EP1181361A2 - Derives d'oligonucleotides diriges contre les arn messagershumains bcl-xl et bcl-2 - Google Patents

Derives d'oligonucleotides diriges contre les arn messagershumains bcl-xl et bcl-2

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Publication number
EP1181361A2
EP1181361A2 EP00925239A EP00925239A EP1181361A2 EP 1181361 A2 EP1181361 A2 EP 1181361A2 EP 00925239 A EP00925239 A EP 00925239A EP 00925239 A EP00925239 A EP 00925239A EP 1181361 A2 EP1181361 A2 EP 1181361A2
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EP
European Patent Office
Prior art keywords
oligonucleotide derivative
human bcl
derivative according
oligonucleotide
base sequence
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP00925239A
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German (de)
English (en)
Inventor
Uwe Zangemeister-Wittke
Gerd Lüdke
Dieter Huesken
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
STAHEL, ROLF
Zangemeister-Wittke Uwe
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Universitaet Zuerich
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Publication of EP1181361A2 publication Critical patent/EP1181361A2/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • 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
    • C12N2310/33—Chemical structure of the base
    • 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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • 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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

  • the present invention relates to antisense oligonucleotide derivatives directed against human bcl-xL mRNA and being capable of modulating the biosynthesis of human bcl-xL protein Furthermore, the present invention relates to antisense oligonucleotide derivatives directed against both human bcl-xL mRNA and human bcl-2 mRNA, and being capable of modulating the biosynthesis of both human bcl-xL protein and human bcl-2 protein The present invention further relates to a pharmaceutical composition comprising such oligonucleotide derivatives, uses thereof and methods of treatment and diagnosis utilizing such oligonucleotide derivatives
  • Human bcl-xL and human bcl-2 are proteins which are closely associated with the process of programmed cell death (apoptosis)
  • Apoptosis is an active, tightly regulated physiological process involved in development, normal cell turnover, and hormone-induced tissue athropy
  • Lack of programmed cell death plays an important role in cancer and other hyperpro ferative diseases like re- stenosis, fibrosis, psoriasis or certain types of allergic diseases, in particular in tumor progression and, importantly, might contribute to the clinical problem of resistance to anti-neoplastic regimens, in particular standard chemotherapeutic drugs
  • Human bcl-xL and human bcl-2 have distinct patterns of tissue expression In contrast to most normal tissues, in malignant tumors, such as a small cell lung cancer (SCLC) and non-small lung cancer (NSCLC), bcl-xL and bcl-2 are often co-expressed
  • SCLC small cell lung cancer
  • NSCLC non-small lung cancer
  • Oligonucleotides or, in particular, oligonucleotide derivatives directed against human bcl-xL mRNA or human bcl-2 mRNA may be used in an antisense technology strategy interfering with expression of bcl- xL and bcl-2, respectively
  • oligonucleotides or oligonucleotide derivatives showing high efficacy, preferably improved efficacy, in modulating the biosynthesis or expression of human bcl-xL and/or human bcl-2, in particular for the treatment of hyperprohferative diseases, e g those as mentioned above
  • the present invention relates to antisense oligonucleotide derivatives directed against these regions
  • the oligonucleotide derivatives according to the present invention show the capability to modulate human bcl-xL biosynthesis in a cell
  • some of the oligonucleotide derivatives of the present invention, mentioned below, in addition to be capable of modulating human bcl-xL biosynthesis are able to modulate the biosynthesis of human bcl-2
  • the present oligonucleotide derivatives are therefore appropriate for the therapeutic treatment of diseases that respond to this modulation, especially inhibition, of bcl-xL or, in addition, bcl-2 biosynthesis
  • Such an inhibition may result, as one effect, in an induction of apoptosis, resulting in inhibition of cell (hy- per)prol ⁇ ferat ⁇ on
  • an oligonucleotide derivative which is specifically hyb ⁇ dizable to a region ranging from base position no 687 (5') to no base position 706 (3') of the human bcl-xL mRNA encoding human bcl-xL protein
  • the oligonucleotide derivative additionally is specifically hybndizable to a region ranging from base position no 2032 (5') to base position no 2051 (3') of the human bcl-2 mRNA encoding human bcl-2 protein
  • an oligonucleotide derivative is preferred which is capable of modulating the biosynthesis of the human bcl-xL protein
  • an oligonucleotide derivative is preferred which is capable of modulating the biosynthesis of both the human bcl-xL protein and the human bcl-2 protein
  • An oligonucleotide derivative according to the present invention has a length of 8 to 25 consecutive building blocks, preferably of 15 to 20 consecutive building blocks, and most preferably of 20 consecutive building blocks
  • oligonucleotide derivative is familiar to the skilled person and will only be explained here for the sake of completeness Within the context of the present invention, the expression “oligonucleotide derivative” denotes, in particular, a de ⁇ vatized oligonucleotide
  • An oligonucleotide per se is preferably an oligomer which consists of a sequence of natural nucleoside building blocks which are connected to each other by way of natural mternucleosidic bridging groups
  • a natural nucleoside building block as such preferably consists of a sugar, in particular a ⁇ -D-erythropentofuranose, in particular ⁇ -D- ⁇ bose or ⁇ -D-2'-deoxyr ⁇ bose, together with a natural nucleic acid base which is linked to it in the ⁇ position
  • nucleic acid base preferably denotes a base which, in relation to a naturally occurring nucleoside, is capable of forming Watson
  • modulation of the biosynthesis or expression of the human bcl-xL protein or human bcl-2 protein denotes an interference with the biosynthesis or expression, in particular a partial or complete inhibition thereof, in particular in connection with the translation or transcription process
  • an inhibition in particular due to partial or complete degradation of the target nucleic acid, due to the process for translating the target nucleic acid being completely or partially inhibited, or due to the transcription process being completely or partially inhibited, can be determined by means of known methods, for example by means of the Northern blot technique at the level of the target nucleic acid, or by means of the Western blot technique at the protein level (cf , for example, Sambrook, J , F ⁇ tscn, E F and Maniatis, T "Molecular Cloning A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, 1989)
  • the term “hybridization” in particular denotes binding by way of hydrogen
  • Specific hybridization can be determined, for example, by means of an in-vitro hybridization assay, preferably under physiological conditions, between an oligonucleotide derivative according to the invention and a target nucleic acid
  • Appropriate reaction conditions are known (cf , for example, Sam- brook, J , F ⁇ tsch, E F and Maniatis, T "Molecular Cloning A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, Vol 2, 9 47 to 9 58) In this context, further reference is made to those methods as described in the Examples, below
  • the sequence of the human bcl-xL mRNA as referred to herein is accessible in the EMBL Nucleotide Sequence Database as HSBCLXL under accession no Z23115
  • the sequence of the human bcl-2 mRNA as referred to herein is accessible in the GenBank Data Base as HUMBCL2A under accession number M13994 (The sequence of the human bcl-2 mRNA is also available in the GeneBank Data Base under accession number NM 000633 In this case said region encompasses nucleotide no 605 (5') to no 624 (3') of the human bcl-2 mRNA)
  • the numbering of nucleic acids, in particular of mRNA or corresponding cDNA sequences relates to the respective numbering of the human bcl-xL mRNA or bcl-2 mRNA as contained in said data bases under said accession numbers
  • a corresponding cDNA sequence can be deduced from the mRNA sequence in particular by ex
  • the mRNA sequence ranging from nucleotide nos 687 (5') to 706 (3') of the human bcl-xL mRNA reads as follows ⁇ '-AACGGCGGCUGGGAUACUUU-S' (SEQ ID NO 1 )
  • the mRNA sequence ranging from nucleotide nos 2032 (5') to 2051 (3') of the human bcl-2 mRNA reads as follows
  • a preferred embodiment of the present invention is directed to an oligonucleotide derivative as mentioned above, comprising a base sequence which is complementary to at least a part of the said region of the human bcl-xL mRNA or the human bcl-2 mRNA, or wherein such base sequence contains up to 3 mispai ⁇ ng building blocks, or wherein such base sequence contains up to 3 abasic building blocks
  • a further preferred embodiment thereof is directed to an oligonucleotide derivative as mentioned above, wherein said base sequence is selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO 3), the base sequence 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO 4) and the base sequence 5'-AAAGCATCCCAGCCTCCGTT-3' (SEQ ID NO 5)
  • an oligonucleotide derivative of the present invention consisting of a base sequence selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO 3), the base sequence 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO 4) and the base sequence 5'-AAAGCATCCCAGCCTCCGTT-3' (SEQ ID NO 5)
  • an analogous base sequence denotes a base which, although being different from the corresponding classical complementary base, upon introduction into an oligonucleotide derivative of the present invention, does not alter the oligonucleotide derivative's capability of specific hybridization with the target nucleic acid
  • cytosine can be exchanged by 5-methylcytos ⁇ ne, or adenine by 2-am ⁇ noaden ⁇ ne
  • a given base can be replaced by an inert base like hypoxanthine, as long as the function of the oligonucleotide derivative is not impaired
  • analogous or inert bases are known to
  • abasic building block denotes a building block of said oligonucleotide derivative which bears a nucleic acid base which is not the complementary base with respect to the respective position of the target nucleic acid strand in the sense of Wat- son-C ⁇ ck base pairing
  • the complementary base of the oligonucleotide derivative would be the base T
  • a mispanng building block can, for example, be the base C or the base G
  • abasic building block denotes a building block of said oligonucleotide derivative bearing no nucleic acid base capable of hybridization with the complementary base with respect to the respective position of the target nucleic acid strand in the sense of Watson-Crick base pairing
  • an abasic building block can be a nucleoside unit solely consisting of a backbone (i e a sugar and an intern
  • the incorporation of such a m ⁇ spa r ⁇ ng or abasic building block into an oligonucleotide derivative can be tolerated as long as the resulting oligonucleotide derivative is capable of specific hybridization with the target nucleic acid and, preferably, capable of modulating the biosynthesis of human bcl-xL protein or of both the human bcl-xL and human bcl-2 proteins
  • the oligonucleotide derivative according to the present invention comprises not more than up to 3 mispai ⁇ ng building blocks and/or not more that up to 3 abasic building blocks
  • An oligonucleotide derivative according to the present invention i e of any of the types as mentioned above, preferably comprises at least one building block of formula (I)
  • Q is H, -OCH 3 , -OCH 2 CH 2 NR 1 R 2 , wherein R, and R 2 are, independently of each other, H or -CH 3 , or, preferably, -0(CH 2 CH 2 ) n OCH 3 , wherein n is 1 , 2 or 3, preferably 1 ,
  • V and W are, independently of each other, the same or different radicals of an internucleosidic bridging group selected from the following group 5'-O-P(0)(0H)-0-3' (phosphodiester), 5'-0-P(0)(SH)-0-3' (phosphorothioate), 5'-0-P(S)(SH)-0-3' (phosphodithioate), 5'-0-P(0)(CH 3 )-0-3' (methylphosphonate), 5'-0-P(0)(NH-R 7 )-0-3' (phosphoamidate) in which R 7 is C r C 3 alkyl, 5'-0-P(0)(0R 8 )-0-3' (phosphotn- ester) in which R 8 is d-Caalkyl, 5'-0-S(0) 2 -CH 2 -3' (sulfonate), 5'-0-S(0) 2 -NH-3' (sulfamate), 5'-NH- S(0) 2 -CH 2 -3' (sulfonamide
  • V and W is such an internucleosidic bridging group and the other is a terminal radical selected from the group consisting of -OH and -NH 2 , preferably -OH;
  • B is a radical of a nucleic acid base
  • V or W is an internucleosidic bridging group other than 5'-0-P(0)(OH)-0-3' (phosphodiester).
  • Q is preferably -OCH 2 CH 2 NR 1 R 2 , wherein one of R, and R 2 is H and the other is -CH 3 , or each of R, and R 2 is H, or, more preferably, Q is -OCH 2 CH 2 OCH 3 .
  • Oligonucleotide derivatives according to the present invention which comprise a building block of formula (I), wherein Q is other than H, can be prepared in accordance with the methods as described, for example, by P. Martin, Helv. Chem. Acta, 78 (1995), pp. 486 - 504, or in an analogous way.
  • V for example is a radical 5'-CH 2 -C(0)-NH-3' (amide)
  • the corresponding nucleoside building block of the above-defined formula (I) has the following structure (1.1):
  • oligonucleotide derivative also encompasses those tautomeric forms which are familiar to the skilled person
  • V and W as the radical of an internucleosidic bridging group, are selected, independently, from the following group 5'-0-P(0)(OH)-0-3' (phosphodiester), 5'-0-P(0)(SH)-0-3' (phosphorothioate) and 5'-CH 2 -C(0)-NH-3' (amide).
  • one of the radicals V or W as the radical of an internucleosidic bridging group, is 5'-0-P(0)(OH)-0-3' (phosphodiester) and the other radical is 5'-0-P(0)(SH)-0-3' (phosphorothioate)
  • V and W are also preferably, as the radical of an internucleosidic bridging group, in each case 5'-0-P(0)(OH)-0-3' (phosphodiester) or, mostly preferred, in each case 5'-0-P(0)(SH)-0-3' (phosphorothioate).
  • oligonucleotide derivative according to the present invention, comprising only building blocks of formula (I), wherein V and W each are phosphorothioate and Q is H
  • nucleic acid base in particular a nucleic acid base B of formula (I), is understood as being, in particular, natural nucleic acid bases and known analogues (cf , for example, Accounts of Chem. Res. 28 (1955), pp. 366-374; Sanghvi, Y.S. in: Antisense Research and Applications, Crooke, ST. and Lebleu, B. (Ed.), CRC Press, Boca Raton (1993), pp. 273-288)
  • nucleic acid bases B can exist in tautomeric forms depending on the ambient conditions. According to the invention, such tautomeric forms are also encompassed by the oligonucleotide derivatives according to the invention, including the preferred embodiments.
  • the invention preferably relates to an oligonucleotide derivative according to the invention, including the said preferences, in which
  • said nucleic acid base in particular said nucleic acid base B of formula (I), is a radical of the formula (V1 ), (V2), (V3), (V4) or (V5)
  • R b1 is -NH 2 , -SH or -OH
  • R b2 is H, -NH 2 or -OH
  • R b3 is H, Br, I, -CN, -C ⁇ C-CH 3 , -C(0)NH 2 or -CH 3 ;
  • R M is -NH 2 or -OH
  • R b5 is H, F, Br, I, -CN, -C ⁇ C-CH 3 , -C(0)NH 2 or -CH 3 .
  • said nucleic acid base in particular said nucleic acid base B of formula (I), is a radical of the formula (V1 ) or (V5)
  • R bl is -NH 2 , -SH or -OH
  • R b2 is H, -NH 2 or -OH
  • R b4 is -NH 2 or -OH
  • R b5 is H, F, Br, I, -CN, -C ⁇ C-CH 3 , -C(0)NH 2 or -CH 3 .
  • said nucleic acid base in particular said nucleic acid base B of formula (I), is selected from the group of the following radicals: xanthine, hypoxanthine, adenine, 2-aminoadenine, guanine, 6-thioguanine, uracil, thymine, cytosine, 5-methylcytosine, 5-propynyluracil, 5-fluorouracil and 5-propynylcytosine.
  • oligonucleotide derivatives which (in their nucleotide/nucleotide derivative sequence) comprise at least one building block of formula (I), wherein B and Q are as defined herein, including the respective preferences and embodiments, and V and W are selected from the group consisting of the following radicals: 5'-0-P(0)(OH)-0-3' (phosphodiester), 5'-0-P(0)(SH)-0-3' (phosphorothioate) and 5'-CH 2 -C(0)-NH-3' (amide),
  • the oligonucleotide derivative comprises preferably the following dimeric unit (bivalent radicals) of the formula (II)
  • Q a and Q b independently of each other, are H, -OCH 3 or -OCH 2 CH 2 OCH 3 , or preferably Q a is -OCH 3 and Q b is H, or more preferably, Q a and Q b are each H;
  • V and W are as defined herein, inclusive of the respective preferences and embodiments.
  • an oligonucleotide derivative according to the present invention consisting of a base sequence selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 3) and the base sequence 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO: 4), wherein each V and each W as radicals of an internucleosidic bridging group of the building blocks according to formula (I) are of the 5'-0-P(0)(SH)- 0-3' (phosphorothioate) type and wherein each Q according to formula (I) is -H.
  • a "chimeric structure” also termed a “chimera” is to be understood as meaning an oligonucleotide derivative which contains 2 or more chemically different regions which are in each case synthesized from one type of nucleic acid building block.
  • Such chimeric oligonucleotide derivatives typically comprise at least one region of modified nucleic acid building blocks which confer one or more advantageous property/properties (for example increased resistance to nucleases, increased binding affinity or diminished occurrence of sequence-independent side-effects) on the oligonucleotide derivative, the so-called "wing”, also designated the M region in that which follows, and a region which enables RNAse H-mediated cleavage of the target nucleic acid to take place, i.e. the so-called "RNAse H window”, also designated the U region in that which follows.
  • wing also designated the M region in that which follows
  • RNAse H window also designated the U region in that which follows.
  • the affinity of an oligonucleotide or an oligonucleotide derivative is customarily determined by measuring the T m value of the oligonucleotide (derivative)Aarget nucleic acid hybrid.
  • the T m value is the temperature at which the oligonucleotide, or its derivative, and the target nucleic acid dissociate from a previously formed hybrid The dissociation is determined spectrophotomet ⁇ cally
  • Methods for determining the T m value belong to the state of the art (cf , for example, F ⁇ tsch and Maniatis, "Molecular Cloning -A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, 1989)
  • increased resistance to nucleases denotes decreased or slowed-down degradation of the oligonucleotide derivatives according to the invention by exonucleases
  • a chimeric oligonucleotide derivative according to the present invention comprises at least one M region, which consists of at least one nucleic acid building block of the formula (I) as mentioned above, and at least one U region, which enables RNAse H-mediated cleavage of the target nucleic acid to take place
  • the U region consists, in particular, of customary 2'-deoxyr ⁇ bonucle ⁇ c acid building blocks which are linked to each other by way of phosphodiester bonds, or preferably phosphorothioate bonds, as the internucleosidic group
  • the M region of a chimeric oligonucleotide derivative according to the present invention consists, in particular, of nucleic acid building blocks of the formula (I) in which Q is as defined, preferably -OCH 2 CH 2 OCH 3 , in which W and V, as the radical of an internucleosidic bridging group, are a phosphodiester bond, a phosphorothioate bond or
  • Chimeric oligonucleotide derivatives according to the invention of the above-mentioned type which preferably consist of a total of 8 to 25, in particular of 8 to 20, and preferably of 20 consecutive building blocks, preferably of the nucleoside type, and preferably comprise one or more, preferably one, U rehaving, preferably 4 to 13, for example 10 or 13, nucleoside building blocks of formula III
  • V and W in each case are a phosphodiester group or phosphorothioate group, preferably a phosphorothioate group, as the radical of an internucleosidic bridging group,
  • M reg ⁇ on(s) comprising the remaining nucleoside building blocks, which are of formula (I), in which V and W are, as the radical of an internucleosidic bridging group, in particular, in each case, a phosphodiester, phosphorothioate or amide group, preferably phosphodiester or phosphorothioate, in particular phosphodiester, and in which Q and B are as defined above, including the respective preferences and specific embodiments, Q being in particular - 0-CH 2 CH 2 OCH 3
  • the M and U regions in chimeric oligonucleotide derivatives according to the invention are preferably present in one of the following arrangements
  • oligonucleotide derivatives according to the invention are conjugated with other units, for example a micelle-forming group, an antibody, a carbohydrate, a receptor-binding group, a steroid such as cholesterol, a polypeptide, an intercalating agent, such as an acridine derivative, a long-chain alcohol, a dendrimer, a phospholipid and other lipophilic groups.
  • Conjugating in this way confers advantageous properties with regard to the pharmacokinetic characteristics on the oligonucleotide derivative according to the invention. In particular, conjugating in this way achieves increased cellular uptake
  • an oligonucleotide derivative according to the present invention consists exclusively of nucleoside building blocks of the formula (I) which are connected to each other by way of phosphodiester bonds as the internucleosidic bridging groups V and/or W.
  • an oligonucleotide derivative according to the invention exclusively comprises nucleoside building blocks of the formula III which are connected to each other by way of phosphorothioate bonds as the internucleosidic bridging groups V and/or W.
  • the present invention furthermore relates to an oligonucleotide derivative as mentioned herein, consisting of a base sequence selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO 3), the base sequence
  • each V and each W as radicals of an internucleosidic bridging group of all building blocks according to formula (I) are of the 5'-0-P(0)(SH)- 0-3' (phosphorothioate) type, and wherein each Q according to formula (I) of the nucleotides being underlmed is -OCH 2 CH 2 OCH 3 and wherein each Q according to formula (I) of the remaining nucleo- tides is -H
  • oligonucleotide derivative also encompasses salts, in particular acid addition salts, salts with bases or, if several salt-forming groups are present possibly also mixed salts or internal salts
  • Salts of oligonucleotide derivatives according to the invention are, in particular pharmaceutically tolerated salts, i e essentially non-toxic salts
  • Such salts are formed, for example, from the oligonucleotide derivatives according to the invention which possess an acidic group, for example a carboxyl group, a phosphodiester group or a phosphorothioate group, and are, for example, salts with suitable bases
  • These salts include, for example, non- toxic metal salts which are derived from metals of groups la, lb, lla and lib of the Periodic System of the elements, in particular suitable alkali metal salts, for example lithium, sodium or potassium salts, or alkaline earth metal salts, for example magnesium or calcium salts
  • suitable alkali metal salts for example lithium, sodium or potassium salts, or alkaline earth metal salts, for example magnesium or calcium salts
  • They furthermore include zinc and ammonium salts and also salts which are formed with suitable organic amines, such as unsubstituted or hydroxyl-substituted mono-, di- or tri-alkylammes, in particular mono-, di-
  • Lithium salts, sodium salts, magnesium salts, zinc salts or potassium salts are preferred, with sodium salts being particularly preferred
  • Oligonucleotide derivatives according to the invention which possess a basic group, for example an ammo group or imino group can form acid addition salts, for example with inorganic acids, for example with a hydrohalic acid, such as hydrochloric acid, sulfu ⁇ c acid or phosphoric acid, or with organic car- boxylic acids, sulfonic acids, sulfo acids or phospho acids or N-substituted sulfamic acid, for example acetic acid, propionic acid, glyco c acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fuma ⁇ c acid, malic acid, tarta ⁇ c acid, gluconic acid, gluca ⁇ c acid, glucuronic acid, citric acid, ben- zoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-am ⁇ nosal ⁇ cyl ⁇ c acid, 2-phenoxybenzo ⁇ c acid, 2-acetoxybenzo
  • Oligonucleotide derivatives according to the invention which possess both acidic and basic groups can also form internal salts
  • Oligonucleotide conjugates according to the invention which possess more than one group which is suitable for salt formation can also form mixed salts
  • the present invention relates to a process for preparing an oligonucleotide derivative according to the present invention, said process comprising incorporating at least one building block of formula (I) as mentioned above into the oligonucleotide derivative during oligonucleotide synthesis
  • the compounds of the formula (I), wherein V and W each are a terminal group as defined above, are employed as nucleoside building blocks in the synthesis of the oligonucleotide derivatives according to the invention
  • the oligonucleotide derivatives according to the invention can be prepared, in a manner known per se, in accordance with a variety of methods, in DNA synthesis equipment which can be automated and which can be obtained commercially in conjunction with method protocols
  • the phosphot ⁇ ester method, the phosphite triester method or the H-phosphonate method which are familiar to the skilled person, can be used (cf , for example, Eckstein, F , "Oligonucleotides and Analogues, A Practical Approach", IRL Press (1991 ))
  • the approach can, for example, be to react, for example, a nucleoside building block of the formula (I), in which V a and W a are in each case -OH, with a protecting group reagent, for example 4,4'-d ⁇ methoxytr ⁇ phenylmethyl chloride, to give a nucleoside of the formula (lb)
  • a nucleoside building block of the formula (I) in which V a and W a are in each case -OH
  • a protecting group reagent for example 4,4'-d ⁇ methoxytr ⁇ phenylmethyl chloride
  • V a is a protected hydroxyl group and Q and B are defined as above for the compound of the formula I including the said preferences, reactive groups thereof being protected by a suitable protective group where necessary, and to bind the compound of the formula lb with the aid of a linker, for example succinic anhydride, to a solid support material, for example to "Controlled Pore Glass” (CPG), which contains long-chain alkylamino groups
  • CPG Controlled Pore Glass
  • the hydroxyl group of another nucleoside building block of the formula (lb) is denvatized, for example using R x O-P[N( ⁇ -propyl) 2 )] 2 to give a phosphoramidite of the formula (lc)
  • R x is a customary protecting group, for example ⁇ -cyanoethyl, wherein B and Q are defined above
  • Protecting groups for the radical V a as protected hydroxyl group in compounds of the formulas (lb) and (lc) preferably are of the t ⁇ tyl-type, in particular t ⁇ tyl (Tr), 4-monomethoxyt ⁇ tyI (MMTr), preferably 4,4'-d ⁇ methoxyt ⁇ tyl (DMTr) and, likewise preferably, 4,4',4"-tr ⁇ s-tert-butylt ⁇ tyI (TTTr)
  • Oligonucleotide derivatives according to the present invention which do not contain, or which do not exclusively contain, phosphodiester groups or phosphorothioate groups as the internucleosidic bridging groups can be prepared in a manner known per se (cf , for example, the above-mentioned publications of De Mesmaeker, A , or Crooke, S T )
  • the oligonucleotide derivatives according to the invention possess a number of advantageous properties These include, in particular, a high binding affinity for a target nucleic acid and a high resistance to nucleases Furthermore, they are capable of a sequence-specific effect, are taken up satisfactorily by a cell and have adequate bioavailability These properties make the oligonucleotides according to the invention particularly suitable for pharmaceutical applications, in particular for modulating the biosynthesis or expression of the human bcl-xL protein or both of the human bcl-xL and bcl-2 proteins Consequently, in a further aspect the present application relates to an oligonucleotide derivative according to the present invention for use in medicine
  • an oligonucleotide derivative according to the invention can be used, in particular, as an antisense oligonucleotide
  • the expression "antisense” is known to the skilled person and, in the context of the present invention, characterizes, in particular, the relationship between an oligonucleotide derivative according to the invention and the sequence, which is complementary to it, of a target nucleic acid (i e human bcl-xl mRNA or bcl-2 mRNA), namely that the oligonucleotide derivative and the complementary sequence are able to hybridize to each other
  • a target nucleic acid i e human bcl-xl mRNA or bcl-2 mRNA
  • the identification of a suitable antisense oligonucleotide is a multi-step process First of all, a target nucleic acid is identified which underlies the protein whose expression characterizes a pathological state in humans, and is to be modulated In the present case,
  • the invention also provides the use of an oligonucleotide derivative according to the invention, including the said preferences, as an antisense oligonucleotide
  • oligonucleotide derivatives are preferred which are capable of modulating the expression (biosynthesis) of the human bcl-xL protein. Even more preferred are oligonucleotide derivatives which are capable of modulating the expression (biosynthesis) of both the human bcl-xL protein and the human bcl-2 protein.
  • the invention furthermore relates to a pharmaceutical composition which comprises an oligonucleotide derivative according to the invention, or a pharmaceutically tolerated salt thereof, in a pharmaceutically effective quantity, if desired together with a pharmaceutically tolerated excipient and/or auxiliary substance.
  • a pharmaceutical composition preferably is suitable for administration to humans suffering from a disease that responds to the modulation of human bcl-xL expression or that responds to the modulation of human bcl-xl and human bcl-2 expression.
  • compositions according to the invention can be used, for example, for the therapeutic or prophylactic treatment of hyper- plastic or neoplastic states, for example of cancer, in particular solid tumors, or of restenosis, fribrosis or psoriasis, preferably in a cancer selected from the group consisting of colorectal cancer, gastric cancer, prostate cancer, neuroblastoma, melanoma, thyroid cancer, renal cancer, breast cancer and, preferably, lung cancer, the letter being, for example, in particular NSCLC (non-small cell lung cancer) or, most preferably, SCLC (small cell lung cancer).
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • compositions according to the invention are capable of killing cancer cells, preferably by induction of apoptosis, and/or of reverting multidrug resistance of tumors.
  • compositions which are preferred in accordance with the invention comprise preferred oligonucleotide derivatives as described above.
  • compositions according to the invention are preferably present in the form of preparations which can be administered parenterally or of infusion solutions.
  • Aqueous solutions of the active substance in water-soluble form for example in the form of one of the above-mentioned water- soluble salts, in the presence or absence of salts, such as NaCI, and/or pharmaceutically tolerated excipient materials, such as sugar alcohols, for example mannitol, are suitable, in particular, for par- enteral administration, for example for intravenous or intraperitoneal administration.
  • Aqueous suspensions for injection which comprise viscosity-increasing substances, such as sodium carboxymethyl cellulose, sorbitol and/or dextran, are also suitable for parenteral administration.
  • compositions according to the invention can be sterilized and/or comprise auxiliary substances, for example preservatives, stabilizers, wetting agents and/or emulsifying agents, solubilizmg agents, salts for regulating the osmotic pressure and/or buffers
  • the pharmaceutical preparations which, if desired, can comprise additional pharmacologically (or pharmaceutically) active compounds, for example antibiotics, are prepared in a manner known per se, for example by means of conventional solubilizmg or iyophilizing methods, and comprise from about 0 0001 % by weight to about 95 % by weight, preferably from about 0 1 % by weight, to about 90 % by weight, in particular from about 0 5 % by weight to about 30 % by weight, for example from 1 % by weight to 5 % by weight, of active compound(s)
  • Dosage forms in the form of individual doses comprise, for example, from about 0 001 % by weight to about 20 % by weight, of active compound(s) dosage forms which are not in the form of individual doses comprise, for example, from about 0 001 % by weight to about 10 % by weight of active compound(s)
  • Dose units preferably comprise from about 0 0005 mg to about 0 5 mg, preferably from about 0 005
  • compositions according to the invention can comprise cationic lipids
  • compositions according to the invention are also preferred which additionally comprise a customary cytostatic agent Such combination preparations are preferably employed for treating hy- perplastic or neoplastic states such as cancer
  • the present invention furthermore relates to an oligonucleotide derivative according to the invention, including the above-mentioned preferences, or a pharmaceutically tolerated salt thereof, for use in the prophylactic or therapeutic treatment of humans, in particular of a pathological state, which is characterized by the expression or biosynthesis of human bcl-xL protein or of both human bcl-xL and human bcl-2 proteins
  • the present invention furthermore relates to the use of an oligonucleotide derivative according to the invention, including the above-mentioned preferences, for preparing a pharmaceutical composition for the prophylactic or therapeutic treatment of a pathological state in humans, which is characterized by the expression or biosynthesis of human bcl-xL protein of both human bcl-xL and human bcl-2 proteins
  • the present invention relates to a method for the prophylactic or therapeutic treatment of a pathological state in a mammalian subject, including man, which state is characterized by the expression or biosynthesis of human Bcl-xL protein or of both human bcl-xL and human bcl-2 proteins, which method comprises administering a pharmaceutical composition according to the inven ⁇
  • the invention relates to a method for modulating the expression of human Bcl-xl protein or of both human bcl-xL and human bcl-2 proteins in a cell, which comprises bringing the cell, or a tissue or body fluid which contains this cell, into contact with an oligonucleotide denvat ve according to the invention, including the above-mentioned preferences or with a pharmaceutical composition according to the invention
  • a process for modulating the expression or biosynthesis of a protein in a cell can be advantageously applied both in vitro and in vivo
  • oligonucleotide derivatives according to the invention are also suitable for use as diagnostic agents and can be employed, for example, in a manner known per se, as gene probes for detecting genetically determined diseases or viral infections by means of selective interaction at the level of single-stranded or double-stranded target nucleic acids
  • a diagnostic application is possible in vivo as well as in vitro, due to the increased stability towards nucleases
  • the diagnosis can take place, for example, on isolated tissue samples, blood plasma, blood serum or other body fluids, and, in the case of m-vivo diagnosis, on tissues, cells or body fluids in the patient to be investigated as well In a preferred embodiment thereof, such diagnosis takes place under physiological conditions
  • Another aspect of the present invention consequently relates to an oligonucleotide derivative according to the invention, including the above-mentioned preferences, for use in a diagnostic method
  • the oligonucleotide derivatives according to the invention are suitable both for m-vivo and for in-vitro diagnostic methods
  • the SW2 SCLC cell line is obtained from the Dana Farber Cancer Institute Boston, MA and The NSCLC cell line NCI-H125 cell line is obtained from the American Type Tissue Culture Collection (ATCC, Rockville, MD) Lung cancer cell lines are cultured in RPMI-1640 medium (Gibco Life Technologies Ltd, Paisley, UK) supplemented with 2 mM L-glutamine, 10% fetal calf serum (FCS Hy-Clone II Fetal Clone II), 50 lU/mL penicillin and 50 ⁇ g/mL streptomycin at 37°C in a humidified atmosphere with 5% CO2 SW2 cells are grown as floating aggregates, H125 cells are grown as monolayers
  • oligonucleotides of the present invention can be prepared according to methods known in the art
  • a Perseptive Expedite MOSS synthesizer is used The syntheses are performed on Polystyrene Primer support (Pharmacia) loaded with the 3'-end residue Solutions at 0 05M in Acetonitrile of ⁇ -cyanoethyl deoxynbonucleosides phosphoramidites (Amersham Pharmacia Biotech) or ⁇ -cyanoethyl 2'-methoxyethylr ⁇ bonucleos ⁇ des phosphoramidites (P Martin, Helvetica Chimica Acta, 78 (1995), 486 - 504) are used In the coupling step, phosphoramidites are activated by Benzimidazolium T ⁇ flate (0 2M in Acetonitrile, R Noyo ⁇ , J Org Chem 61 , 1996, 7996 - 7997) Sulfu ⁇ - zation can be performed by a 0 5 M solution of
  • Crude product of approximately 70 % purity is further purified by reverse phase chromatography using C18 stationary phases.
  • the purified material is lyophilized, redissolved in water, quantified by UV absorption at 260 nm and checked using standard procedures by capillary gel electrophoresis for purity and by MALDI-TOF MS for identification.
  • the purified material is re- dissolved in 100 nriM NaCI and further desalted by ultrafiltration. The oligonucleotides are finally lyophilized and stored as dry materials.
  • oligonucleotide derivatives are synthesized, all being 20-mer phosphorothioates purified by high pressure liquid chromatography (HPLC). The sequences are
  • OD1 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 3)
  • OD2 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO: 4)
  • OD3 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 3)
  • OD4 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO: 4)
  • OD5 5'-AAAGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 5)
  • the underlined nucleotides of OD3, OD4 and OD5 denote building blocks bearing a 2'-methoxyethoxy substituent. These oligonucleotides OD1 , OD2, OD3, OD4 and OD5 are the most preferred embodiment of the present invention
  • oligonucleotide derivative C01 for use as scrambled control oligonucleotide derivative to OD2 is likewise synthesised
  • oligonucleotide derivative C02 targets the translation initiation site of the bcl-2 mRNA and is used as a bcl-2 specific control
  • oligonucleotide derivatives C03 and C04 are synthesized in the same manner C03 and C04 are used as scrambled controls to OD3 and OD4, respectively
  • the underlined nucleotides of C3 and C4 designate building blocks bearing a 2'-methoxyethoxy sub- stituent
  • Antisense oligonucleotide derivatives OD1 and OD2 and the C01 control oligonucleotide derivative are delivered to cells in the form of complexes with the cationic lipid N-[1-(2,3-d ⁇ oleoyloxy)propyl]-N,N,N- trimethylammonium methylsulfate (DOTAP) (Boehnnger Mannheim, Germany) under serum free conditions Equal volumes of ODNs (6 ⁇ M) and DOTAP (0 2 mM) in HEPES-buffered saline are mixed and allowed to complex for 10 mm at room temperature Different dilutions of the stock solutions are prepared in RPMI-1640 depending on the experiment, and the mixtures are further diluted into 9 volumes of cell suspension to achieve a final density of 2 x 10 ⁇ cells/ml in the cultures After 4 h of incubation serum is added to the cultures to a final concentration of 10% Control cultures ware treated with equivalent concentrations of DOTAP or medium alone (untreated
  • Total RNA is prepared from 5 x 10 6 SW2 cells by use of the RNeasy Mini Kit (Qiagen GmbH, Hilden Germany) and QIAshredder (Quiagen GmbH) for homogenization
  • 5 ⁇ g total RNA are electrophoresed on a denaturing 1 % formaldehyde-agarose gel, transferred to a NYTRAN NY 13 N membrane (Schleicher & Schuell, Dassel, Germany) according to standard procedures (Anony- mous1994), and cross-linked using a Stratalinker (Stratagene, La Jolla, USA)
  • Equal loading and blotting of the RNA is confirmed and documented by staining with 0,03% methylenblue in 0,3M sodium acetate, pH 5,2 Membranes are pre-hyb ⁇ dized for 1 h at 68°C in QuickHyb Solution (Stratagene) Membranes are hybridized for 2 hours at 68°C with an EcoRI restriction fragment composed of nucleotides 1410
  • OD1 efficiently reduces the level of the bcl-2 mRNA and, despite the 3-base mismatches compared with the natural base sequence of bcl-xL mRNA, also effectively reduces the level of the bcl- xL mRNA
  • OD1 can tolerate mismatches, in particular those which comprise an A to G, a T to C, and a G to T substitution at positions 3, 5, and 15 of OD1 , respectively, said oligonucleotide derivative still being effective in modulating the biosynthesis both of bcl-xL and bcl- 2
  • OD2 only downregulates bcl-xL expression and does not reduce the level of the bcl-2 mRNA species As expected for the control oligonucleotide derivative C02, it selectively down- regulates the expression of bcl-2
  • Example 6 Western Blot analysis using the 2'-methoxyethoxy oligonucleotide derivatives OD3, OD4 and OD5
  • the antisense oligonucleotide derivative OD3 reduces the level of the bcl-2 protein to 40 1 % and of the bcl-xL protein to 32.6% of the respective untreated controls OD5 reduces the level of the bcl-2 protein to 48 3% and of the bcl-xL protein to 29.1 %.
  • Example 7 Caspase-3 activation assay following treatment with the 2'-methoxyethoxy modified oligonucleotide derivatives OD3, OD4 and OD5
  • Caspase activity in cells is analyzed by use of a colorimet ⁇ c test system
  • SW2 cells are treated with the 2'-methoxyethoxy modified oligonucleotide derivatives 0D3, OD4 and OD5, respectivley, as described above
  • the cells are lysed in buffer by freeze/thawing essentially as described (Olie et al , Curr Biol 8 (1998), pp 955 - 958) and lysates are centifuged at 13000 rpm at 4°C for 15 mm
  • Cytosolic protein is mixed with 50 ⁇ M of the caspase-3 specific substrate DEVC-pNA (Asp-Glu-Val-Asp-p-nitroaniiides) (Bachem, Dubendorf, Switzerland) and incubated at 37°C Subsequently, substrate cleavage is monitored at 405nm using a SPECTRAmax 340 microplate reader and analyzed using SOFTmax PRO software (Molecular Devices)
  • SW2 SCLC and the H125 NSCLC cell lines both co-express bcl-2 and bcl-xL, although bcl-2 expression in H125 cells is rather low This, however, does not necessarily mean that both death antagonists are equally important survival factors for the different lung cancer cell types
  • SW2 and H125 cells are treated with the antisense oligonucleotide derivatives OD1 or OD2, and for control purposes, with C01 or C02, and the cytotoxic effects are measured in colo ⁇ metric cell viability assays
  • H125 cells The viability of H125 cells is determined by use of a standard MTT For each treatment, 100 ⁇ L of cells/oligonucleotide derivative mixture are plated in triplicates in 96-well plates Cells are incubated for 96 h under cell culture conditions before 10 ⁇ L of MTT reagent are added per well and allowed to react for 2 h Cells are lysed for 3 hours under 37°C with 100 ⁇ l lysis buffer, containing 20% SDS in 50% dimethyl-formamide and 50% H 2 0, pH 4,7 Absorbance at 570 nm is measured by use of an enzyme- linked immunosorbent assay reader (2550 EIA reader, Bio Rad Laboratories Ltd , Hercules, CA) Data are expressed as percent of the absorbance of untreated control cells
  • the H125 cell line is highly susceptible to treatment with oligonucleotide derivative OD2 and also to treatment with oligonucleotide derivative OD1 Since the control oligonucleotide derivative C02 is less effective in reducing the viability of these cells, it can be concluded that bcl-xL is the more critical survival factor for this NSCLC cell line and that bcl-2 only plays a minor protective role
  • Example 9 Detection of apoptotic cells based on light scattering properties and electron microscopy

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Abstract

Cette invention a trait à des dérivés d'oligonucléotide antisens dirigés contre l'ARN messager humain bcl-xL et capables de moduler la biosynthèse de la protéine humaine bcl-xL. Elle concerne, de surcroît, des dérivés d'oligonucléotide antisens dirigés à la fois contre l'ARN messager humain bcl-xL et l'ARN messager humain bcl-2 et capables de moduler la biosynthèse des deux protéines humaines bcl-xL et bcl-2. Elle porte également sur des compositions pharmaceutiques renfermant ces dérivés d'oligonucléotide, sur l'usage qui en est fait ainsi que sur des méthodes thérapeutiques et diagnostiques utilisant ces dérivés d'oligonucléotide.
EP00925239A 1999-04-30 2000-04-26 Derives d'oligonucleotides diriges contre les arn messagershumains bcl-xl et bcl-2 Withdrawn EP1181361A2 (fr)

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FR2832154B1 (fr) 2001-11-09 2007-03-16 Centre Nat Rech Scient Oligonucleotides inhibiteurs et leur utilisation pour reprimer specifiquement un gene
DE10258677A1 (de) * 2002-12-13 2004-06-24 Elez, Vera, Dr. Kombinations-antisense-Oligonukleotid-Krebstherapie
FR2852606A1 (fr) * 2003-03-18 2004-09-24 Inst Nat Sante Rech Med Moyens pour inhiber simultanement l'expression de plusieurs genes impliques dans une pathologie
US9993472B2 (en) 2014-01-28 2018-06-12 Unity Biotechnology, Inc. Treatment for osteoarthritis in a joint by administering a means for inhibiting MDM2

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US5470974A (en) * 1985-03-15 1995-11-28 Neu-Gene Development Group Uncharged polynucleotide-binding polymers
ATE286125T1 (de) * 1993-09-20 2005-01-15 Univ Pennsylvania Regulation der genexpression von bcl-2
WO1995031470A2 (fr) * 1994-05-13 1995-11-23 Merck Frosst Canada Inc. Inhibiteurs antisens de l'expression de genes
US5874566A (en) * 1996-10-25 1999-02-23 Hisamitsu Pharmaceutical Co. Inc. Il-15 triplex oligonucleotides
CA2336384A1 (fr) * 1998-07-02 2000-01-13 The Trustees Of Columbia University In The City Of New York Oligonucleotides inhibiteurs de bcl-xl
US6214986B1 (en) * 1998-10-07 2001-04-10 Isis Pharmaceuticals, Inc. Antisense modulation of bcl-x expression

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