% guanines and adenines.
  • compositions of the invention comprise an oligonucleotide, or derivative thereof, substantially
  • oligonucleotide does not form a substantially stable
  • Intermolecular triple helix formation with the target region is measured at conditions comprising
  • oligonucleotides of the invention comprise at least about 8 nucleotides, preferably about 8 to about 30 nucleotides. In a further preferred embodiment, the oligonucleotides of the invention comprise at least about 65% cytosines and thymines.
  • the oligonucleotides of the invention are circular. researchers have found that circular oligodeoxyribonucleotides bind to single-stranded target regions more strongly than linear DNA complementary
  • a circular oligonucleotide may form an intermolecular triple helix with the putative single-stranded region of the target gene. See, for example, Prakash and Kool, J. Chem . Soc , Chem . Commun . 1991, 1161- 1163 and Figure 3.
  • the oligonucleotides of the invention have a stem-loop functioning structure.
  • the stem- loop functioning structure is defined as a sequence that has two arms having substantial mirror symmetry connected by a linker.
  • the stem-loop functioning oligonucleotide thus can form an intermolecular triple helix with a complementary single-stranded DNA.
  • circular oligonucleotides Like circular oligonucleotides,
  • oligonucleotides having a stem-loop functioning structure may form both Watson-Crick and Hoogsteen bonds with a single- stranded target. See, for example, Giovannangeli et al., J. Am . Chem . Soc. 1991, 113, 7775-7777.
  • the length of the stems of such structures are preferably at least 5 nucleotides.
  • the loop is at least 3 nucleotides long, and preferably less than 10. In a preferred embodiment, the loop is 3-7
  • the linker may be any of a number of compounds, as would be understood by one skilled in the art, once armed with the present disclosure. Such compounds include, for example, nucleotides, polyethylene glycol, polyamides, and
  • the oligonucleotide need not match the target sequence exactly; it may span only a portion of it and some mismatches are contemplated.
  • the phrase "at least a portion of" represents preferably at least about 8
  • oligonucleotide is preferably from about 8 to about 30 nucleotides.
  • the oligonucleotides should be a size which is long enough to bind specifically to the target region, but not too large to prohibit entry into a cell.
  • the number of mismatches contemplated are those which do not prevent binding of the oligonucleotide to the extent necessary to reduce transcription.
  • oligonucleotide While any length oligonucleotide may be utilized, it is preferable to use a length that confers specificity.
  • the length of the oligonucleotide required for specificity depends upon the content of the bases, as discussed, for example, in Herschlag, Proc . Natl . Acad . Sci . USA 1991, 85, 6921-6925. Specifically, it has been demonstrated that adding more bases to an oligonucleotide may decrease
  • oligonucleotides having at least about 8 nucleotides are preferred.
  • the size of the oligonucleotide is limited by its ability to enter the target cell. Large oligonucleotides may be somewhat less effective in
  • interference with expression means that there is a detectable decrease in transcription.
  • substantially complementary refers to an oligonucleotide designed to generally have a C for every G, a G for every C, an A for every T and a T for every A in the purine-rich strand. It will be understood by one skilled in the art that the oligonucleotide need not be perfectly complementary to the purine strand to be capable of
  • Some mismatches can be included, so long as there remains a detectable decrease in transcription of the target gene.
  • oligonucleotides of the present invention differ from
  • the oligonucleotides of the invention are "anti- gene.”
  • the targeted region in the DNA is a transcriptional regulatory region, this area of the DNA is not transcribed into a corresponding mRNA.
  • viral gene targets it will be understood by one skilled in the art, once armed with the present disclosure, that the same
  • oligonucleotide of the invention may be capable of both decreasing transcription and performing an antisense function by binding to the corresponding mRNA and thus decreasing expression also.
  • the oligonucleotide can be a synthetic
  • ribonucleotide derivatives has been reported by Shibahara et al., Nucleic Acids Research 1989, 17, 239-252.
  • Ribonucleotides may confer more binding stability than deoxyribonucleotides, as discussed, for example, in Roberts and Crothers, Science 1992, 258, 1463-1466. However,
  • deoxyribonucleotides are generally more stable to nuclease attack.
  • oligonucleotides of the present invention can be synthesized by any of the known chemical oligonucleotide synthesis methods. See for example. Gait, M.J., Ed. (1984), Oligonucleotide Synthesis (IRL, Oxford). The
  • oligonucleotides may also be synthesized through recombinant expression from an appropriate vector.
  • derivatives of the oligonucleotides known in the art is also within the scope of the present invention, including derivatives such as methylphosphonates, phosphotriesters, phosphorothioates and phosphoroamidates. Additionally, beta-anomers may be replaced with alpha- anomers. Sun et al., Triple-Helix Formation by ⁇
  • Oligodeoxynucleotides and ⁇ Oligodeoxynucleotide-Intercalator Conjugates Proc . Natl . Acad . Sci . 1991, 88, 6023-6027. See also C.A. Stein & J.S. Cohen, Oligodeoxynucleotides as
  • PNA polyamide nucleic acid
  • oligonucleotide derivatives include the use of linkers attached to the 5' and/or 3' termini along with a modifying group selected for its ability to damage DNA, such as an intercalator. See, for example, Nguyen et al., U.S. Patent No. 4,835,263, which is hereby incorporated by
  • labeling groups such as psoralen, chemiluminescent groups, cross-linking agents, intercalating agents such as acridine, or groups capable of cleaving the targeted position of the targeted DNA such as molecular scissors like o-phenanthrolinecopper or EDTA-iron may be incorporated in the oligonucleotides. See, for example, Ts'o, WO 90/15884, which is hereby incorporated by reference.
  • DNA modifying groups may also be used to derivatize the oligonucleotides of the invention.
  • Such groups include groups that cross-link, alkylate, cleave, degrade, or
  • oligonucleotides of the invention may also be modified, for example, by covalent binding on either the 3' or the 5' end to a ligand or a ligand mimic for receptor- mediated endocytosis.
  • the oligonucleotides may be mixed with such a ligand.
  • the oligonucleotides may be mixed with such a ligand.
  • oligonucleotides of the invention may be substituted on the 3 ' end with a thiol coupled to 6-phosphomannosylated proteins via a difulside bridge according to Bonfils et al., Nucleic Acids Res . 1992, 4621-4629. Such modifications would be found in the literature.
  • oligonucleotide as used herein includes both ribonucleotides and deoxyribonucleotides, and includes molecules which may be long enough to be termed
  • polynucleotides Oligodeoxyribonucleotides are preferred since oligoribonucleotides are more susceptible to enzymatic attack by ribonucleotides than deoxyribonucleotides.
  • bases, sugars or internucleotide linkages may be substituted or chemically modified by methods known in the art. Modifications may be made, for example, to improve stability and/or lipid solubility. For instance, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting a methyl group or sulfur atom for a phosphate oxygen in the internucleotide phosphodiester linkage.
  • suppositories may also be useful. Use of the
  • oligonucleotides and their derivatives in prophylaxis is also contemplated.
  • Use of pharmaceutically acceptable carriers is preferred for some embodiments.
  • compositions of this invention comprise a pharmaceutically acceptable carrier or diluent and an effective quantity of one or more of the oligonucleotides or their derivatives, or an acid or base salt thereof.
  • the carrier or diluent can take a wide variety of forms depending on the form of preparation desired for administration, e.g., sublingual, rectal, nasal, oral, or parenteral.
  • any of the usual pharmaceutical media can be employed, for example, waters, oils, alcohols, flavoring agents,
  • preservatives, and coloring agents to make an oral liquid preparation (e.g., suspension, elixir, or solution) or with carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents, to make an oral solid preparation (e.g., powder, capsule, or tablet).
  • an oral liquid preparation e.g., suspension, elixir, or solution
  • carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents
  • an oral solid preparation e.g., powder, capsule, or tablet
  • Controlled release forms or enhancers to increase bioavailability can also be used. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are employed. If desired, tablets can be sugar coated or enteric coated by standard techniques.
  • the carrier will usually be sterile water, although other ingredients to aid
  • oligonucleotides can be combined with a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • suitable liquid vehicle or excipients are
  • oligonucleotides and their derivatives can also be administered locally at a lesion by topical application of a solution or cream.
  • the oligonucleotides or their derivatives can be administered in liposomes or microspheres (or microparticles).
  • Methods for preparing liposomes and microspheres for administration to a patient are known to those skilled in the art.
  • U.S. Patent No. 4,789,734 describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary.
  • a review of known methods is provided by G. Gregoriadis, Drug Carriers in
  • Microspheres formed of polymers are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the bloodstream. Alternatively, the nucleotides or their derivatives can be incorporated therein and the microspheres, or composite of microspheres, implanted for slow release over a period of time, ranging from days to months. See, for example, U.S. Patents Nos. 4,906,474, 4,925,673 and
  • oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques. For example, oligonucleotides have been successfully
  • oligonucleotides can be carried into the cell by exploitation of folate receptor- mediated endocytosis. Leamon and Low, Proc. Nat 'l . Acad .
  • oligonucleotides can be administered by vector-mediated delivery. Such delivery systems are within the scope of one skilled in the art once armed with the present disclosure. Preferred methods of gene therapy include, for example, the incorporation of the gene encoding the therapeutic
  • the gene is then transferred into the stem cells of the patient's bone marrow ex vivo .
  • the patient's own bone marrow is treated, for example, with irradiation, chemotherapy or ablation.
  • the treated bone marrow is then transplanted into the patient.
  • vector-mediated delivery systems see,
  • oligonucleotides of the invention can also be delivered locally in vivo in high concentrations, for
  • oligonucleotides or derivatives
  • the oligonucleotides can be administered in an amount effective to reduce the symptomology of disease. It is within the scope of a person skilled in the art to determine optimum dosages and treatment schedules for such treatment regimens.
  • the oligonucleotides can be administered in an amount effective to reduce the symptomology of disease.
  • administered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, health and sex of the patient, the route of administration, and other factors.
  • oligonucleotides ex vivo by isolating white blood cells from peripheral blood, treating them with the oligonucleotides, then returning the cells to the donor's blood.
  • Ex vivo techniques have been used in the treatment of cancer patients with interleukin-2 activated lymphocytes. Rosenberg et al., New England Journal of Medicine , 1990, 323, 570-578
  • the oligonucleotide therapeutics can be any suitable oligonucleotide therapeutics.
  • the oligonucleotide therapeutics can be
  • viability of normal cells may vary depending on the nature and extent of the disease, the particular oligonucleotide utilized, the relative sensitivity of the disease to the oligonucleotide, and other factors.
  • the present invention also provides methods for decreasing transcription of a target gene having a region of DNA that is significantly involved in transcription and which region includes at least about 10 nucleotides, at least about 65% of which are purines on one strand and which region has substantial mirror symmetry in at least two palindromic arms having at least about 5 nucleotides each, the arms being either contiguous or having about 1 to about 50 nucleotides between them.
  • a preferred method of the invention comprises the administration of an oligonucleotide or derivative thereof substantially complementary to one of the palindromic arms of the purine strand and, in some embodiments, the sequence between the arms when they are not contiguous, and which does not form a substantially stable intermolecular triple helix with the target gene at physiological pH.
  • a target gene is selected in which the gene has a region of DNA that is significantly involved in transcription and which comprises at least about 8 nucleotides, at least about 65% of which are purines on one strand.
  • the oligonucleotide is circular. In further preferred embodiments, the
  • oligonucleotide has a stem-loop functioning structure. Further, the present invention provides methods of treating a vertebrate having a disease involving the
  • oligonucleotide of the invention comprises administering to the vertebrate an oligonucleotide of the invention.
  • the oligonucleotide is administered in an a blood-level concentration corresponding to an in vitro concentration of about 1 ⁇ M.
  • target genes that are expressed upon virus infection and the oligonucleotides of the
  • invention may be used to decrease the transcription of the viral genes and viral replication in the treatment of
  • Additional preferred embodiments pertain to target genes involved in autoimmunity and transplant
  • Triplex binding is less stable than duplex binding—in contrast to stem loop oligonucleotide binding to a single stranded target, which involves Watson-Crick binding as well as Hoogsten base pairing and, thus, both specificity and stability are higher than Watson-Crick Base binding alone.
  • oligonucleotides of the present invention targeting regions capable of forming a putative H-DNA structure in vivo are expected to have a substantial effect only when the gene is transcriptionally active.
  • oligonucleotide which can be used to inhibit transcription since, unlike the conventional antisense RNA approach, the target is a single- copy DNA sequence.
  • certain oligonucleotides of the invention may be capable of decreasing transcription of a target gene in tissue culture cells in concentrations as low as 1 ⁇ M, and even as low as .1 ⁇ M, as seen in Figure 4a. It is believed that modifications to the oligonucleotides will decrease the required
  • C-myc is an example of a gene having a purine-rich region with substantial mirror symmetry.
  • the upstream sequences of this gene have two potential sites of H-DNA formation, as illustrated in the mirror symmetry present in nucleotides 12-17 and 22-27 as well as 34-41 and 49-56 of SEQ ID NO: 1.
  • the expression of c-myc has been implicated in many cancers including leukemias. Examples of
  • oligonucleotides of the invention targeted against c-myc include SEQ ID NOS : 2-6 and SEQ ID NOS: 26-27.
  • oligonucleotides, and their derivatives can be used, for example, in the treatment of many cancers including
  • HL60 and K562 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum. All reagents used for tissue culture were obtained from Gibco Laboratories. Transfection was carried out using lipofectin reagent
  • oligonucleotide ranged from 0.1 to 5 ⁇ m. Cells were
  • oligonucleotide/lipofectin mixture incubated with the oligonucleotide/lipofectin mixture for 4 hours, supplemented with FBS to 10%, and harvested 24 hours post transfection.
  • the adherent cell lines LS180 and HuH7 were grown in alpha MEM supplemented with 10% fetal bovine serum.
  • oligonucleotides were mixed with 2 ⁇ g of lipofectin in OPTIMEM and added to the 96- well plate containing the oligonucleotide 5000 cells per well in 100 ⁇ l. The concentration ranges of oligonucleotides were as above. Cells were incubated with the
  • oligonucleotide/lipofectin mixture for 4 hours, medium was aspirated, and cells were refed with medium containing the same concentration of the oligonucleotides as initially, supplemented with FBS to 10% for 5 days, after which cells were harvested. Further additions were made to the cells during that time as required due to evaporation effects. Thymidine Uptake and Cell Counts.
  • H660 and K562 cells were determined by performing an assay measuring thymidine uptake. After the H660 and K562 cells were treated with lipofectin, 0.5 ⁇ Ci of 3 Hthymidine (New England Nuclear, specific
  • LS180 and HuH7 cells were incubated with 0.5 mg/ml MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) for 2 hours. The medium was aspirated and 100 ⁇ l dimethylsulfoxide was added to each well. The plates were shaken for 30 minutes using a plate shaker at room
  • the absorbance was then read immediately at a wavelength 540 nm with a Vmax microtiter plate reader.
  • RNA levels of c-myc and ⁇ -actin were analyzed as follows. Cells were incubated with various oligonucleotides under identical conditions as described above, lysed in 0.5% NP40 solution containing 140 mM NaCl, 1.5 mM MgCl 2 and 10 mM Tris-HCl at pH 8.0. After lysis, RNasin was immediately added to 1000 U/ml. RNA was then reverse-transcribed by using 400 units of Moloney murine leukemia virus reverse transcription (RT) and 0.2 ⁇ g of random primer for 1 hour at 42°C. The resulting cDNA
  • Duplex formation was effected by combining equimolar concentrations of oligonucleotides in a buffer containing 0.15 M NaCl, 10 mM MgCl 2 , and 5 mM Tris-HCl, pH 7.0, heating at 70°C for 15 minutes, slowly cooling, and then incubating at various temperatures.
  • oligonucleotides were added to a fixed concentration of duplex target and incubated overnight. The concentration of duplex was constant at 5x10 -8 M in each reaction in a total volume of 10 ⁇ l. Concentrations of oligonucleotides ranged from 2x10 -9 M to 2x10 -6 M. Electrophoresis was carried out on 12% polyacrylamide gels (17x18x0.8 cm) in a buffer containing 50 mM boric acid, 5 mM MgCl 2 and 50 mM Tris-HCl, pH 8.3 at room temperature. In general, the electrophoresis was performed at a constant voltage of 10 V/cm with a current of 20 to 30 mA. After electrophoresis was halted, the gel was dried and the radioactivity contained in each band was quantitated using a Phosphorolmager. EXAMPLE 1
  • the upstream regulatory region of the c-myc gene beginning at -125 bp from the initiation of transcription, contains a region of high purine/pyrimidine sequence
  • NSE nuclease sensitive element
  • This C-myc element is a cis-acting positive transcription element. Davis et al., Proc . Natl . Acad. Sci . USA 1989, 86, 9682-9686.
  • oligonucleotides were tested in the present assay for their ability to inhibit c-myc transcription .
  • the oligonucleotides tested are listed in Table II and comprised the following: 1) G-rich oligonucleotides designed to bind the duplex target in antiparallel manner (G:GC, A:AT or
  • T:AT 2) G-rich oligonucleotides with a scrambled sequence; 3) C-rich oligonucleotides complementary to the target duplex DNA in parallel or antiparallel manner (C+:GC); and 4) C-rich oligonucleotides with a scrambled sequence.
  • An "(AP)" preceding the oligonucleotide designation indicates anti- parallel orientation. As “(P)" indicates parallel
  • the c-myc substrate comprised two inverted repeats of two arms each.
  • the regions of symmetry comprising the arms of the inverted repeats on the pyrimidine-rich strand are underlined in the substrate sequence (SEQ ID NO: 1) and designated "S1", “S2", “T1” and “T2” respectively. They comprise nucleotides 12-17 (S1) and 22-27 (S2); and 34- 41 (T1) and 49-56 (T2).
  • SEQ ID NO: 1 substrate sequence
  • SEQ ID NO: 1 substrate sequence
  • Oligonucleotides complementary to Loop 2 are highlighted in a similar manner. Oligonucleotides were incubated with HL60 and K562 cells at concentrations ranging from 0.5 to 5 ⁇ M, in the presence or absence of lipofectin, for 4 hours in OPTI-MEM prior to the addition of bovine serum. Thymidine uptake was measured 24 hours
  • oligonucleotides were tested and compared, as indicated in Table II.
  • the oligonucleotides of the present invention demonstrated an unanticipated superiority over a conventional triplex-forming oligonucleotide, specifically, PU1 (SEQ ID NO: 30).
  • oligonucleotides was also demonstrated by cell counts.
  • the cell counts of both HL60 and K562 are provided in Table III. Cells were incubated with 1 ⁇ M oligonucleotide in lipofectin as described above and counted upon harvest. Viable cells only, as measured by Trypan blue exclusion, were counted. Cells treated with G-rich oligonucleotides doubled their number in a 24 hour period post-treatment, while cells treated with C-rich oligonucleotides did not show any
  • a quantitative calorimetric assay for mammalian cell survival and cell proliferation was utilized for quantifying cells from the two adherent cell lines LS180 and HuH7.
  • the assay is dependent on the reduction of tetrazolium salt MTT by the mitochondrial dehydrogenase of viable cells to form a blue formazan product.
  • the assay measures cell respiration and the amount of formazan produced, which is proportional to the number of living cells present in culture.
  • phosphorothioate oligonucleotides of the same sequence were tested. Phosphorothioate oligonucleotides exhibited much better inhibitory effect in cellular
  • oligonucleotide 8-mers (designated "KY100N8-PT” in Figures 7 and 8) were synthesized using equal amounts of A, G, C, and T at each position and exhibited no effect.
  • oligonucleotides of SEQ ID NOS: 2, 3, 5, 6, and 28. inhibited cell growth to various degrees in the cell lines tested and, thus, demonstrated therapeutic capability. Tests showed that the shorter length oligonucleotides such as the 18-mer of SEQ ID NO: 6 perform as well as the longer 27-mer of SEQ ID NO: 2. Although SEQ ID NO: 2 demonstrated inhibition, the oligonucleotides of the present invention preferably do not include this sequence. Notably, SEQ ID NO: 28, designated "C Scramble", shares homology with SEQ ID NO: 2, but with several deletions and mismatches, yet still inhibited cell growth.
  • C Scramble shares homology with SEQ ID NO: 2, but with several deletions and mismatches, yet still inhibited cell growth.
  • C-rich oligos may be any transcriptional inhibitory effect of C-rich oligos.
  • C-rich oligos may be any transcriptional inhibitory effect of C-rich oligos.
  • sequence of the oligonucleotide KY10025 (SEQ ID NO: 2) is entirely complementary, in anti-parallel orientation, to the G-rich loop in the putative H-DNA.
  • Erb-B2 is another example of a gene having a purine- rich segment with substantial mirror symmetry.
  • a potential site of H-DNA formation is exemplified by the mirror symmetry seen in SEQ ID NO: 8, which begins 69 nucleotides upstream of the transcription initiation site. Specifically, two amino acids having a purine- rich segment with substantial mirror symmetry.
  • substantially palindromic arms are located at nucleotides 1-9 and 17-25 of SEQ ID NO: 8.
  • the overexpression of Erb-B2 is particularly associated with breast cancer.
  • One example of an oligonucleotide of the invention targeted against Erb-B2 is that of SEQ ID NO: 13. This oligonucleotide and its derivatives may be used, for example, in the treatment of breast cancer, erythroleukemia, and sarcoma, and more
  • HBV ppt polypurine-rich tract
  • Hepatitis B virus is an example of a human virus having a purine-rich segment with substantial mirror
  • oligonucleotides of the invention are therefore potentially therapeutic in the treatment of diseases involving hepatitis B virus.
  • One example of an oligonucleotide of the invention complementary to the purine-rich region with substantial two-fold symmetry is SEQ ID NO: 14. This oligonucleotide and its derivatives may be used, for example, in the treatment of hepatitis.
  • N-myC is another example of a gene having a purinerich segment with substantial mirror symmetry.
  • a potential site of H-DNA formation is exemplified by the mirror symmetry seen in the sequence listed in SEQ ID NO: 10, at nucleotides 5-14 and 21-30.
  • N-myc has been implicated, for example, in neuroblastoma.
  • One example of an oligonucleotide of the invention targeted against N-myc is that of SEQ ID NO: 15. This oligonucleotide and its derivatives may be used, for example, in the treatment of neuroblastoma, and more
  • HIV is another example of a human virus having a purine-rich segment with substantial mirror symmetry.
  • the palindromic arms of the LTR are seen in nucleotides 2-7 and 11-16 of SEQ ID NO: 11, which begins at position -293 in the HIV genome. This region is characteristic of a nuclear factor activating T-cells (NFAT, Durand et al., Molecular Cellular Biol . , 1988, 8, 1715-1724) binding site.
  • NFAT nuclear factor activating T-cells
  • oligonucleotide of the invention targeted against the HIV LTR is that of SEQ ID NO: 16. This
  • oligonucleotide and its derivatives may be used, for example, in the treatment of HIV infection.
  • EGFR Epithelial Growth Factor Receptor
  • EGFR is another example of a gene having a purine-rich region with substantial mirror symmetry.
  • the palindromic arms are evident in nucleotides 9-17 and 25-33 of SEQ ID NO: 12, which represents an area of the gene beginning at
  • oligonucleotide of the invention targeted against EGFR is that of SEQ ID NO: 17.
  • This oligonucleotide and its derivatives may be used, for example, in the treatment of lung cancer, and other cancers involving fast-growing solid tumors and, more generally, any disease involving the expression of EGFR.
  • K-ras also exemplifies substantial mirror symmetry in an upstream transcriptionally important region that is purine-rich.
  • the palindromic arms are visible in nucleotides 9-20 and 24-35 of SEQ ID NO: 18, which murine sequence has been published in Pestov et al., Nuc . Acids Res . 1991, 19, 6527-6532.
  • This oligonucleotide and its derivatives may be used, for example, in the treatment of sarcoma and erythroleukemia, solid tumors, and any cancer or other disease involving the expression of K-ras.
  • IL2 has been implicated in autoimmune disorders, such as AIDS, and the upstream regulatory region of this gene likewise illustrates an area of substantial mirror symmetry in a purine-rich region.
  • the two palindromic arms can be seen in nucleotides 4-8 and 10-14 of SEQ ID NO: 20, which represents an area of the gene beginning at position -292. This region is characteristic of an NFAT binding site.
  • An oligonucleotide of the invention targeted to this region is illustrated by SEQ ID NO: 21. This oligonucleotide may be used, for example, in the treatment of HIV infection other diseases involving autoimmunity and diseases requiring transplantation and, more generally, any disease involving the expression of IL2.
  • Herpes Simplex Virus I is another example of a human virus having a purine-rich segment with substantial mirror symmetry. The palindromic arms are seen in
  • This sequence represents a promoter region for the genes encoding viral polymerase and the DNA binding protein.
  • One example of an oligonucleotide of the invention targeted against this region is that of SEQ ID NO: 23. This oligonucleotide and its derivatives may be used, for example, in the treatment of HSV I infection.
  • the polypurine-rich tract of HIV present in the 3 ' Long Terminal Repeat (LTR) is also an example of a
  • the HIV ppt is an example of a sequence in which the palindromic arms are not identical.
  • the palindromic arms can be seen in nucleotides 11-19 and 24-32 of SEQ ID NO: 24.
  • One example of an oligonucleotide of the invention targeted against this region is that of SEQ ID NO: 25.
  • oligonucleotide may be used, for example, in the treatment of HIV infection.
  • CAGCCGCTCC CTCCCTCCCT CCTTCCCTCC CTCCCGCGCGCG CGCGG 45 (2) INFORMATION FOR SEQ ID NO: 19:

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Abstract

La présente invention se rapporte à des oligonucléotides ou à des dérivés permettant de réduire la transcription d'un gène cible comprenant une région d'ADN riche en purine qui est impliquée de manière importante dans la transcription, et qui présente essentiellement une symétrie spéculaire. Les oligonucléotides de la présente invention sont sensiblement complémentaires au brin de purine. Selon certains modes préférés de réalisation, les oligonucléotides ne forment pas une hélice triple intramoléculaire pratiquement stable, in vitro, et au pH physiologique, avec le gène cible. Selon d'autres modes préférés de réalisation, des oligonucléodies présentant une structure fonctionnelle à boucle et tige ou circulaire et qui peuvent former à la fois des liaisons de Watson-Crick et des liaisons de Hoogstean avec l'ADN cible sont décrits. L'invention se rapporte également à des procédés de préparation d'une composition permettant de réduire la transcription d'un gène cible, à des procédés de réduction de la transcription d'un gène cible et à des procédés de traitement d'un vertébré dont on suppose qu'il souffre d'une maladie impliquant l'expression d'un gène cible.
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WO1996018732A2 (fr) * 1994-12-15 1996-06-20 Board Of Trustees Of The University Of Illinois Inhibition specifique des sequences de la synthese de l'adn a l'aide d'oligonucleotides formant des triplex
WO1996030384A1 (fr) * 1995-03-30 1996-10-03 Research Corporation Technologies, Inc. Oligonucleotides circulaires simple brin
WO1996032474A1 (fr) * 1995-04-12 1996-10-17 Hybridon, Inc. Oligonucleotides cooperants
EP0759693A1 (fr) * 1994-05-19 1997-03-05 The Trustees Of The University Of Pennsylvania PROCEDE D'IDENTIFICATION DE COMPOSES CAPABLES D'ANNULER LES INTERACTIONS DE LIAISON DE Vpr-rip-1 DE VIH
WO1997038097A1 (fr) * 1995-04-12 1997-10-16 Hybridon, Inc. Oligonucleotides cooperatifs
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FR2794755A1 (fr) * 1999-06-14 2000-12-15 Inst Nat Sante Rech Med Procede de circularisation d'oligonucleotides autour d'un acide nucleique en double brin, les structures obtenues et leurs applications
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EP1484394A1 (fr) * 2002-02-20 2004-12-08 Sysmex Corporation Amorces pour l'amplification d'acides nucleiques servant a la detection de l'arnm d'un gene domestique et methode d'essai faisant intervenir ces amorces
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EP0759693A1 (fr) * 1994-05-19 1997-03-05 The Trustees Of The University Of Pennsylvania PROCEDE D'IDENTIFICATION DE COMPOSES CAPABLES D'ANNULER LES INTERACTIONS DE LIAISON DE Vpr-rip-1 DE VIH
EP0759693A4 (fr) * 1994-05-19 1999-06-09 Univ Pennsylvania PROCEDE D'IDENTIFICATION DE COMPOSES CAPABLES D'ANNULER LES INTERACTIONS DE LIAISON DE Vpr-rip-1 DE VIH
WO1996018732A2 (fr) * 1994-12-15 1996-06-20 Board Of Trustees Of The University Of Illinois Inhibition specifique des sequences de la synthese de l'adn a l'aide d'oligonucleotides formant des triplex
WO1996018732A3 (fr) * 1994-12-15 1997-02-13 Univ Pennsylvania Inhibition specifique des sequences de la synthese de l'adn a l'aide d'oligonucleotides formant des triplex
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