WO1994023755A1 - Nouveaux procedes et compositions destines au traitement du cancer active par ras au moyen d'oligonucleotides heterotypiques anti-raf et antisens - Google Patents

Nouveaux procedes et compositions destines au traitement du cancer active par ras au moyen d'oligonucleotides heterotypiques anti-raf et antisens Download PDF

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WO1994023755A1
WO1994023755A1 PCT/US1994/004091 US9404091W WO9423755A1 WO 1994023755 A1 WO1994023755 A1 WO 1994023755A1 US 9404091 W US9404091 W US 9404091W WO 9423755 A1 WO9423755 A1 WO 9423755A1
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raf
oligonucleotide
seq
ras
activated
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PCT/US1994/004091
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English (en)
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Patrick L. Iversen
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Board Of Regents Of The University Of Nebraska
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Priority to AU66336/94A priority Critical patent/AU6633694A/en
Publication of WO1994023755A1 publication Critical patent/WO1994023755A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • 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
    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates to novel methods and compositions for killing cancer causing cells expressing an activated ras oncogene, and for the treatment of associated mammalian cancers. More particularly, the present invention relates to the novel use of heterotypic oligodeoxyribonucleo- tides which are complementary and antisense to a raf target gene sequence contained in the cells of ras-activated cancers, and which can be used as active ingredients in new anticancer therapeutic compositions.
  • heterotypic antisense oligonucleotide a ⁇ used herein, is meant a therapeutic antisense oligonucleotide which is specifically targeted to and binds a complementary nucleotide sequence of DNA or transcribed messenger RNA of a gene which is completely different from the cancer-causing oncogene which is known to be actively expressed in the cancer cells being treated.
  • Cancer is a one of the most-devastating and dreaded of human diseases.
  • Much of the horror engendered by this disease derives not only from the severe debilitation often associated with its advanced stages, but also from the pain and disfigurement which frequently accompanies its clinical management. This is because much of the current therapeutic modalities (surgery, radiation treatments, intensive chemotherapy) are not able to specifically and with a high degree of accuracy kill only cancer cells; rather, by their very nature, their actions are so broad that they also kill healthy, non-cancerous bystander cells.
  • RNA nucleotide bases which are complementary to the "sense" (information bearing) strand of nucleic acids have become widely recognized in recent years for their ability to inhibit the expression of specific genes (Cohen JS [editor], Oliqodeoxyribonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL, 1989).
  • "Antisense” oligonucleotides are single-stranded nucleic acids which, by hybridizing either to the complemen ⁇ tary DNA nucleotide sequence in a target gene, or, more commonly, to the messenger RNA (mRNA) transcribed from that gene, are able to completely abrogate the function of the targeted gene. Because antisense oligonucleotides target RNA or DNA rather than proteins, they are drugs that can be orders of magnitude more selective than traditional drugs, a factor which should very significantly reduce problems of unwanted side effects.
  • the current thinking in antisense oligonucleotide therapy is to utilize homologous DNA-based oligonucleotides a ⁇ therapeutic agents; i.e., as agents whose nucleotide base sequence is complementary to all or part of the nucleotide sequence of the cancer gene believed to be responsible for causing the disease.
  • the dramatic discovery of the present invention is that a heterotypic antisense oligonucleotide directed to a target gene completely unrelated to a ras oncogene can actually kill cancer cells which contain an activated ras oncogene.
  • many (although not all) of the mutated viral-related gene sequences identified a ⁇ being re ⁇ ponsible for the transformed state of the cancer cell were found to be identical with (or related to) genes normally found in many, if not all, healthy cells.
  • ra ⁇ genes have become one of the mo ⁇ t intere ⁇ ting and intensely ⁇ tudied oncogene explore ⁇ in human malignancie ⁇ (Barbacid M, Ann. Rev. Biochem. 56: 779-827, 1987). While the function of ra ⁇ oncogene ⁇ in facilitating the development of human cancer is not clear, it is known that native ras proto-oncogene ⁇ code for the ⁇ ynthesis of nearly 30 different single-chain regulatory proteins (approximate molecular weight of 21,000 daltons each, and, therefore, called "p21" proteins) which bind guanine nucleotides on the cytoplasmic side of the outer cell membrane.
  • the 30 or more ras-related small GTP-binding proteins are highly conserved in structure, whether derived from yeast cells or human tissue cells. At least three ras proteins .H-ras, Ki-ras. and N-ras) are expres ⁇ ed in most, if not all, mammalian cell types (Bos JL, Cancer Research 49: 4682-4689, 1989).
  • the raf family of proteins which have serine/threo- nine- ⁇ pecific protein kinase activity, are well known a ⁇ intracytopla ⁇ mic ⁇ ignal tran ⁇ ducers.
  • the A-raf-1 gene which is located on chromosome Xpll.3, is only expre ⁇ sed in certain tissues. These gene ⁇ code for cytosolic proteins of approximately 74,000 and 68,000 daltons, respectively.
  • FIGURE 1 i ⁇ a histogram drawing showing survival of various NIH/3T3 target cell lines on the left axis plotted against dose of a heterotypic anti-raf antisense oligonucleotide (SEQ ID NO.l) with which the target cell ⁇ were co-incubated in vitro.
  • FIGURE 2 i ⁇ made up of 3 ⁇ maller ⁇ ub-figure ⁇ .
  • FIGURE 2A i ⁇ a line drawing of cytotoxicity curves, which shows the cytotoxic effect when the heterotypic anti-raf antisense oligonucleotide of the present invention (SEQ ID NO.l) was co- incubated with NIH-3T3 cells.
  • FIGURE 2B and FIGURE 2C are drawings of control cytotoxicity curves in which different phosphorothioate oligonucleotides, antisense in sequence to an unrelated gene (coding for metallothionein) .
  • FIGURE 3 is a drawing similar to FIGURE 1, in which a histogram is presented which shows target cell survival on the left axis plotted against dose of the anti-raf oligonucleotide identified a ⁇ SEQ ID NO.2.
  • FIGURE 4 i ⁇ a similar drawing in which a histogram is presented which shows survival of the various NIH/3T3 target cells on the left axis plotted against dose of a different heterotypic anti-raf oligonucleotide, identified as SEQ ID NO.3.
  • cytotoxicaUy-effective amount an administered amount of a therapeutic oligonucleotide preparation which is well below the cytotoxic endpoint of the oligonucleotide preparation, but which is sufficient to kill ra ⁇ -activated target cell ⁇ in preference to other cell ⁇ .
  • the present invention also provides novel methods for treating an individual who has ra ⁇ -activated cancer.
  • Thi ⁇ treatment involves the use of heterotypic antisense oligonucleotide therapies, in which a cytotoxicaUy-effective amount of a preparation containing an anti-raf antisense oligonucleotide, or combination of selected anti-raf antisense oligonucleotides, or one' or more pharmaceutically-effective analog ⁇ thereof, is administered as specific drug therapy of cancers expressing an activated ras oncogene.
  • the oligonucleotide preparation is admini ⁇ tered ⁇ ystemically to the individual.
  • autologou ⁇ bone marrow cell ⁇ (or peripheral blood- derived ⁇ tem cells) from an individual having ra ⁇ -activated cancer are treated ex vivo with ⁇ pecific anti-raf anti ⁇ en ⁇ e oligonucleotides in order to kill any and all of the ras- activated malignant cell ⁇ which may be contained in the bone marrow or stem cell tran ⁇ plant specimen.
  • the treated autologou ⁇ bone marrow cells (or peripheral blood-derived stem cells) are infused back into the patient who has, in the meanwhile, received appropriate surgical, radiation, immuno- and/or chemotherapy.
  • the method for removing contaminating ras- activated cancerous cell ⁇ from the marrow cell ⁇ uspension i ⁇ straight forward comprises the steps of (i) collecting an appropriate amount of bone marrow (preferably about 1500 cc from multiple points in the pelvic iliac crest, although as little a ⁇ 500 cc and as much a ⁇ 2000 cc can be u ⁇ ed) from the individual who has the ra ⁇ -activated cancer, and isolating the nucleated cells from the bone marrow sample; (ii) contacting the nucleated bone marrow cells ex vivo (in culture) with a cytotoxically-effective amount of an anti-raf antisense oligonucleotide which ha ⁇ a base sequence complementary to the DNA or transcribed messenger RNA of a raf target gene also present in the cell ⁇ of the ra ⁇ -activated cancer (thi ⁇ incubation take ⁇ from about 12 hour
  • Thi ⁇ form of intensive therapy can be further improved by the additional step of administering systemically to the individual, after the bone marrow transplant ha ⁇ engrafted, a therapeutic preparation of this invention containing anti-raf antisense oligonucleotide, administered in an amount sufficient to kill the few ras-activated cancerous cells which remain in the individual.
  • the anti-raf antisense oligonucleotides of the present invention can be of significant clinical utility when administered systemically to individuals who have ras- activated cancers, concomitant with or following primary tumor ablation with surgery, radiation and/or chemotherapy. Additional therapeutic gains can be obtained by systemic administration of anti-raf antisense oligonucleotides to recipients of autologous bone marrow cell suspensions, after the bone marrow — itself purged of contaminating ras- activated cancer cells by treatment with anti-raf oligonucleotides — has engrafted in the individual.
  • the anti-raf antisense oligonucleotides are administered in vivo as a systemic therapy, and they can also be administered in vitro t as a procedure for eliminating contaminating ras-activated tumor cell ⁇ from a ⁇ uspension of autologous peripheral blood stem cells or autologous bone marrow cell ⁇ .
  • the physical form of the therapeutic preparation may vary, as discussed more fully hereinafter.
  • the size of the oligonucleotide i.e., the number of bases in the oligonucleotide sequence, is an important consideration.
  • the length (in base numbers) of a therapeutic oligonucleotide of the present invention ranges from at least about 8 bases to as many as about 50 bases.
  • the longer the antisen ⁇ e oligonucleotide the higher i ⁇ it ⁇ affinity for a target sequence when it binds with an exact complementarity.
  • the longer the antisense sequence being utilized the more unique i ⁇ the targeted sequence.
  • these advantages are off-set by the fact that the longer oligonucleotides are also more difficult and more costly to prepare and more difficult to handle.
  • RNA to which the selected oligonucleotide i ⁇ designed to hybridize is an important variable that will affect the practice of this invention.
  • Several criteria upon which the targeted region can be selected are: (i) thermal stability of the hybrid complex, which i ⁇ influenced by the guanine- cytosine (GC) content of the region bound; (ii) secondary structure in the mRNA, such as stem-loop ⁇ ; (iii) region ⁇ of intron-exon splicing; and (iv) sequences which are not self- complementary or palindromic.
  • certain regions of gene sequence ⁇ are more important for the proper functioning of the gene product than are other regions of that same genome. At least four critical coding regions on the DNA or transcribed mes ⁇ enger RNA mu ⁇ t be con ⁇ idered for targeting.
  • the coding region( ⁇ ) required for initiation of protein synthesis; (2) the coding region(s) which control how the protein will fold in three dimen ⁇ ions; (3) the coding region( ⁇ ) which code for the active ⁇ ite of the protein; and (4) the coding region(s) which code for termination of the protein synthe ⁇ is.
  • oligonucleotide ⁇ complementary to a genetic region which include ⁇ the initiation coding region of the raf gene are particularly effective in blocking the function of that gene.
  • an anti-raf oligonucleotide ha ⁇ a nucleotide ba ⁇ e ⁇ equence part of which is complementary to the 3-base initiation coding region of the target gene.
  • the most common initiation coding region in the mes ⁇ enger RNA i ⁇ the 3-ba ⁇ e nucleotide ⁇ equence AUG, particularly the AUG coding region neare ⁇ t the 5'-end of the mRNA (usually within 10 to 100 nucleotides of the beginning of the gene) .
  • initiation sequence in an mRNA i ⁇ a 7-ba ⁇ e ⁇ equence which incorporate ⁇ the AUG sequence comprising either AXXAUGG or GXXAUGG, where "X" is any of the four base nucleotides.
  • AUG sequence comprising either AXXAUGG or GXXAUGG, where "X" is any of the four base nucleotides.
  • GUG i ⁇ utilized a ⁇ an initiation coding region, and, very rarely, the ⁇ equences UUG or CUG perform this initiation function.
  • the corresponding complementary sequence ⁇ in the antisense DNA molecules would then be most commonly TAC, very occasionally CAC, and only rarely AAC or GAC, re ⁇ pectively.
  • the natural protein product ⁇ of functional genes have a characteristic size and 3-dimen-sional shape, which is dictated by the amino acid sequence coded by the genome. Any significant departure from thi ⁇ natural size and shape usually interferes with the natural 3-dimensional ⁇ tructure of the protein, thereby altering, if not completely abrogating, the function of that modified protein.
  • the experimental process of "denaturing" a protein in a laboratory protocol is an example of how changing the natural 3-dimensional shape of a protein molecule causes it to lose its natural function.
  • an antisense oligonucleotide which binds to any portion of the mRNA which codes for an amino acid sequence critical for creating and holding the 3- dimen ⁇ ional ⁇ hape of the serine/threonine-specific esterase protein molecule will inhibit, if not completely abrogate, the function of that estera ⁇ e molecule.
  • an antisense oligonucleotide which binds to a portion of the DNA or transcribed mRNA which codes for the active site of the raf e ⁇ tera ⁇ e molecule can al ⁇ o completely block the function of that raf protein.
  • antisense oligonucleotides complementary to a genetic region which includes the termination coding region of the raf gene being targeted will also be effective in modifying, if not completely abrogating, the function of that raf gene.
  • the mRNA coding regions which code for termination of a protein are commonly the 3-ba ⁇ e sequences of UAA, UAG, and UGA. Much less commonly the sequence ⁇ AGA and AGG code for a termination.
  • the corre ⁇ ponding complementary ⁇ equence ⁇ in the anti ⁇ en ⁇ e DNA molecules would then be ATT, ATC, ACT, TCT, and TCC, respectively.
  • anti-raf oligonucleotides of the present invention have a nucleotide base sequence part of which is complementary to the 3-base termination coding region( ⁇ ) of the targeted gene sequence.
  • Anti-raf antisense oligonucleotide selected. A ⁇ discu ⁇ ed el ⁇ ewhere herein, there are two known raf gene ⁇ in the human (see Heidecker et al., In: Gene ⁇ and Signal Tran ⁇ duction in Multi ⁇ ta ⁇ e Carcino ⁇ ene ⁇ is [NH Colburn, editor], New York: Marcel Dekker, Inc., pp 339-374, 1989). One i ⁇ designated A- raf-1, and is a gene expressed in select tissues.
  • c-raf-1 located on chromosome 3p25, it i ⁇ expres ⁇ ed in all tissue ⁇ , i ⁇ approximately 2977 nucleotide base ⁇ in length, and code ⁇ for another cyto ⁇ olic ⁇ erine/threonine kinase with an approximate molecular weight of 74,000 daltons.
  • a number of anti- raf . antisense oligonucleotides can be utilized to kill cancer cells which contain an activated ras oncogene.
  • the ⁇ equence ⁇ are herein identified with a SEQUENCE IDENTIFICATION NUMBER, and are antisen ⁇ e to portion ⁇ of either the A-raf-1 gene, or the c-raf-1 gene, as indicated.
  • nucleotide base sequences for each of the anti-raf antisense oligonucleotides listed in the 5' to 3' reading direction for the anti-raf antisense oligo-nucleotide, and, next to each of the antisense sequences, the numerical position of the base sequence in the corresponding portion of the human mes ⁇ enger RNA transcribed from the appropriate human raf gene (where position 1 i ⁇ the beginning of the gene) .
  • anti-raf antisense oligonucleotide sequence ⁇ of the pre ⁇ ent invention are described in conjunction with preferred e bodiment ⁇ and specific examples, the listing of these selected sequences is not meant to imply that they are the only ones which may be utilized in practicing this invention.
  • one of ordinary skill in the art can, in order to modify the teaching of the present invention, delete one or more nucleotide bases from a listed anti-raf antisense oligonucleotide sequence and retain complete functional capacity to kill the ras-activated cancer cells with that modified anti-raf oligonucleotide; in practicing thi ⁇ invention, one can shorten the antisense oligonucleotide by deleting nucleotide bases from the listed sequence until the killing function in an in vitro assay is lost.
  • one of ordinary skill in the art can modify the teaching of the present invention by lengthening the anti- raf antisense oligonucleotide by adding one or more nucleotide bases to a listed anti-raf oligonucleotide sequence and po ⁇ ibly retain funtional capacity to kill the ras-activated cancer cells with the longer anti-raf (modified) oligonucleo- tide.
  • nucleotide sequence of antisense oligo EC-2C 5' T C T T G G T G A G G T C G C A C T 3'
  • SEQ ID NO.3 (nucleotide sequence of antisense oligo EC-3C): 5' C A A G A A T A T A T C G A A T G A 3'
  • SEQ ID NO.4 (nucleotide sequence of antisense oligo DK-1)
  • Site 89 through 72 of the human mRNA for c-raf-1 oncogene.
  • SEQ ID NO.11 (nucleotide sequence of antisen ⁇ e oligo DK-8)
  • nucleotide ⁇ equence of anti ⁇ ense oligo DK-10 5' A C G G T A A A T G G G A A T 3' Site: 1235 through 1221 of the human mRNA for c-raf-1 oncoge
  • SEQ ID NO.15 (nucleotide sequence of antisense oligo DK-12) 5' A G G A A A A C A G T A C A T G G G G T A 3
  • SEQ ID NO.25 (nucleotide sequence for antisense oligo DK-22)
  • SEQ ID NO.26 (nucleotide sequence for anti ⁇ en ⁇ e oligo DK-23) 5' C A A A C A A A C A A A C A A A C A A T
  • Site 106 through 99 of the human mRNA for A-raf-l oncogene.
  • SEQ ID NO.28 (nucleotide sequence for anti ⁇ ense oligo DK-25)
  • Site 950 through 928 of the human mRNA for A-raf-l oncogene.
  • Nuclease-re ⁇ istant backbone structure in the preferred embodiment of the present invention.
  • the "normal" structure of an oligonucleotide is a defined sequence of nucleotide bases built upon a sugar-pho ⁇ phate backbone containing pho ⁇ phodie ⁇ ter linkage ⁇ .
  • considerable experience indicates that the normal phosphodie ⁇ ter linkage i ⁇ highly susceptible to rapid degradation by a variety of nuclease ⁇ found in abundance in tissues and cellular fluids.
  • an antisense oligonucleo-tide For an antisense oligonucleo-tide to be useful as a therapeutic agent following sy ⁇ temic administration, it must survive in solution long enough to reach its designated target gene in the body and block the activity of that target gene.
  • the anti-raf antisense oligonucleotides are those analogs which contain a nuclease-resi ⁇ tant backbone linkage structure.
  • a number of these nuclease-re ⁇ i ⁇ tant linkage ⁇ tructure ⁇ are known in the art (for example, ⁇ ee the discussion of nuclease-resistant linkage ⁇ in: Stein CA, et al., Nucleic Acid ⁇ Re ⁇ earch 16: 3209-3221, 1988).
  • One such linkage i ⁇ the pho ⁇ phorothioate linkage.
  • Pho ⁇ phorothioate ⁇ are compounds well known in the art, and are those in which one of the non-bridging oxygen atoms in the phosphate portion of a nucleotide i ⁇ replaced by sulfur.
  • the use of oligo- nucleotide analogs which contain a backbone of phophorothioate linkages is based on the known resistance of thi ⁇ internucleotide linkage to degradation by nucleases of many types. Since phosphorothioates also have the same number of charges a ⁇ normal phosphodiester-linked oligonucleotides, they have good aqueous solubility.
  • Antisense phosphorothioate analogues have been used by several groups in as ⁇ ay ⁇ for measuring antisense activity, and evidence indicates that thi ⁇ nuclea ⁇ e-re ⁇ istant backbone linkage doe ⁇ not diminish the potential for sequence specific recognition by the oligonucleotide analog of its target gene. Furthermore, the unmodified (normal) oligonucleotide has a half-life in vivo of about 2 hours, whereas more than 95% of the phophorothioate bond ⁇ are still intact after 10 days in vivo.
  • the antisen ⁇ e oligonucleotides selected for practice of the invention may have nuclea ⁇ e-re ⁇ istant ethyl- or methylphos- phonate linkages between nucleotide bases.
  • oligonucleotide analogs with these type ⁇ of linkage ⁇ are le ⁇ efficient at hybridization with a complementary DNA ⁇ equence than are the corre ⁇ ponding analog ⁇ which incorporate phorphorothioate linkage ⁇ .
  • oligonucleotide ⁇ having a methylphosphonate backbone are more lipophilic than are the other analogs, and this may prove advantageous in certain circumstance .
  • ribozy e structure ⁇ (Greene JJ, Clinical Biotechnology 2: 75-76, 1990), incorporating methyl-pho ⁇ phonate oligonucleotide analogs, have a long half-life in vivo becau ⁇ e the lipophilic structure reduces the rate of renal clearance of the compound while the ribozyme structure facilitates cleavage of the target RNA message (Gerlach, Nature 334: 585, 1988).
  • nuclease- re ⁇ i ⁇ tant backbone linkages other than those mentioned above are readily available for incorporation into all or part of a newly-synthe ⁇ ized oligonucleotide.
  • nuclea ⁇ e-resi ⁇ ting linkage ⁇ are continually being being developed. It is the intent of the present invention that any anti-raf antisense oligonucleotide used alone or in combination with other therapies, and which contains such nuclease-resistant backbone linkage ⁇ be included within the scope of the present invention.
  • anti-raf anti ⁇ en ⁇ e oli ⁇ onucleotide ⁇ in pharmaceutical formulation ⁇ .
  • the therapeutic anti-raf antisen ⁇ e oligonucleotides must be formulated into suitable pharmaceutical compositions; the protocol for ⁇ ystemic administration would use a therapeutic approach compatible with the particular formulation selected.
  • Pharmaceutical compositions within the scope of the pre ⁇ ent invention include those composition ⁇ where the anti-raf oligonucleotide i ⁇ contained in an effective amount sufficient to kill the ras- activated cells of the cancer without causing unacceptable toxicity for the patient.
  • the therapeutic amount which represents a cytotoxicaUy-effective dose sufficient for treatment of each of the various types of ras-activated tumor remains to be determined empirically by those skilled in the art of designing and administering chemotherapy.
  • a preferred dosage comprises that which is sufficient to achieve an effective blood concentration of from about 0.1 to about 200 micromolar.
  • the anti-raf antisen ⁇ e oligonucleotide compounds of the present invention may be administered in a pharmaceutical composition which contains, in addition to the active ingredient, any of a number of pharmaceutically-acceptable excipients which facilitate proces ⁇ ing of the active compound into suitable pharmaceutical preparations.
  • the preparations are designed for parenteral administration.
  • pharmaceutical compositions designed for oral admini ⁇ stration in such forms a ⁇ tablets, capsules, and dragees, or for rectal administration in the form of suppositories are also considered to fall within the ⁇ cope of the pre ⁇ ent invention.
  • Appropriate formulations of the therapeutic oligo ⁇ nucleotide for parenteral admini ⁇ tration include aqueous solutions of the active compound prepared in a water-soluble or water-dispersible form.
  • the active com- pounds may be administered as suspensions in appropriate oily injection carriers, i.e., in suitable lipophilic carriers, such as fatty oils (sesame oil being an example) , or synthetic fatty acid e ⁇ ter ⁇ (ethyl oleate or triglyceride ⁇ being example ⁇ ) .
  • Pharmaceutical formulation ⁇ prepared for aqueous injection may contain substance ⁇ which increase the viscosity of the su ⁇ pen ⁇ ion such as, for example, ⁇ odium carboxymethyl cellulo ⁇ e, ⁇ orbitol, and/or dextran.
  • the therapeutic anti-raf oligonucleotide ⁇ of the pre ⁇ ent invention may also be administered encapsulated in liposomes.
  • the anti-raf oligonucleotides are contained in corpuscle ⁇ which consist of concentric aqueous layers interspersed between hydrophobic lipidic layers.
  • the oligonucleotides depending upon their solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspen ⁇ ion.
  • the hydrophobic layer generally but not exclusively, comprises phospholipids such as lecithin and ⁇ phingomyelin, ⁇ teroids such as cholesterol, more or les ⁇ ionic ⁇ urfactant ⁇ ⁇ uch a ⁇ a diacetylpho ⁇ phate, ⁇ tearylamine, or phosphatidic acid, and/or other material ⁇ of a hydrophobic nature which are generaly well known in the art.
  • Purging bone marrow su ⁇ pen ⁇ ion ⁇ of contaminating tumor cell ⁇ is presently accomplished either by in vitro incubation of the transplanted marrow cells with potent anti-cancer chemotherapeutic agents, or by contacting the bone marrow cells with immunotherapeutic agents which recognize certain structures unique to the surface membrane of tumor cell ⁇ .
  • a major difficulty with immunotherapy i ⁇ the fact that many tumor cell ⁇ fail to expre ⁇ the tumor-a ⁇ sociated membrane ⁇ tructure, and thereby go unrecognized by the immunother ⁇ apeutic agent.
  • the immunotherapeutic agent binds to its target but fail ⁇ to kill the cell.
  • chemotherapetic agents most of the agents are highly toxic and mu ⁇ t be u ⁇ ed at relatively high do ⁇ e ⁇ in order to maximize tumor cell kill.
  • thi ⁇ can lead to death of a large number of normal marrow cells and, in some instances, to graft failure.
  • the pre ⁇ ent invention provide ⁇ ⁇ uch an agent for u ⁇ e with ra ⁇ -activated cancer ⁇ .
  • anti-raf antisen ⁇ e oligonucleotides are used to kill any and all ras-activated cancer cell ⁇ which may be present in a su ⁇ pen ⁇ ion of bone marrow cell ⁇ obtained from the afflicted individual.
  • bone marrow cells are obtained from an individual who ha ⁇ a ra ⁇ -activated cancer, using standard procedures, which include aspiration from the pelvic iliac crest of a donor, as described, for example, in U.S. Patents No. 4,481,946 and No. 4,486,188.
  • the sample of autologou ⁇ bone marrow cell ⁇ i ⁇ then immediately treated with the anti-raf oligonucleotide, a ⁇ discussed below, and reinfused into the donor as soon a ⁇ is appropriate.
  • the autologou ⁇ bone marrow i ⁇ purged of contaminating cancer cell ⁇ by expo ⁇ ure ex vivo to a cytotoxicaUy-effective amount of an anti ⁇ en ⁇ e oligonucleo ⁇ tide which ha ⁇ a nucleotide ⁇ equence complementary to that of an RNA message transcribed from a raf target gene present in the cell ⁇ of the ra ⁇ -activated cancer.
  • the time of expo ⁇ ure required to obtain complete kill of the targeted cell ⁇ in the bone marrow specimen varie ⁇ depending on the tumor cell target and mu ⁇ t be determined empirically; however, exposure times varies from 1 hour to 4 days or longer.
  • the purged bone marrow cell ⁇ can be frozen and ⁇ tored until needed.
  • Procedure ⁇ for preparing and ⁇ toring bone marrow ⁇ ample ⁇ frozen in a viable ⁇ tate are discus ⁇ ed in detail in U.S. Patent ⁇ No. 4,107,937 and No. 4,117,881.
  • Anti ⁇ ense oligonucleotide treatment of peripheral blood- derived ⁇ tem cell ⁇ is an important component of peripheral blood- derived ⁇ tem cell ⁇ .
  • peripheral blood There are in the circulating peripheral blood a substantial number of mononuclear cells which have the potential to regenerate the complete function of the bone marrow compartment of a host organism, such a ⁇ a human.
  • These peripheral "stem” cells can be isolated, concentrated, and reintroduced via injection into the peripheral circulation a ⁇ a " ⁇ tem cell tran ⁇ plant.”
  • Autologou ⁇ peripheral blood ⁇ tem cell tran ⁇ plantation ha ⁇ been found important in facilitating recovery of functional bone marrow after high-do ⁇ e therapy for a variety of malignant di ⁇ ea ⁇ e ⁇ .
  • Autologou ⁇ peripheral blood ⁇ tem cell tran ⁇ plantation offer ⁇ certain advantage ⁇ to autologou ⁇ bone marrow tran ⁇ plantation, ⁇ ince the general ane ⁇ thesia used during bone marrow harvesting can be avoided, the collections of peripheral stem cells can be made in an outpatient setting, and the risk of contamination of the transplanted product with malignant cells appears to be less.
  • Purging the peripheral stem cell ⁇ u ⁇ pension of contaminating tumor cells are very similar, if not identical, to the procedure ⁇ outlined above for purging bone marrow cell ⁇ with anti-raf anti ⁇ en ⁇ e oligonucleotides.
  • another embodiment of the present invention is to provide a course of ⁇ ystemically- ad ini ⁇ tered anti ⁇ en ⁇ e oligotherapy a ⁇ an adjunct therapy to the individual who received the tran ⁇ plant of autologou ⁇ bone marrow cells or peripheral stem cells.
  • NIH/3T3 mou ⁇ e embryo fibrobla ⁇ t ⁇ ATCC Culture # CCL-92, American Type Culture Collection, Rockville, MD
  • a ⁇ well as several NIH/3T3 transformant ⁇ and virally infected cell line ⁇ were grown in Dulbecco-modified Eagle' ⁇ minimal essential medium supplemented with 10 percent (volume/volume) heat-inactivated calf serum, 50 ug/ml each of gentamicin sulfate antibiotic, and exogenous glutamine to a final concentration equivalent to 2 mmol per liter.
  • NIH/3T3 cell line ⁇ Six different long-term cultured NIH/3T3 cell line ⁇ were incubated for 24 hours in the presence of an anti-raf antisen ⁇ e oligonucleotide, de ⁇ ignated SEQ ID NO.l (al ⁇ o referred to a ⁇ "EC-IC" in the laboratory experiments).
  • SEQ ID NO.l de ⁇ ignated SEQ ID NO.l
  • the nucleotide base sequence is complementary to a portion of the mRNA transcribed from the c-raf-1 oncogene, site positions 1269-1252, as follows:
  • the oligonucleotide was con ⁇ tructed with a pho ⁇ phorothioate backbone, a ⁇ described hereinafter.
  • Oligonucleotide synthesis and purification Oligo ⁇ nucleotide EC-IC (SEQ ID NO.l) was synthesized on an Applied Biosystems Model 380A DNA ⁇ ynthesizer. Each synthetic cycle resulted in a phosphite linkage which was oxidized with
  • Cytotoxicity Assay Eight different concentrations of SEQ ID NO.l ("EC-IC”) anti-raf oligonucleotide, prepared by serial 1:1 dilutions in the wells of 96-well microtest plates, were examined for their capacity to produce death of the cell lines with which it had been contacted in vitro for 24 hours. Cell death was determined by the los ⁇ of the ability of cell mitochondria to reduce MTT dye. Thi ⁇ i ⁇ a quantitative colorimetric a ⁇ say for mammaliam cell survival and proliferation, employing MTT (3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide). Only living cell ⁇ with active mitochondria can reduce MTT, a process which generates a colored formazan dye.
  • MTT 3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide
  • FIGURE 1 is a drawing in which a hi ⁇ togram is presented. The drawing show ⁇ cell ⁇ urvival on the left axis plotted against dose of the anti-raf oligonucleotide. The data were analyzed by fitting the log dose of oligonucleotide versus percent cell ⁇ urvival to a ⁇ igmoid curve.
  • the LD-50 (a mea ⁇ ure of potency) values are presented: open bars, NIH/3T3 control (untreated) cells; right diagonal hatching, NIH/3T3 cells with a "normal” ras proto-oncogene ⁇ tably in ⁇ erted into the cellular genome; left diagonal hatching, NIH/3T3 cell ⁇ (al ⁇ o de ⁇ ignated "S80" cell ⁇ ) with "activated” Harvey-ras-1 oncogene sequence stably inserted into the cellular genome; horizontal hatching, NIH/3T3 ("453") cells; vertical hatching, NIH/3T3 ("485") cell ⁇ ; and cros ⁇ hatching, NIH/3T3 ("504") cells.
  • the 504 cells were more sensitive to the SEQ ID NO.l oligonucleotide than were the other cell ⁇ , but were more ea ⁇ ily wa ⁇ hed off the plates during the as ⁇ ay than the other cells.
  • FIGURE 1 A ⁇ ⁇ hown in FIGURE 1, the SEQ ID NO.l pho ⁇ phorothioate oligonucleotide with ⁇ equence anti ⁇ en ⁇ e to a select portion of the oncogene c-raf-1 wa ⁇ found to be greater than .
  • Thi ⁇ is also shown in another manner in FIGURE 2A.
  • FIGURE 2A is a drawing of cytotoxicity curve ⁇ .
  • the triangles repre ⁇ ent NIH/3T3 murine cell ⁇ which ⁇ how 60% cell ⁇ urvival at 1 uM do ⁇ e
  • the open circle ⁇ represent NIH/3T3 with the human ras proto-oncogene ⁇ tably integrated and show 55% cell survival at 1 uM dose
  • the closed circles represent NIH/3T3 cells with activated human ras oncogene stably integrated and show only 5% cell survival at l uM dose.
  • the oligonucleotide was con ⁇ tructed with a phosphorothioate backbone, a ⁇ de ⁇ cribed hereinafter.
  • Cytotoxicity Assay In a manner similar to that de ⁇ cribed above in Example 1, eight different concentration ⁇ of SEQ ID NO.2 ("EC-2C") anti-raf oligonucleotide were prepared by ⁇ erial 1:1 dilution ⁇ in the well ⁇ of 96-well microte ⁇ t plate ⁇ , and examined for their capacity to produce death of the cell lines co-cultured in vitro with the anti-raf antisense oligonucleotide for 24 * hours. Cell death wa ⁇ determined by the lo ⁇ s of the ability of cell mitochondria to reduce MTT dye, a ⁇ outlined above in Example 1.
  • FIGURE 3 is a drawing in which a histogram is presented.
  • the drawing shows cell survival on the left axis plotted against do ⁇ e of the anti-raf oligonucleotide.
  • the data were analyzed by fitting the log do ⁇ e of oligonucleotide ver ⁇ u ⁇ percent cell ⁇ urvival to a ⁇ igmoid curve.
  • the LD-50 (a mea ⁇ ure of potency) value ⁇ are pre ⁇ ented in the figures: the different vertical bars in the histogram represent the same target cell types a ⁇ de ⁇ cribed in the hi ⁇ togram of FIGURE 1.
  • the SEQ ID NO.2 phosphorothioate oligonucleotide with an antisense nucleotide sequence uniquely complementary to a select portion of the oncogene c-raf-1 were found to be about eight times more lethal in NIH/3T3 mouse fibrobla ⁇ t cell ⁇ with activated ra ⁇ expressed than in the NIH/3T3 cells which contained normal ras proto-oncogene.
  • eight different concentration ⁇ of SEQ ID NO.3 (“EC-3C") anti-raf oligonucleotide were prepared by ⁇ erial 1:1 dilution ⁇ in the wells of 96-well microte ⁇ t plate ⁇ , and examined for their capacity to produce death of the cell line ⁇ co-cultured in vitro with the anti-raf antisense oligonucleotide for 24 hours. Cell death was determined by the loss of the ability of cell mitochondria to reduce MTT dye, a ⁇ outlined above in Example 1.
  • FIGURE 4 i a drawing in which a hi ⁇ togram i ⁇ pre ⁇ ented.
  • the drawing shows cell survival on the left axis plotted against dose of the anti-raf oligonucleotide.
  • the data were analyzed by fitting the log do ⁇ e of oligonucleotide ver ⁇ us percent cell survival to a sigmoid curve.
  • the LD-50 (a measure of potency) values are presented in the figures: the different vertical bars in the hi ⁇ togram repre ⁇ ent the same target cell types a ⁇ described in the histogram of FIGURE 1.
  • the SEQ ID NO.3 phosphorothioate oligonucleotide with an antisen ⁇ e nucleotide ⁇ equence uniquely complementary to a ⁇ elect portion of the oncogene c-raf-1 were found to be about twenty time ⁇ more lethal in NIH/3T3 ou ⁇ e fibrobla ⁇ t cell ⁇ with activated ras expressed than in the NIH/3T3 cells which contained normal ras proto-oncogene.
  • the anti-raf anti ⁇ en ⁇ e oligonucleo- tide ⁇ of the pre ⁇ ent invention may be admini ⁇ tered a ⁇ ⁇ y ⁇ temic oligotherapy, or it may be u ⁇ ed in vitro to purge contaminating tumor cell ⁇ from an autologou ⁇ peripheral blood ⁇ tem cell preparation to be u ⁇ ed in an autologou ⁇ tran ⁇ plantation procedure.
  • the following prophetic example illu ⁇ trates how the anti-raf oligonucleotide ⁇ of the present invention can be used to purge contaminating ras-activated cancer cells from a su ⁇ pension of peripheral blood stem cells.
  • the peripheral ⁇ tem cell ⁇ are collected with a Haemonetic ⁇ Model V50 apheresis device (Haemonetic ⁇ , Braintree, MA).
  • the ⁇ tem cell ⁇ are collected in a technique called a component collection lymphocytophere ⁇ i ⁇ auto ⁇ urge protocol de ⁇ cribed in the operating manual for the aphere ⁇ i ⁇ in ⁇ trument. Venou ⁇ blood i ⁇ withdrawn into a Latham aphere ⁇ i ⁇ bowl.
  • the mononuclear cell fraction is then further fractionated in the apheresis device u ⁇ ing a Ficoll- diazitroate den ⁇ ity gradient to remove contaminating red blood cell ⁇ .
  • the red cell depleted product i ⁇ washed twice by centrifugation and resu ⁇ pended in Hank , ⁇ balanced ⁇ alt solution without calcium or magnesium with 20% (volume/volume) autologou ⁇ ⁇ eru and 0.6% citrate formula B (Fenwal, Deerfield, IL) .
  • Approximately eight collection ⁇ are made to harve ⁇ t the stem cells used for a patient's stem-cell transplant. All stem cell collected are cryopreserved in a buffered salt solution containing a 10% concentration of dimethylsulfoxide (DMSO) as a cryoprotectant.
  • DMSO dimethylsulfoxide
  • the ⁇ tem cell ⁇ are thawed, warmed to normal body temperature (37°C), and infused intravenou ⁇ ly.
  • the ⁇ eparated peripheral ⁇ tem cell ⁇ are then re ⁇ u ⁇ pended at a concentration of approximately 10 7 cell ⁇ per ml in a culture medium made up of the following: RPMI 1640 balanced ⁇ alt ⁇ olution, ⁇ upplemented with a ⁇ ource of growth factors (such as "5637 Conditioned Medium”); 5 uM per liter hydrocortisone hemi ⁇ uccinate; 250 ug per ml catala ⁇ e; 2 mM per liter mannitol; 1% (volume/volume) of a 100X sodium pyruvate solution; 1% (v/v) of a 100X vitamin solution; 1% (v/v) of a 50X amino acid solution; 0.5% (v/v) of a 200X nones ⁇ ential amino acid ⁇ ⁇ olution; L-glutamine to a final concentration of 200mM per liter.
  • RPMI 1640 balanced ⁇ alt ⁇ olution ⁇ upplemented with a ⁇
  • the cultured cell ⁇ are fed by a change of 2/3 of the medium, and by a dilution of the cell ⁇ into additional culture ve ⁇ el ⁇ to near the original cell den ⁇ ity.
  • the pre ⁇ ence of progenitor stem cells is determined in a ⁇ ay ⁇ for Colony-Forming Unit ⁇ - Granulocyte/Macrophage (CFU-GM), and a ⁇ ays for Blast-Forming Units - Erythrocyte (BFU-E) .
  • the ⁇ tem cell cultures are harvested when it is determined that no ras-activated tumor cells remain among the peripheral stem cell ⁇ . U ⁇ ing radiolabeled probe ⁇ to the ras oncogene and polymerase chain reaction (PCR) amplification of test sample ⁇ , thi ⁇ determination i ⁇ rapid and accurate.
  • PCR polymerase chain reaction
  • the treated ⁇ tem cell ⁇ are then concentrated and tran ⁇ ferred into infu ⁇ ion bags, where they are infused back into the donor.
  • the recipient of the autologou ⁇ tran ⁇ plant i ⁇ then followed to verify that the tran ⁇ planted cells have successfully engrafted in the host marrow.
  • additional anti-raf antisense oligonucleotide is administered systemically to the patient in a low dose maintenance shcedule, a ⁇ discu ⁇ sed above, to be certain that any remaining ras-activated tumor cell ⁇ in the patient i ⁇ eliminated.
  • Thi ⁇ pharmaceutical preparation contain ⁇ one or more of the anti-raf antisense oligonucleotides described as useful in the present invention.
  • the anti-raf antisense oligonucleo- tides of the present invention may be administered as sy ⁇ temic oligotherapy, or u ⁇ ed in vitro to purge tumor cell ⁇ from an autologous bone marrow preparation.
  • the following prophetic example illustrate ⁇ how the anti-raf oligonucleotides of the pre ⁇ ent invention can be u ⁇ ed to purge contaminating ra ⁇ - activated cancer cell ⁇ from a ⁇ u ⁇ pen ⁇ ion of bone marrow cell ⁇ .
  • the ⁇ eparated bone marrow cells are then resuspended at a concentration of approximately 107 cells per ml in a culture medium made up of the following: RPMI 1640 balanced salt solution, supplemented with a source of growth factors (such as "5637 Conditioned Medium”); 5 uM per liter hydrocortisone hemi ⁇ uccinate; 250 ug per ml catalase; 2 mM per liter mannitol; 1% (volume/volume) of a 100X sodium pyruvate solution; 1% (v/v) of a 100X vitamin solution; 1% (v/v) of a 50X amino acid solution; 0.5% (v/v) of a 200X nones ⁇ ential amino acids ⁇ olution; L-glutamine to a final concentration of 200mM per liter. To this is then added ultra-filtered horse serum (Hyclone Laboratories, Logan, Utah) to a final concentration of 12.5%, and ultra-filtered fetal calf
  • anti-raf antisense oligonucleotide i ⁇ added to a concentration of approximately 50 uM.
  • the cultures are harvested when it is determined that no ras-activated tumor cell ⁇ remian in the bone marrow culture. U ⁇ ing radiolabeled probe ⁇ to the ra ⁇ oncogene and polymera ⁇ e chain reaction (PCR) amplification of test sample ⁇ , thi ⁇ determination i ⁇ rapid and accurate.
  • the treated marrow cell ⁇ are then concentrated and transferred into infusion bags, where they are infu ⁇ ed back into the donor.
  • the recipient of the autologou ⁇ transplant i ⁇ then followed to verify that the marrow ha ⁇ succe ⁇ sfully engrafted.
  • additional anti-raf antisense oligonucleotide i ⁇ administered ⁇ y ⁇ temically to the patient in a low do ⁇ e maintenance ⁇ hcedule, a ⁇ di ⁇ cus ⁇ ed above, to be certain that any remaining ra ⁇ -activated tumor cell ⁇ in the patient are eliminated.
  • This pharmaceutical preparation contains one or more of the anti-raf antisen ⁇ e oligonucleo ⁇ tide ⁇ de ⁇ cribed a ⁇ useful in the present invention.
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE yes
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • Site 89 through 72 of the human mRNA for c-raf-1 oncogene.
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • Site 203 through 191 of the human mRNA for c-raf-1 oncogene.
  • MOLECULE TYPE cDNA to mRNA
  • TYPE deoxyribonucleic acid (DNA)
  • STRANDEDNESS ⁇ ingle, anti-sen ⁇ e
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE CDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE yes
  • ORIGINAL SOURCE DNA synthesizer, Applied Biosystems, Inc.
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE CDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE CDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • Site 106 through 99 of the human mRNA for A-raf-l oncogene.
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL no
  • ANTI-SENSE yes
  • ORIGINAL SOURCE DNA synthesizer, Applied Biosystems, Inc.
  • MOLECULE TYPE cDNA to mRNA
  • Site 1190 through 1173 of the human mRNA for A-raf-l oncogene.
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • Site 1454 through 1434 of the human mRNA for A-raf-l oncogene.
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • a method for killing cells expres ⁇ ing an activated ras oncogene comprising contacting said cells in vivo or in vitro with a cytotoxically-effective amount of a heterotypic antisense oligonucleotide, or combination of antisense oligonucleotide ⁇ , or pharmaceutically-effective analog ⁇ thereof, said antisense oligonucleotide having a base sequence complementary to the DNA or transcribed me ⁇ enger RNA of a raf gene in said cells.
  • nuclease- resistant backbone linkage i ⁇ a pho ⁇ phorothioate linkage.
  • nuclease- resistant backbone linkage i ⁇ a methylpho ⁇ phonate linkage.

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Abstract

La présente invention se rapporte à un procédé permettant de détruire des cellules cancérigènes exprimant un oncogène ras activé, et consistant à mettre ces cellules activées par ras en contact, in vivo ou in vitro, avec une dose cytotoxique efficace d'un oligonucléotide hétérotypique antisens ou avec une combinaison d'oligonucléotides antisens ou des analogues pharmaceutiquement efficaces de ceux-ci, lesdits oligonucléotides ou analogues présentant des séquences de base complémentaires à une séquence de l'ADN ou de l'ARN messager transcrit d'un gène raf également présent dans les mêmes cellules cancérigènes. La présente invention se rapporte également à de nouveaux procédés permettant de traiter un individu souffrant d'un cancer activé par ras. Ce traitement consiste à utiliser des thérapies basées sur l'oligonucléotide hétérotypique antisens, selon lesquelles une dose cytotoxique efficace d'une préparation contenant un oligonucléotide antisens et anti-raf, ou une combinaison d'oligonucléotides antisens et anti-raf choisis, ou au moins un analogue pharmaceutiquement efficace de ces oligonucléotides, est administrée sous forme d'une pharmacothérapie spécifique des cancers exprimant un oncogène ras activé.
PCT/US1994/004091 1993-04-09 1994-04-11 Nouveaux procedes et compositions destines au traitement du cancer active par ras au moyen d'oligonucleotides heterotypiques anti-raf et antisens WO1994023755A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995032987A1 (fr) * 1994-05-31 1995-12-07 Isis Pharmaceuticals, Inc. MODULATION OLIGONUCLEOTIDIQUE ANTISENS DE L'EXPRESSION DU GENE raf
FR2733500A1 (fr) * 1995-04-28 1996-10-31 Centre Nat Rech Scient Nouveaux antisens diriges contre ras, preparation et utilisations
WO1996039415A1 (fr) * 1995-06-05 1996-12-12 Isis Pharmaceuticals, Inc. MODULATION PAR OLIGONUCLEOTIDES ANTISENS DE L'EXPRESSION DE GENES raf
WO1997032604A1 (fr) * 1996-03-07 1997-09-12 Novartis Ag Combinaisons antiproliferatives contenant des oligonucleotides cibles sur raf et des composes chimiotherapeutiques
US5744460A (en) * 1996-03-07 1998-04-28 Novartis Corporation Combination for treatment of proliferative diseases
US5981731A (en) * 1994-05-31 1999-11-09 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of B-raf gene expression
WO2001061030A2 (fr) * 2000-02-14 2001-08-23 Bollon Arthur P Bibliotheques de regions de sous-sequences optimales d'arnm et d'adn genomique pour la regulation de l'expression genetique
US6391636B1 (en) 1994-05-31 2002-05-21 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of raf gene expression
US6957148B2 (en) 2001-05-21 2005-10-18 The Board Of Regents, University Of Texas System Next-nearest-neighbor sequence determinants of antisense DNA
US7262173B2 (en) 1997-03-21 2007-08-28 Georgetown University Chemosensitizing with liposomes containing oligonucleotides
US7838541B2 (en) 2002-02-11 2010-11-23 Bayer Healthcare, Llc Aryl ureas with angiogenesis inhibiting activity
US7897623B2 (en) 1999-01-13 2011-03-01 Bayer Healthcare Llc ω-carboxyl aryl substituted diphenyl ureas as p38 kinase inhibitors
US8124630B2 (en) 1999-01-13 2012-02-28 Bayer Healthcare Llc ω-carboxyaryl substituted diphenyl ureas as raf kinase inhibitors
US8796250B2 (en) 2003-05-20 2014-08-05 Bayer Healthcare Llc Diaryl ureas for diseases mediated by PDGFR

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CANCER GENE THERAPY, Volume 1, No. 1, issued March 1994, B.Y. TSENG et al., "Antisense Oligonucleotides Technology in the Development of Cancer Therapeutics", pages 65-71. *
CHEMICAL REVIEWS, Volume 90, No. 4, issued June 1990, E. UHLMANN et al., "Antisense Oligonucleotides: A New Therapeutic Principle", pages 543-584. *
JOURNAL OF CELL BIOLOGY, Volume 266, No. 23, issued 15 August 1991, M. CARROLL et al., "Erythropoietin Induces Raf-1 Activation and Raf-1 is Required for Erythropoietin-Mediated Proliferation", pages 14964-14969. *
JOURNAL OF CELLULAR BIOCHEMISTRY, Supplement 16B, issued February 1992, A. CUADRADO et al., "Functional Link Between Ras and Raf in Oncogenic Transformation", page 245. *
NATURE, Volume 349, issued 31 January 1991, W. KOLCH et al., "Raf-1 Protein Kinase is Required for Growth of Induced NIH/3T3 Cells", pages 426-428. *
SCIENCE, Volume 243, issued 10 March 1989, U. KASID et al., "Effect of Antisense C-Raf-1 on Tumorigenicity and Radiation Sensitivity of a Human Squamous Carcinoma", pages 1354-1356. *
SCIENCE, Volume 261, issued 20 August 1993, C.A. STEIN et al., "Antisense Oligonucleotides as Therapeutic Agents - Is the Bullet Really Magical?", pages 1004-1012. *

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981731A (en) * 1994-05-31 1999-11-09 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of B-raf gene expression
WO1995032987A1 (fr) * 1994-05-31 1995-12-07 Isis Pharmaceuticals, Inc. MODULATION OLIGONUCLEOTIDIQUE ANTISENS DE L'EXPRESSION DU GENE raf
US6391636B1 (en) 1994-05-31 2002-05-21 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of raf gene expression
FR2733500A1 (fr) * 1995-04-28 1996-10-31 Centre Nat Rech Scient Nouveaux antisens diriges contre ras, preparation et utilisations
WO1996034008A1 (fr) * 1995-04-28 1996-10-31 Institut National De La Sante Et De La Recherche Medicale (Inserm) Nouveaux antisens diriges contre ras, preparation et utilisations
WO1996039415A1 (fr) * 1995-06-05 1996-12-12 Isis Pharmaceuticals, Inc. MODULATION PAR OLIGONUCLEOTIDES ANTISENS DE L'EXPRESSION DE GENES raf
WO1997032604A1 (fr) * 1996-03-07 1997-09-12 Novartis Ag Combinaisons antiproliferatives contenant des oligonucleotides cibles sur raf et des composes chimiotherapeutiques
US5744460A (en) * 1996-03-07 1998-04-28 Novartis Corporation Combination for treatment of proliferative diseases
US7262173B2 (en) 1997-03-21 2007-08-28 Georgetown University Chemosensitizing with liposomes containing oligonucleotides
US7897623B2 (en) 1999-01-13 2011-03-01 Bayer Healthcare Llc ω-carboxyl aryl substituted diphenyl ureas as p38 kinase inhibitors
US8124630B2 (en) 1999-01-13 2012-02-28 Bayer Healthcare Llc ω-carboxyaryl substituted diphenyl ureas as raf kinase inhibitors
US8841330B2 (en) 1999-01-13 2014-09-23 Bayer Healthcare Llc Omega-carboxyaryl substituted diphenyl ureas as raf kinase inhibitors
WO2001061030A2 (fr) * 2000-02-14 2001-08-23 Bollon Arthur P Bibliotheques de regions de sous-sequences optimales d'arnm et d'adn genomique pour la regulation de l'expression genetique
WO2001061030A3 (fr) * 2000-02-14 2002-08-29 Arthur P Bollon Bibliotheques de regions de sous-sequences optimales d'arnm et d'adn genomique pour la regulation de l'expression genetique
US6957148B2 (en) 2001-05-21 2005-10-18 The Board Of Regents, University Of Texas System Next-nearest-neighbor sequence determinants of antisense DNA
US7838541B2 (en) 2002-02-11 2010-11-23 Bayer Healthcare, Llc Aryl ureas with angiogenesis inhibiting activity
US8242147B2 (en) 2002-02-11 2012-08-14 Bayer Healthcare Llc Aryl ureas with angiogenisis inhibiting activity
US8618141B2 (en) 2002-02-11 2013-12-31 Bayer Healthcare Llc Aryl ureas with angiogenesis inhibiting activity
US8796250B2 (en) 2003-05-20 2014-08-05 Bayer Healthcare Llc Diaryl ureas for diseases mediated by PDGFR

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