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WO1994028720A1 - Oligomeres de modulation de l'oncogene ras - Google Patents

Oligomeres de modulation de l'oncogene ras Download PDF

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Publication number
WO1994028720A1
WO1994028720A1 PCT/US1994/006620 US9406620W WO9428720A1 WO 1994028720 A1 WO1994028720 A1 WO 1994028720A1 US 9406620 W US9406620 W US 9406620W WO 9428720 A1 WO9428720 A1 WO 9428720A1
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PCT/US1994/006620
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Walter Lima
Brett Monia
Susan Freier
David Ecker
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Isis Pharmaceuticals, Inc.
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Priority to AU72067/94A priority Critical patent/AU7206794A/en
Publication of WO1994028720A1 publication Critical patent/WO1994028720A1/fr

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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
    • C12N15/1135—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 oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA

Definitions

  • This invention is directed to compounds that are not polynucleotides yet which bind in a complementary fashion to DNA and RNA strands.
  • the invention concerns compounds wherein naturally-occurring nucleobases or other nucleobase-binding moieties are covalently bound to a polyamide backbone.
  • This invention further provides methods for the inhibition of expression of the ras gene, a naturally occurring gene which occasionally converts to an activated form which has been implicated in tumor formation.
  • This invention is also directed to the specific inhibition of expression of the activated form of the ras gene.
  • This invention is further directed to the detection of both normal and activated forms of the ras gene in cells and tissues, and can form the basis for research reagents and kits both for research and diagnosis. Furthermore, this invention is directed to treatment of such conditions as arise from activation of the ras gene.
  • Oligodeoxyribonucleotides as long as 100 base pairs (bp) are routinely synthesized by solid phase methods using commercially available, fully automatic synthesis machines. The chemical synthesis of oligoribonucleotides (RNAs) , however, is far less routine. Oligoribonucleotides are also much less stable than oligodeoxyribonucleotides, a fact which has contributed to the more prevalent use of oligodeoxyribonucleotides in medical and biological research directed to, for example, gene therapy or the regulation of transcription or translation.
  • mRNA messenger RNA
  • tRNA transfer RNAs
  • RNA- synthesizing enzyme RNA polymerase
  • RNA polymerase RNA- synthesizing enzyme
  • sequence-specific binding of protein transcription factors to the promoter Other proteins which bind to the promoter, but whose binding prohibits action of RNA polymerase, are known as repressors .
  • repressors proteins which bind to the promoter, but whose binding prohibits action of RNA polymerase.
  • gene activation is typically regulated positively by transcription factors and negatively by repressors.
  • synthetic oligodeoxynucleotides could be used as antisense probes to block and eventually lead to the breakdown of mRNA.
  • synthetic DNA could suppress translation in vivo.
  • It also may be possible to modulate the genome of an animal by, for example, triple helix formation using oligonucleotides or other DNA recognizing agents.
  • triple helix formation there are a number of drawbacks associated with triple helix formation. For example, it can only be used for homopurine sequences and it requires unphysiologically high ionic strength and low pH.
  • oligonucleotides are impractical both in the antisense approach and in the triple helix approach because they have short in vivo half-lives, and are difficult to prepare in more than milligram quantities and, thus, are prohibitively costly. They are also poor penetrators of the cell membrane.
  • methylphosphonates monothiophosphates, dithiophosphates, phosphoramidates, phosphate esters, bridged phosphoro- amidates, bridged phosphorothioates, bridged methylene- phosphonates, dephospho internucleotide analogs with siloxane bridges, carbonate bridges, carboxymethyl ester bridges, acetamide bridges, carbamate bridges, thioether, sulfoxy, sulfono bridges, various "plastic" DNAs, ⁇ -anomeric bridges, and borane derivatives.
  • PNAs denominated peptide nucleic acids
  • ligands are linked to a polyamide backbone through aza nitrogen atoms.
  • peptide nucleic acids are disclosed in which their recognition moieties are linked to the polyamide backbone additionally through amido and/or ureido tethers.
  • PCT/EP 92/01219 filed May 22, 1992 also discloses protein nucleic acids.
  • peptide nucleic acids are synthesized by adaptation of certain peptide synthesis procedures, either in solution or on a solid phase.
  • the synthons used are certain monomer amino acids or their activated derivatives, protected by standard groups.
  • These oligonucleotide analogs also can be synthesized by using the corresponding diacids and diamines.
  • Peptide nucleic acid oligomers have been found to be superior to prior reagents in that they have significantly higher affinity for complementary single stranded DNA (ssDNA) . These compounds are also able to form triple helices wherein a first PNA strand binds with RNA or ssDNA and a second PNA strand binds with the resulting double helix or with the first PNA strand.
  • PNAs generally possess no significant charge and are water soluble, which facilitates cellular uptake.
  • PNAs contain amides of non- biological amino acids, making them biostable and resistant to enzymatic degradation, for example, by proteases.
  • PNAs can ideally be used to target RNA and ssDNA to produce antisense-type gene regulating moieties.
  • Reagents that bind sequence-specifically to dsDNA, RNA, or ssDNA have applications as gene targeted drugs useful for modulating metabolic processes such as metabolic regulatory dysfunctions, such as cancer. Alterations in the cellular genes which directly or indirectly control cell growth and differentiation are considered to be the main cause of cancer.
  • oncogenes which are implicated in human tumor formation.
  • the ras gene family are frequently found to be mutated in human tumors. In their normal state, proteins produced by the ras genes are thought to be involved in normal cell growth and maturation.
  • ras gene causing an amino acid alteration at one of three critical positions in the protein product, results in conversion to a form which is implicated in tumor formation.
  • a gene having such a mutation is said to be "activated.” It is thought that such a point mutation leading to ras activation can be induced by carcinogens or other environmental factors .
  • pancreatic adenocarcinomas about 50% of adenomas and adenocarcinomas of the colon, about 50% of adenocarcinomas of the lung and carcinomas of the thyroid, and a large fraction of malignancies of the blood such as acute myeloid leukemia and myelodysplastic syndrome have been found to contain activated ras oncogenes.
  • some 10 to 20% of human tumors have a mutation in one of the three ras genes (H-ras, K-ras, or N-ras) .
  • Antisense oligonucleotide inhibition of oncogenes has proven to be a useful tool in understanding the roles of various oncogene families.
  • Antisense oligonucleotides refers to small oligonucleotides which are complementary to the "sense” or coding strand of a given gene, and as a result are also complementary to, and thus able to specifically hybridize with, the mRNA transcript of the gene. Holt et al., Mol . Cell Biol .
  • PCT/US88/01024 discloses phosphorothioate oligonucleotide analogs hybridizable to the translation initiation region of the amplified c-myc oncogene to inhibit HL-60 leukemia cell growth and DNA synthesis in these cells. Tidd et al .
  • Anti - Cancer Drug Design 3, 117-127, 1988, evaluated antisense oligonucleotide methylphosphonate analogs hybridizing specifically to the activated N-ras oncogene and found that while they were resistant to biochemical degradation and were nontoxic in cultured human HT29 cells, they did not inhibit N-ras gene expression and had no effect on these cells.
  • Chang et al . Anti -Cancer Drug Design, 4, 221-232, 1989, showed that both methylphosphonate and phosphorothioate analogs of oligonucleotides hybridizing specifically to mRNA transcripts of the Balb-ras gene could inhibit translation of the protein product of this gene in vi tro .
  • the H-ras gene has recently been implicated in a serious cardiac arrhythmia called long Q-T syndrome, a hereditary condition which often causes sudden death if treatment is not given immediately. Frequently there are no symptoms prior to the onset of the erratic heartbeat. Whether the H-ras gene is precisely responsible for long Q-T syndrome is unclear. However, there is an extremely high correlation between inheritance of this syndrome and the presence of a particular variant of the chromosome 11 region surrounding the H-ras gene. This makes the H-ras gene an excellent indicator of increased risk of sudden cardiac death due to the long Q-T syndrome.
  • compositions of matter which can modulate the expression of the ras gene and particularly to provide compositions of matter which specifically modulate the expression of the activated form of the ras gene. It is greatly desired to provide methods of diagnosis and detection of the ras gene in animals. It is also desired to provide methods of diagnosis and treatment of conditions arising from ras gene activation. In addition, improved research kits and reagents for detection and study of the ras gene are desired.
  • the present invention provides oligomers comprising peptide nucleic acids (PNAs) , that bind complementary ssDNA and RNA strands through their oligoribonucleotide ligands which are linked to a peptide backbone.
  • PNAs peptide nucleic acids
  • the sequence of the oligoribonucleotide ligands specifies the target to which they bind.
  • oligomers are provided that are specifically hybridizable with DNA or RNA deriving from the human ras gene. Such oligomers are conveniently and desirably presented in a pharmaceutically acceptable carrier.
  • aspects of the invention are directed to methods for modulating the expression of the human ras gene in cells or tissues and for specifically modulating the expression of the activated ras gene in cells or tissues suspected of harboring a mutation leading to such activation. Additional aspects of the invention are directed to methods of detection of the ras gene in cells or tissues and specific detection of the activated ras gene in cells or tissues suspected of harboring said mutated gene. Such methods comprise contacting cells or tissues suspected of containing the human ras gene with oligomers in accordance with the invention in order to interfere with the effect of or detect said gene. Other aspects of the invention are directed to methods for diagnostics and therapeutics of animals suspected of having a mutation leading to activation of the ras gene. Such methods comprise contacting the animal or cells or tissues or a bodily fluid from the animal with oligomers in accordance with the invention in order to modulate the expression of this gene, to treat conditions arising from activation of this gene, or to effect a diagnosis thereof.
  • Such cancer-causing genes, or oncogenes are believed to acquire transformation-inducing properties through mutations leading to changes in the regulation of expression of their protein products.
  • changes occur in non-coding DNA regulatory domains, such as promoters and enhancers, leading to alterations in the transcriptional activity of oncogenes, resulting in over- or under-expression of their gene products.
  • gene mutations occur within the coding regions of oncogenes, leading to the production of altered gene products that are inactive, overactive, or exhibit an activity that is different from the normal (wild- type) gene product.
  • more than 30 cellular oncogene families have been identified. These genes can be categorized on the basis of both their subcellular location and the putative mechanism of action of their protein products.
  • the ras oncogenes are members of a gene family which encode related proteins that are localized to the inner face of the plasma membrane. ras proteins have been shown to be highly conserved at the amino acid level, to bind GTP with high affinity and specificity, and to possess GTPase activity. Although the cellular function of ras gene products is unknown, their biochemical properties, along with their significant sequence homology with a class of signal- transducing proteins known as GTP binding proteins, or G proteins, suggest that ras gene products play a fundamental role in basic cellular regulatory functions relating to the transduction of extracellular signals across plasma membranes.
  • H-ras Three ras genes, designated H-ras, K-ras, and N- ras, have been identified in the mammalian genome. Mammalian ras genes acquire transformation-inducing properties by single point mutations within their coding sequences. Mutations in naturally occurring ras oncogenes have been localized to codons 12, 13, and 61. The most commonly detected activating ras mutation found in human tumors is in codon 12 of the H-ras gene in which a base change from GGC to GTC results in a glycine-to-valine substitution in the GTPase regulatory domain of the ras protein product.
  • the present invention provides oligomers for inhibition of human ras gene expression. Oligomers of the present invention are comprised of at least one subunit having the formula:
  • L is one of the adenine, thymine, cytosine or guanine heterocyclic bases of the oligomer
  • C is (CR 6 R 7 ) y where R 6 is hydrogen and R 7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R 6 and R 7 are independently selected from the group consisting of hydrogen, (C 2 -C 6 ) alkyl, aryl, aralkyl, heteroaryl, hydroxy, alkoxy, (C x - C 6 ) alkylthio, NR 3 R 4 and SR 5 , where each of R 3 and R 4 is independently selected from the group consisting of hydrogen, (C 1 -C 4 ) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C 1 -C 4 ) alkyl, hydroxy, alkoxy, alkylthio and amino; and R 5 is hydrogen, (C ⁇ -Cg) alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C- L -Cg) alkyl, or R 6 and R 7 taken together complete an
  • D is (CR 6 R 7 ) Z where R 6 and R 7 are as defined above; each of y and z is zero or an integer from 1 to 10, the sum y + z being greater than 2 but not more than 10;
  • G is -NR 3 CO-, -NR 3 CS-, -NR 3 SO- or -NR 3 S0 2 -, in either orientation, where R 3 is as defined above; each pair of A and B is selected such that : (a) A is a group of formula (Ila) , (lib) or (lie) and B is N or R 3 N ⁇ - or (b) A is a group of formula (lid) and B is CH;
  • X is O, S, Se, NR 3 , CH 2 or C(CH 3 ) 2 ;
  • Y is a single bond, 0, S or NR 4 ; each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; each of r and s is zero or an integer from 1 to 5,, the sum r+s being not more than 10; and each R 1 and R 2 is independently selected from the group consisting of hydrogen, (C- L -C J ) alkyl which may be hydroxy- or alkoxy- or alkylthio- substituted, hydroxy, alkoxy, alkylthio, amino and halogen.
  • PNA subunit peptide nucleic acid subunits
  • Preferred oligomers of the present invention are oligomers in which substantially all subunits of the oligomer are subunits as described in Formula I, i.e. PNA subunits. Oligomers of the present invention may also comprise one or more subunits which are naturally occuring nucleotides or nucleotide analogs as long as at least one subunit satisfies Formula I. Thus, oligomers as used herein may refer to a range of oligomers from oligomers comprising only one PNA subunit as defined in Formula I to oligomers in which every subunit is a PNA subunit as defined in Formula I.
  • Those subunits which are not PNA subunits preferably comprise naturally occuring bases, sugars, and intersugar (backbone) linkages as well as non-naturally occurring portions which function similarly to naturally occurring portions. Sequences of oligomers of the present invention are defined by reference to the L group (for PNA subunits) or nucleobase (for nucleotide subunits) at a given position.
  • the nomeclature is modeled after traditional nucleotide nomenclature, identifying each PNA subunit by the identity of its L group such as the heterocycles adenine (A) , thymine (T) , guanine (G) and cytosine (C) and identifying nucleotides or nucleosides by these same heterocycle residing on the sugar backbone.
  • L group such as the heterocycles adenine (A) , thymine (T) , guanine (G) and cytosine (C) and identifying nucleotides or nucleosides by these same heterocycle residing on the sugar backbone.
  • the sequences are conveniently provided in traditional 5' to 3' or amino to carboxy orientation.
  • Oligomers of the present invention may range in size from about 5 to about 50 subunits in length. In other embodiments of the present invention, oligomers may range in size from about 10 to about 30 subunits in length. In still other embodiments of the present invention oligomers may range in size from about 10 to about 25 subunits in length. In yet further embodiments of the present invention, oligomers may range in size from about 12 to about 20 subunits in length.
  • Solid- Phase Synthesis the principle of anchoring molecules onto a solid matrix, which helps in accounting for intermediate products during chemical transformations, is known as Solid- Phase Synthesis or Merrifield Synthesis (see, e . g. , Merrifield, J “ . Am . Chem . Soc , 1963, 85, 2149 and Science, 1986, 232 , 341) .
  • Established methods for the stepwise or fragmentwise solid-phase assembly of amino acids into peptides normally employ a beaded matrix of slightly cross- linked styrene-divinylbenzene copolymer, the cross-linked copolymer having been formed by the pearl polymerization of styrene monomer to which has been added a mixture of divinylbenzenes. A level of 1-2% cross-linking is usually employed.
  • Such a matrix also can be used in solid-phase PNA synthesis in accordance with the present invention.
  • benzhydrylamino functionality (Pietta, et al . , J. Chem. Soc , 1970, 650) are the most widely applied. Regardless of its nature, the purpose of the functionality is normally to form an anchoring linkage between the copolymer solid support and the C-terminus of the first amino acid to be coupled to the solid support. As will be recognized, anchoring linkages also can be formed between the solid support and the amino acid N-terminus. It is generally convenient to express the "concentration" of a functional group in terms of millimoles per gram (mmol/g) . Other reactive functionalities which have been initially introduced include 4-methylbenzhydrylamino and 4-methoxybenzhydrylamino.
  • Preferred methods for PNA synthesis employ aminomethyl as the initial functionality, in that aminomethyl is particularly advantageous with respect to the incorporation of "spacer” or “handle” groups, owing to the reactivity of the amino group of the aminomethyl functionality with respect to the essentially quantitative formation of amide bonds to a carboxylic acid group at one end of the spacer-forming reagent.
  • spacer- or handle-forming bifunctional reagents have been described (see, Barany, et al . , Int . J. Peptide Protein Res . , 1987, 30 , 705) , especially reagents which are reactive towards amino groups such as found in the aminomethyl function.
  • bifunctional reagents include 4- (haloalkyl) aryl-lower alkanoic acids such as 4- (bromomethyl)phenylacetic acid, Boc-aminoacyl-4- (oxymethyl) aryl-lower alkanoic acids such as Boc-aminoacyl-4- (oxymethyl)phenylacetic acid, N-Boc-p-acylbenzhydrylamines such as N-Boc-p-glutaroylbenzhydrylamine, N-Boc-4' -lower alkyl-p-acylbenzhydrylamines such as N-Boc-4' -methyl-p- glutaroylbenzhydrylamine, N-Boc-4' -lower alkoxy-p-acylbenz- hydrylamines such as N-Boc-4' -methoxy-p-glutaroyl-benzhy- drylamine, and 4-hydroxymethylphenoxyacetic acid.
  • One type of spacer group particularly relevant within the context of the present invention is the phenylacetamidomethyl (Pam) handle (Mitchell and Merrifield, J. Org. Chem. , 1976, 41 , 2015) which, deriving from the electron withdrawing effect of the 4-phenylacetamidomethyl group, is about 100 times more stable than the classical benzyl ester linkage towards the Boc-amino deprotection reagent trifluoroacetic acid (TFA) .
  • benzhydrylamino, 4- methylbenzhydrylamino and 4-methoxybenzhydrylamino which may be incorporated for the purpose of cleavage of a synthesized PNA chain from the solid support such that the C-terminal of the PNA chain is in amide form, require no introduction of a spacer group. Any such functionality may advantageously be employed in the context of the present invention.
  • spacer or handle groups An alternative strategy concerning the introduction of spacer or handle groups is the so-called "preformed handle” strategy (see, Tarn, et al . , Synthesis, 1979, 955- 957) , which offers complete control over coupling of the first amino acid, and excludes the possibility of complications arising from the presence of undesired functional groups not related to the peptide or PNA synthesis.
  • spacer or handle groups of the same type as described above, are reacted with the first amino acid desired to be bound to the solid support, the amino acid being N-protected and optionally protected at the other side-chains which are not relevant with respect to the growth of the desired PNA chain.
  • the first amino acid to be coupled to the solid support can either be coupled to the free reactive end of a spacer group which has been bound to the initially introduced functionality (for example, an aminomethyl group) or can be reacted with the spacer- forming reagent.
  • the space-forming reagent is then reacted with the initially introduced functionality.
  • Other useful anchoring schemes include the "multidetachable" resins (Tarn, et al . , Tetrahedron Lett . , 1979, 4935 and J. Am. Chem. Soc , 1980, 102, 611; Tarn, J. Org. Chem. , 1985, 50 , 5291) , which provide more than one mode of release and thereby allow more flexibility in synthetic design.
  • Suitable choices for N-protection are the tert- butyloxycarbonyl (Boc) group (Carpino, J " . Am. Chem . Soc . ,
  • the optical integrity, if any, of the incoming amino acid should preferably be highly preserved upon coupling.
  • side-chain protecting groups in general, depends on the choice of the amino protecting group, since the protection of side-chain functionalities must withstand the conditions of the repeated amino deprotection cycles. This is true whether the overall strategy for chemically assembling PNA molecules relies on, for example, differential acid stability of amino and side-chain protecting groups (such as is the case for the above- mentioned "Boc-benzyl” approach) or employs an orthogonal, that is, chemoselective, protection scheme (such as is the case for the above-mentioned "Fmoc-tBu” approach) ,
  • Novel monomer synthons may be selected from the group consisting of amino acids, diacids and diamines having general formulae:
  • the temporary protecting group, such as a Boc or Fmoc group, on the last-coupled amino acid is quantitatively removed by a suitable treatment, for example, by acidolysis, such as with trifluoroacetic acid, in the case of Boc, or by base treatment, such as with piperidine, in the case of Fmoc, so as to liberate the N-terminal amine function.
  • a suitable treatment for example, by acidolysis, such as with trifluoroacetic acid, in the case of Boc, or by base treatment, such as with piperidine, in the case of Fmoc, so as to liberate the N-terminal amine function.
  • the next desired N-protected amino acid is then coupled to the N-terminal of the last-coupled amino acid.
  • This coupling of the C-terminal of an amino acid with the N- terminal of the last-coupled amino acid can be achieved in several ways. For example, it can be bound by providing the incoming amino acid in a form with the carboxyl group activated by any of several methods, including the initial formation of an active ester derivative such as a 2,4,5- trichlorophenyl ester (Pless, et al . , Helv. Chim. Acta, 1963, 46, 1609) , a phthalimido ester (Nefkens, et al . , J. Am . Chem.
  • the carboxyl group of the incoming amino acid can be reacted directly with the N-terminal of the last-coupled amino acid with the assistance of a condensation reagent such as, for example, dicyclohexylcarbodiimide (Sheehan, et al . , J. Am. Chem . Soc , 1955, 77, 1067) or derivatives thereof.
  • a condensation reagent such as, for example, dicyclohexylcarbodiimide (Sheehan, et al . , J. Am. Chem . Soc , 1955, 77, 1067) or derivatives thereof.
  • BOP Benzotriazolyl N- oxytrisdimethylaminophosphonium hexafluorophosphate
  • Castro's reagent see, e . g. , Rivaille, et al .
  • stepwise chain building of achiral PNAs such as those based on aminoethylglycyl backbone units can start either from the N-terminus or the C-terminus, because the coupling reactions are free of racemization.
  • syntheses commencing at the C-terminus typically employ protected amine groups and free or activated acid groups
  • syntheses commencing at the N-terminus typically employ protected acid groups and free or activated amine groups.
  • PEPS polyethylene
  • PS pendant long-chain polystyrene
  • the loading capacity of the film is as high as that of a beaded matrix, but PEPS has the additional flexibility to suit multiple syntheses simultaneously.
  • the PEPS film is fashioned in the form of discrete, labeled sheets, each serving as an individual compartment. During all the identical steps of the synthetic cycles, the sheets are kept together in a single reaction vessel to permit concurrent preparation of a multitude of peptides at a rate close to that of a single peptide by conventional methods. It was reasoned that the PEPS film support, comprising linker or spacer groups adapted to the particular chemistry in question, should be particularly valuable in the synthesis of multiple PNA molecules, these being conceptually simple to synthesize since only four different reaction compartments are normally required, one for each of the four "pseudo- nucleotide" units.
  • the PEPS film support has been successfully tested in a number of PNA syntheses carried out in a parallel and substantially simultaneous fashion.
  • the yield and quality of the products obtained from PEPS were comparable to those obtained by using the traditional po ⁇ lystyrene beaded support.
  • experiments with other geometries of the PEPS polymer such as, for example, non ⁇ woven felt, knitted net, sticks or microwellplates have not indicated any limitations of the synthetic efficacy.
  • solid supports which may be of relevance are: (1) Particles based upon copolymers of dimethylacrylamide cross-linked with N,N'- bisacryloylethylenediamine, including a known amount of N- tertbutoxycarbonyl-beta-alanyl-N' - acryloylhexamethylenediamine.
  • spacer molecules are typically added via the beta alanyl group, followed thereafter by the amino acid residue subunits.
  • the beta alanyl-containing monomer can be replaced with an acryloyl sarcosine monomer during polymerization to form resin beads.
  • the polymerization is followed by reaction of the beads with ethylenediamine to form resin particles that contain primary amines as the covalently linked functionali ⁇ ty.
  • the polyacrylamide-based supports are relatively more hydrophilic than are the polystyrene-based supports and are usually used with polar aprotic solvents including dimethyl ⁇ formamide, dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, et al . , J. Am . Chem. Soc , 1975, 97, 6584, Bioorg. Chem.
  • a second group of solid supports is based on silica-containing particles such as porous glass beads and silica gel.
  • silica-containing particles such as porous glass beads and silica gel.
  • One example is the reaction product of trich- loro- [3- (4-chloromethyl)phenyl]propylsilane and porous glass beads (see Parr and Grohmann, Angew. Chem. Internal . Ed. 1972, 11 , 314) sold under the trademark "PORASIL E” by Waters Associates, Framingham, MA, USA.
  • solid- phase PNA synthesis in the context of the present invention is normally performed batchwise. However, most of the syn- theses may equally well be carried out in the continuous-flow mode, where the support is packed into columns (Bayer, et al . , Tetrahedron Lett . , 1970, 4503 and Scott, et al . , J. Chromatogr. Sci . , 1971, 9 , 577) .
  • the rigid poly(dimethylacrylami- de) -Kieselguhr support Atherton, et al . , J. Chem . Soc Chem . Commun .
  • solid-phase technique is presently preferred in the context of PNA synthesis, other methodologies or combinations thereof, for example, in combination with the solid-phase technique, apply as well:
  • PNA molecules may be assembled enzymatically by enzymes such as proteases or derivatives thereof with novel specificities (obtained, for example, by artificial means such as protein engineering) .
  • PNA ligases for the condensation of a number of PNA fragments into very large PNA molecules; (6) since antibodies can be generated to virtually any molecule of interest, the recently developed catalytic antibodies (abzymes) , discovered simultaneously by the groups of Lerner (Tramantano, et al . , Science, 1986, 234 , 1566) and of Schultz (Pollack, et al . , Science, 1986, 234 , 1570) , should also be considered as potential candidates for assembling PNA molecules.
  • there has been considerable success in producing abzymes catalyzing acyl-transfer reactions see for example Shokat, et al .
  • Peptide nucleic acid oligomers hybridizable with, or targeted to, ras genes such as H-ras and K-ras are provided by the present invention.
  • hybridizable is meant that at least 70% sequence homology is present.
  • peptide nucleic acid oligomers have at least 85% sequence homology to a desired target.
  • peptide nucleic acid oligomers of the present invention are at least 95% homologous to a target of interest .
  • the oligomers of this invention are designed to be hybridizable with messenger RNA derived from the ras gene, and especially from the H-ras and K-ras genes. Such hybridization, when accomplished, interferes with the normal roles of the messenger RNA to cause a loss of its function in the cell .
  • the functions of messenger RNA to be interfered with include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and possibly even independent catalytic activity which may be engaged in by the RNA.
  • the overall effect of such interference with the RNA function is to interfere with expression of the ras genes, and particularly the genes of H-ras and K-ras.
  • the nucleic acid sequences of the three ras genes while not identical, are known, and persons of ordinary skill in the art will be able to use this invention as a guide in preparing oligomers hybridizable with all three ras genes.
  • the oligomers of this invention can be used in diagnostics, therapeutics and as research reagents and kits. Since the oligomers of this invention hybridize to the ras gene, sandwich and other assays can easily be constructed to exploit this fact. Furthermore, since the oligomers of this invention hybridize preferentially to the mutant (activated) form of the ras oncogene, such assays can be devised for screening of cells and tissues for ras conversion from wild- type to activated form. Such assays can be utilized for differential diagnosis of morphologically similar tumors, and for detection of increased risk of cancer stemming from ras gene activation. Provision of means for detecting hybridization of oligomers with the ras gene can routinely be accomplished.
  • Kits for detecting the presence or absence of ras or activated ras may also be prepared.
  • a series of PNA oligomers targeted to the H-ras translation initiation codon (AUG) , codon-12 point mutation or the stem and loop of the mRNA hairpin of activated H-ras were identified having specific sequences. The sequences, SEQ ID NO: 1
  • Oligomers of the invention can be formulated in a pharmaceutical composition, which can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the oligonucleotide.
  • Pharmaceutical compositions also can include one or more active ingredients such as antimicrobial agents, anti- inflammatory agents, anesthetics, and the like in addition to oligomer.
  • the pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration can be topically (including opthalmically, vaginally, rectally, intranasally) , orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms may also be useful .
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Formulations for parenteral administration can include sterile aqueous solutions which also can contain buffers, diluents and other suitable additives. Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates.
  • PNA subunits for oligomers of the invention were prepared generally in accordance with the methods disclosed by WO 92/20702, incorporated by reference herein and described above.
  • Benzyhydrylamine resin (initially loaded 0.28 mmol/gm with Boc-L-Lys(2- chlorobenyloxycarbonyl) ) was swollen in DMF and an excess of a monomer to be coupled was added, followed by dicyclohexyl- carbodiimide (0.15M in 50% DMF in dichloromethane) .
  • the Boc deprotection was accomplished by trifluoroacetic acid treatment .
  • the progress of the coupling reactions was monitored by quantitative ninhydrin analysis.
  • the PNA was released from the resin using anhydrous HF under standard conditions.
  • the products were purified using HPLC with acetonitrile-water (0.1%TFA) gradient and structure confirmed by fast atom bombardment mass spectrometry.
  • PNA homopolymer has the structure:
  • the ras-luciferase reporter genes are assembled using PCR technology. Oligonucleotide primers are synthesized for use as primers for PCR cloning of the 5'- regions of exon 1 of both the mutant (codon 12) and non- mutant (wild-type) human H-ras genes.
  • the plasmids pT24-C3, containing the c-H-rasl activated oncogene (codon 12, GGC ⁇ GTC) , and pbc-Nl, containing the c-H-ras proto-oncogene, are obtained from the American Type Culture Collection (Bethesda, MD) .
  • the plasmid pT3/T7 luc containing the 1.9 kb firefly luciferase gene, is obtained from Clontech Laboratories (Palo Alto, CA) .
  • the oligonucleotide PCR primers are used in standard PCR reactions using mutant and non-mutant H-ras genes as templates. These primers produce a DNA product of 145 base pairs corresponding to sequences -53 to +65 (relative to the translational initiation site) of normal and mutant H-ras, flanked by Nhel and HindiII restriction endonuclease sites.
  • the PCR product is gel purified, precipitated, washed and resuspended in water using standard procedures.
  • PCR primers for the cloning of the P. pyralis (firefly) luciferase gene are designed such that the PCR product will code for the full-length luciferase protein with the exception of the amino-terminal methionine residue, which would be replaced with two amino acids, an amino-terminal lysine residue followed by a leucine residue.
  • the oligonucleotide PCR primers used for the cloning of the luciferase gene are used in standard PCR reactions using a commercially available plasmid (pT3/T7-Luc) (Clontech) , containing the luciferase reporter gene, as a template.
  • primers yield a product of approximately 1.9 kb corresponding to the luciferase gene, flanked by unique Hindlll and BssHII restriction endonuclease sites. This fragment is gel purified, precipitated, washed and resuspended in water using standard procedures.
  • the ras and luciferase PCR products are digested with the appropriate restriction endonucleases and cloned by three-part ligation into an expression vector containing the steroid-inducible mouse mammary tumor virus promotor MMTV using the restriction endonucleases Nhel, Hindlll and BssHII.
  • the resulting clone results in the insertion of H-ras 5' sequences (-53 to +65) fused in frame with the firefly luciferase gene.
  • the resulting expression vector encodes a ras-luciferase fusion product which is expressed under control of the steroid-inducible MMTV promoter.
  • These plasmid constructions contain sequences encoding amino acids 1-22 of activated (RA2) or normal (RA4) H-ras proteins fused in frame with sequences coding for firefly luciferase. Translation initiation of the ras- luciferase fusion mRNA is dependent upon the natural H-ras AUG codon. Both mutant and normal H-ras luciferase fusion constructions are confirmed by DNA sequence analysis using standard procedures.
  • a total of 10 ⁇ g or 12 ⁇ g of DNA is added to each dish, of which 1 ⁇ g was a vector expressing the rat glucocorticoid receptor under control of the constitutive Rous sarcoma virus (RSV) promoter and the remainder is ras-luciferase reporter plasmid.
  • RSV Rous sarcoma virus
  • Calcium phosphate-DNA coprecipitates are removed after 16-20 hours by washing with Tris-buffered saline [50 Mm Tris-Cl (pH 7.5), 150 mM NaCl] containing 3 mM EGTA. Fresh medium supplemented with 10% fetal bovine serum is then added to the cells. At this time, cells are pre-treated with PNA oligomers prior to activation of reporter gene expression of dexamethasone.
  • the PNA oligomers will be expected to be more stable than conventional antisense oligonucleotides with respect to degradation.
  • the PNA oligomers also will be expected to be better membrane penetrators than conventional antisense oligonucleotides.
  • plasmid transfection cells are washed with phosphate buffered saline (PBS) prewarmed to 37°C and Opti-MEM containing 5 ⁇ g/mL N- [1- (2, 3-dioleyloxy)propyl] - N,N,N, -trimethylammonium chloride (DOTMA) is added to each plate (1.0 ml per well) .
  • PBS phosphate buffered saline
  • Opti-MEM containing 5 ⁇ g/mL N- [1- (2, 3-dioleyloxy)propyl] - N,N,N, -trimethylammonium chloride (DOTMA) is added to each plate (1.0 ml per well) .
  • GCCCACACCGACGGCGCCCAC (SEQ ID NO: 9) , CACACCGACGGCGCC (SEQ ID NO: 12), CCACACCGACGGCGCCC (SEQ ID NO: 13), CCCACACCGACGGCGCCCA (SEQ ID NO: 14) , CCACACCGACGGCGCC (SEQ ID NO: 21) , CACACCGACGGCCC (SEQ ID NO: 22) , CCCACACCGACGGCGCCC (SEQ ID NO: 23) , CCACACCGACGGCGCCCA (SEQ ID NO: 24) and TATTCCGTCATCGCTCCTCA (SEQ ID NO: 25) , are added from 50 ⁇ M stocks to each plate and incubated for 4 hours at 37°C.
  • Human colon carcinoma cell lines Calu 1, SW480 and SW620 are obtained from the American Type Culture Collection (ATCC) and cultured and maintained as monolayers on 6-well plates in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/ml penicillin.
  • DMEM Dulbecco's Modified Eagle's medium
  • Cells are treated with PNA oligomers prepared as described in Example 1 and having sequences as set forth in Table 3 and K-ras mRNA expression is measured by Northern blot analysis 24 hours later.
  • cells are treated with a single dose of PNA oligomer at day zero and monitored over a five-day period.
  • SW480 cells and HeLa cells are cultured as in Example 6.
  • Cells are treated with a single dose of PNA oligomers prepared as described in Example 1 and having sequence as set forth in Table 3 and mRNA levels are determined by Northern blot analysis 24 hours later.
  • the human urinary bladder cancer cell line T24 is obtained from the American Type Culture Collection (Rockville MD) .
  • Cells are grown in McCoy's 5A medium with L-glutamine (Gibco BRL, Gaithersburg MD) , supplemented with 10% heat- inactivated fetal calf serum and 50 U/ml each of penicillin and streptomycin.
  • Cells are seeded on 100 mm plates. When they reach 70% confluency, they are treated with the aforementioned PNA oligomers directed to H-ras as described in Example 5. Plates are washed with 10 ml prewarmed PBS and 5 ml of Opti-MEM reduced-serum medium containing 2.5 ⁇ l DOTMA.
  • PNA oligomer is then added to the desired concentration. After 4 hours of treatment, the medium is replaced with McCoy's medium. Cells are harvested 48 hours after oligomer treatment and RNA is isolated using a standard CsCl purification method.
  • Kingston, R.E. in Current Protocols in Molecular Biology, (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.A. Smith, J.G. Seidman and K. Strahl, eds.) , John Wiley and Sons, NY.
  • the human epithelioid carcinoma cell line HeLa 229 is obtained from the American Type Culture Collection (Bethesda, MD) . HeLa cells are maintained as monolayers on 6-well plates in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 100 U/ml penicillin. Treatment with the PNA oligomers directed to H- ras as described in Example 5 and isolation of RNA are essentially as described above for T24 cells.
  • Northern hybridization 10 ⁇ g of each RNA is electrophoresed on a 1.2% agarose/formaldehyde gel and transferred overnight to GeneBind 45 nylon membrane (Pharmacia LKB, Piscataway, NJ) using standard methods.
  • RNA is UV-crosslinked to the membrane.
  • Double- stranded 32 P-labeled probes are synthesized using the Prime a Gene labeling kit (Promega, Madison WI) .
  • the ras probe is a SalI-Nhel fragment of a cDNA clone of the activated (mutant) H-ras mRNA having a GGC-to-GTC mutation at codon-12.
  • the control probe is G3PDH.
  • Blots are prehybridized for 15 minutes at 68°C with the QuickHyb hybridization solution (Stratagene, La Jolla, CA) .
  • the heat-denatured radioactive probe (2.5 x 10 6 counts/2 ml hybridization solution) mixed with 100 ⁇ l of 10 mg/ml salmon sperm DNA is added and the membrane is hybridized for 1 hour at 68°C.
  • the blots are washed twice for 15 minutes at room temperature in 2x SSC/0.1% SDS and once for 30 minutes at 60°C with 0.1XSSC/0.1%SDS. Blots are autoradiographed and the intensity of signal is quantitated using an ImageQuant PhosphorImager (Molecular Dynamics, Sunnyvale, CA) .
  • Northern blots are first hybridized with the ras probe, then stripped by boiling for 15 minutes in 0.Ix SSC/0.1%SDS and rehybridized with the control G3PDH probe to check for correct sample loading.
  • Cells are cultured and treated with the aforementioned PNA oligomers directed to ras essentially as described in Example 8. Cells are seeded on 60 mm plates and are treated with PNA oligomer in the presence of DOTMA when they reach 70% confluency.
  • Time course experiment On day 1, cells are treated with a single dose of PNA oligomer at a final concentration of 100 nM. The growth medium is changed once on day 3 and cells are counted every day for 5 days, using a counting chamber.
  • Dose-response experiment Various concentrations of PNA oligomer (10, 25, 50, 100 or 250 nM) are added to the cells and cells are harvested and counted 3 days later.

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Abstract

La présente invention a pour objet des oligomères utiles pour moduler l'expression du gène ras humain dans les formes à la fois normale et activée, les oligomères étant composés de sous-unités, dont l'une au moins est une sous-unité d'acide nucléique protéique. Les oligomères de ce type peuvent être utilisés à des fins de diagnostic ainsi que de recherche. Des procédés sont également décrits pour moduler l'expression du gène ras dans les cellules et les tissus à l'aide des oligomères produits, et pour la modulation spécifique de l'expression du gène ras activé. Des procédés de diagnostic, de détection et de traitement des états résultant de l'activation des gènes ras-H et ras-K sont également divulgués.
PCT/US1994/006620 1993-06-11 1994-06-10 Oligomeres de modulation de l'oncogene ras WO1994028720A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5536750A (en) * 1992-10-29 1996-07-16 Merck & Co., Inc. Inhibitors of farnesyl-protein transferase
US5576313A (en) * 1994-08-29 1996-11-19 Merck & Co., Inc. Inhibitors of farnesyl-protein transferase
US7713738B2 (en) 2003-02-10 2010-05-11 Enzon Pharmaceuticals, Inc. Oligomeric compounds for the modulation of survivin expression
US8173428B2 (en) 2004-11-09 2012-05-08 Santaris Pharma A/S LNA oligonucleotides and the treatment of cancer
US20120295954A1 (en) * 2010-01-04 2012-11-22 Joseph Collard Treatment of interferon regulatory factor 8 (irf8) related diseases by inhibition of natural antisense transcript to irf8

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4871838A (en) * 1985-07-23 1989-10-03 The Board Of Rijks Universiteit Leiden Probes and methods for detecting activated ras oncogenes
US5087617A (en) * 1989-02-15 1992-02-11 Board Of Regents, The University Of Texas System Methods and compositions for treatment of cancer using oligonucleotides
WO1992020702A1 (fr) * 1991-05-24 1992-11-26 Ole Buchardt Acides nucleiques de peptides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4871838A (en) * 1985-07-23 1989-10-03 The Board Of Rijks Universiteit Leiden Probes and methods for detecting activated ras oncogenes
US5087617A (en) * 1989-02-15 1992-02-11 Board Of Regents, The University Of Texas System Methods and compositions for treatment of cancer using oligonucleotides
WO1992020702A1 (fr) * 1991-05-24 1992-11-26 Ole Buchardt Acides nucleiques de peptides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANGEWANDTE CHEMIE, Volume 31, Number 4, issued August 1992, MEIER et al., "Peptide Nucleic Acids (PNAs)-Unusual Properties of Nonionic Oligonucleotide Analogues", pages 1008-1010. *
ANTI-CANCER DRUG DESIGN, Volume 4, issued March 1989, CHANG et al., "Comparative Inhibition of Ras p21 Protein Synthesis with Phosphorus-Modified Antisense Oligonucleotides", pages 221-232. *
CHEMICAL REVIEWS, Volume 90, Number 4, issued June 1990, UHLMANN et al., "Antisense Oligonucleotides: A New Therapeutic Principle", pages 543-584. *
SCIENCE, Volume 254, issued 06 December 1991, NIELSEN et al., "Sequence-Selective Recognition of DNA by Strand Displacement with a Thymine-Substituted Polyamide", pages 1497-1500. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5536750A (en) * 1992-10-29 1996-07-16 Merck & Co., Inc. Inhibitors of farnesyl-protein transferase
US5576313A (en) * 1994-08-29 1996-11-19 Merck & Co., Inc. Inhibitors of farnesyl-protein transferase
US7713738B2 (en) 2003-02-10 2010-05-11 Enzon Pharmaceuticals, Inc. Oligomeric compounds for the modulation of survivin expression
US7741309B2 (en) 2003-02-10 2010-06-22 Enzon Pharmaceuticals Oligomeric compounds for the modulation of survivin expression
US8026355B2 (en) 2003-02-10 2011-09-27 Santaris Pharma A/S Oligomeric compounds for the modulation of survivin expression
US8173428B2 (en) 2004-11-09 2012-05-08 Santaris Pharma A/S LNA oligonucleotides and the treatment of cancer
US20120295954A1 (en) * 2010-01-04 2012-11-22 Joseph Collard Treatment of interferon regulatory factor 8 (irf8) related diseases by inhibition of natural antisense transcript to irf8
US8946181B2 (en) * 2010-01-04 2015-02-03 Curna, Inc. Treatment of interferon regulatory factor 8 (IRF8) related diseases by inhibition of natural antisense transcript to IRF8

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