WO1993024653A1 - Antisense oligonucleotides with antitumoral activity, pharmaceutical compositions which comprise them, and their uses - Google Patents

Antisense oligonucleotides with antitumoral activity, pharmaceutical compositions which comprise them, and their uses Download PDF

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Publication number
WO1993024653A1
WO1993024653A1 PCT/EP1993/001287 EP9301287W WO9324653A1 WO 1993024653 A1 WO1993024653 A1 WO 1993024653A1 EP 9301287 W EP9301287 W EP 9301287W WO 9324653 A1 WO9324653 A1 WO 9324653A1
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gene
antisense oligonucleotides
aodn
antisense
cells
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PCT/EP1993/001287
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French (fr)
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Susanna Morelli
Angelo Nicolin
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Consiglio Nazionale Delle Ricerche
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to antisense oligonucleotides, in particular antisense oligonucleotides which block the synthesis of BCL 2 protein.
  • the traditional obstacle in tumor chemotherapy resides in the difficulty of rigorously identifying cancer-specific targets.
  • the therapeutical use of even active compounds displaying a very selective mechanism of action against specific molecular targets does not succeed in differentiating, to a desired extent, the effect towards neo p l a s t i c c e l l s a n d h e a l t h y c e l l s .
  • molecular anomalies determine the neoplastic outcome by means of one single mechanism, or, more often, through the successive contributions by a plurality of a Iterations.
  • the antisense oligonucleotides are constituted by short chains of single-filament synthetic DNA.
  • the order of sequence of the single bases is so designed as to result complementary with the sequence of messenger RNA which one wants to inhibit.
  • the antisense compounds offer themselves as potential antitumoral compounds in those cases in which the molecular alterations are known.
  • the subject-matter of the present invention are antisense oligonucleotides (aODN's) characterized in that they comprise nucleotidic sequences hybridizing to a sequence of the hybrid gene and its messenger at the splicing region which is formed following a translocation of bcl-2 gene from chromosome 18 to chromosome 14 in a section of the latter which contains Ig-H gene (the gene for immunoglobulin H), and which block the synthesis of human BCL-2 protein.
  • aODN's antisense oligonucleotides
  • the present invention relates to the use of antisense oligonucleotides (aODN's) for the study and therapy of tumors in which BCL 2 protein is produced in excessive amounts owing to an alteration in the genetic code.
  • the alteration consists in the translocation of a portion of chromosome 18 containing bcp-2 gene into a portion of chromosome 14 containing Ig-H gene. The splicing point between said portions forms a nucleotidic sequence only present in tumoral cells.
  • antisense oligonucleotides complementary to the sequence formed owing to the translocation are capable of inhibiting the expression of BCL 2 protein in tumoral cells only, actually causing a growth inhibition and a cellular death of said cells with simultaneously respecting the integrity of the healthy cells.
  • a further subject-matter of the present invention is also a phramaceutical composition containing at least one antisense oligonucleotide as defined hereinabove, or a chemically modified form thereof without that the nucleotidic sequence is altered, and a therapeuticatly acceptable carrier.
  • a last subject-matter of the present invention is the use of such antisense compounds or of modified forms thereof in order to prepare a pharmaceutical composition to be used for the treatment of some pathological forms, in particular some tumor types.
  • the aODN's of the present invention can be designed on the basis of the sequence of mRNA in the splicing region between bcl-2 gene and the gene for the heavy chain of immunoglobulin Ig-H. Said sequence may be either known or determined by sequentiating samples of tumoral lymphocytes according to standardized methodologies as disclosed in a large number of manuals (Molecular Cloning, A Laboratory Manual, 1989, Sambrook, Maniatis, etc.).
  • aODN's are understood as being suitable, which have a specific length suitable for hybridizing in optimal way to mRNA and target DNA.
  • a length may also vary as a function of the chemical modifications introduced into the molecule constituted by natural nucleotides in order to enhance the pharmacological properties thereof.
  • the minimal length preferably is 10 nucleotides, and the maximal length is of 100 nucleotides, preferably 50, still more preferably 30 nucleotides.
  • Antisense compounds may act in various moments of the metabolic history of mRNA, both at nuclear and cytoplasmatic levels. It is furthermore possible that one from the antisense compounds action points is at ribosome level, or they may directly act through a direct interaction with nuclear or mitochondrial DNA.
  • sequence length can also be determined on the basis of considerations known to those skilled in the art, which relate to a sufficient effectiveness of penetration through the cellular membranes. (Locke et al. PNAS 86:3474, 1989, Yakubov et al. PNAS 86: 6454, 1989).
  • a specific sequence defined according to such criteria as cited hereinabove can be synthetized in solid phase by means of well-known methods in the art and described, for example, by Narang (Tetrahedron 39:3, 1983), or by Itakuran (Ann. Rev. Biochem. 53:323, 1984), or in "Oligonucleotide Synthesis; A Practical Approach", Gait M. J. Ed. IRL Press, Oxford, U.K., 1984).
  • such aODN's can be purified by means of known methods in the art, such as, e.g., polyacrylamide gel electrophoresis under denaturing conditions, or by high resolution chromatography, reverse phase chromatography or ion exchange column chromatography, or, finally, by capillary electrophoresis.
  • the aODN's according to the present invention are characterized by the possibility of inhibiting the synthesis of BCL 2 protein in the tumoral cells which display the t(14;18) translocation, by means of the use of specific antibodies, or by taking advantage from the capability of inhibiting the relevant mRNA by means of molecular probes. Also the inhibition of proteinic translation can be caused by means of a translation in cell-free system using lysates of red cells or wheat germ extracts (Sambrook, etc.).
  • the inhibition of synthesis of BCL 2 protein can also be determined according to a dose-effect relationship, by evaluating the growth inhibition of human tumor cells bearing the t(14;18) translocation, by evaluating the number of cells under the optical microscope or by means of the tritiated thymidine incorporation test.
  • the activity of the compounds according to the present invention can also be determined by analysing the induction of orderly cellular deletion in the already cited tumoral cells.
  • the orderly cellular deletion, or apoptosis is determined by analysing the cellular nucleus, or carrying out an electrophoresis in agarose, of the nuclear extract which appears to be fragmented into units of approximately 200 bases.
  • the aODN's according to the present invention can be modified for the purpose of causing the desired characteristics thereof to be improved (for a general survey also as regards general considerations relating to designing antisense oligonucleotides for the purpose of blocking the synthesis of a specific protein, reference is made to Uhlmann E. and Peymann A. Chem. Rev. 90:5543, 1990 or Englisch U. and Gauss D.H. Angewandte Chemie 30:613, 1991).
  • such aODN's can be chemically modified at the phosphodiester linkage, or at t h e terminal 3' ends in order to render them more stable against a cleavage by nuclease enzymes, or in order to improve their penetration or hybridization characteristics, as disclosed in European Patent Application published with Publication No. 386 563.
  • the aODN's can be covalently bound to a lipid, as disclosed, e.g., in International Patent Application published with Publication No. WO/9010448.
  • the concerned aODN can be transported more effectively through the cellular membranes and can be cleft, for example, with cellular cytopl a s ma t i c e n z yme s i n o rd e r t o l i be r a t e a c t i v e a ODN .
  • the aODN's may also be phosphorylated, or they may be linked to cholesterin or to on olesterol derivatives, at their ends (Letsinger et. al. PNAS 86:6553, 1989).
  • the aODN's of the present invention may be modified in order to secure that the drug is specifically delivered to tumoral tissues and consequently the dispersion of administered molecule is limited.
  • the aODN's according to the present invention may be administered in acceptable pharmaceutical forms, in particular in a suitable form for an oral administration. Dosage forms and rhythms can be parallel to those as adopted in clinical administrations of known compounds.
  • the pharmaceutical compositions may contain at least one aODN according to the present invention in association with active substances and with either solid or liquid, pharmaceutically acceptable carriers. Any suitable carriers may be used. Furthermore, the pharmaceutical compositions may contain further pharmaceutically active substances and can be prepared according to known methods in the art.
  • aODN's according to the present invention or modified forms thereof in order to prepare such pharmaceutical compositions and for diagnostic or medical purposes, for example for the treatment of some tumoral forms or of other diseases which may benefit from an inhibition of the synthesis of BCL 2 protein, are also a subject-matter of the present invention.
  • the aODN's of the present invention can be used, after being fixed onto a solid carrier according to well known methods in ther art, in order to detect genetic alterations responsible for various pathological forms, including tumors.
  • the aODN's according to the present invention may be used with a signal portion, for example a radioactive isotope, an enzyme, or a fluorescer according to well known methods in the art.
  • human DHL-4 cells were grown in HB 101 media with a density of 10 4 cells per each well, to which an aODN according to the present invention was added in an amount comprised within the range of from 1 to 10 micromoles (microM).
  • microM micromoles
  • the cultures were admixed with half aODN dose and the activity was tested on from 2 to 7 days by cell count by microscope examination followed by a count of radioactivity incorporated into DNA following the addition of tritiated thymidine.
  • the aODN's demonstrated a clear capability of reducing both the number of cells in the wells and the amount of radioactivity incorporated to cell nuclei.
  • the activity results to be dose-dependent and specific to cells bearing the genetic damage to which aODN was directed.
  • Figure 3 shows the effect of an aODN on the in vitro growth of a human tumor cell line DHL-4.
  • the growth curves respectively relate to the untreated DHL-4 line, to the same line treated with an aODN constituted by a sense sequence, a casual sequence, or the same line DHL-4 treated with an a n t i s e n s e sequence.
  • the reported data relates to the dose of 1 mi croM.
  • the four curves relate to: I: reversed control, i.e., a polynucleotide having a reversed nucleotidic sequence relatively to the antisense nucleotide used in the test; U: untreated cells; S: sense nucleotide (control); AS: antisense nucleotide according to the present invention.
  • Figure 4 displays the dose-dependent effect of aODN on the growth of DHL-4 tumor. The doses are indicated as concentrations (from 10 microM down to 0.001 microM) of aODN used. From the chart of Figure 4, one may understand how a decrease in the activity of inhibition of DHL-4 tumor cell growth is displayed with decreasing antisense oligonucleotide dose administered.
  • Figure 5 shows the specific antiproliferative effect for tumor cells bearing the genetic damage against which aODN is directed. No growth inhibiting effects are exerted against human tumor cells not bearing the molecular alteration which is the target of the present aODN.
  • DHL-4 is the tumor of leukemia B with 14-18 translocation, which is relevant for the purposes of the present invention
  • Raji is a leukemia B tumor without 14-18 translocation
  • LCL represents the tumor of leukemia B without translocation
  • YAC is the Leukemia T tumor without translocation
  • K562 represents the tumor of mieloid leukemia without translocation.
  • the above said cells after being treated with the aODN's according to the present invention, result (a) to modify their morphological appearance, (b) to demonstrate DNA fragmentation and (c) to display an at all abnormal distribution of their DNA content.
  • Figure 6 of the accompanying drawings represents, in that regard, a cytofluorimetric analysis of cellular DNA, relevant to the effect of DNA fragmentation caused by the compounds.
  • the chart “A” relates to the control, in the case of untreated cells.
  • the chart “B” relates to cells treated with an antisense oligonucleotide according to the present invention.
  • the chart “C” relates to the overlapping of the histograms relevant to the cases of untreated cells and of cells treated with the antisense oligonucleotide.
  • the chart “D” is obtained by overlapping the histogram "B” to the histogram of cells treated with sense oligonucleotide.
  • the cells display a fragmented nuclear appearance, and at all recognizable when untreated cells or cells treated with an ODN without antisense activity, or without an activity correlated with the genetic alteration taken into consideration for the purposes of the present invention, are tested for nuclear morphology.
  • the tumor treated as already mentioned is submitted to nuclear DNA extraction according to well known methods in the art and is submitted to electrophoresis in agarose by following a standardized methodology (also described by Maniatis).
  • the DNA of the tumor treated with an aODN according to the present invention appears to have been fragmented into bands which are regularly ditributed in agarose gel and correspond to 200-base segments and are detected by U.V. light examination.
  • the already mentioned cells, after treatment with an aODN according to the present invention appear to be of reduced volume as compared to control cells and, after their DNA being stained with ethidium bromide, when submitted to the same cytofluorimetric examination appear to contain a reduced DNA amount, so that most cells result to emit a lower fluorescence than as corresponding to normal DNA content.
  • aODN's according to the present invention are capable of inducing an orderly cell deletion (apoptosis), and that said Orderly cell deletion results to be important for the pharmacological activity disclosed hereinabove.

Abstract

The present invention relates to antisense oligonucleotides (aODN) which comprise nucleotidic sequences characterized by the possibility of hybridizing, also inside the interior of human cells, to regions of genetic code molecules originated from the translocation of a portion of chromosome 18 to other chromosomes, in particular chromosome 14. Chromosomal translocation is the cause of a large number of diseases, in particular tumors. The inhibition of the biological effects of translocations by antisense oligonucleotides is meaningful from the scientific knowledge, diagnostic and therapeutical viewpoints. The invention relates to the use of such antisense oligonucleotides in order to study the diagnostics and therapy of tumors originated from chromosomal translocation t(14; 18) and the chemical and pharmaceutical modification suitable for rendering them suitable for being administered to human subjects.

Description

"ANTISENSE OLIGONUCLEOTIDES WITH ANTITUMORAL ACTIVITY, PHARMACEUTICAL COMPOSITIONS WHICH COMPRISE THEM, AND THEIR USES"
The present invention relates to antisense oligonucleotides, in particular antisense oligonucleotides which block the synthesis of BCL 2 protein.
The traditional obstacle in tumor chemotherapy resides in the difficulty of rigorously identifying cancer-specific targets. The therapeutical use of even active compounds displaying a very selective mechanism of action against specific molecular targets does not succeed in differentiating, to a desired extent, the effect towards neo p l a s t i c c e l l s a n d h e a l t h y c e l l s .
Du r i n g t h e past years, alterations in genetic code appeared with increasing evidence to be responsible for neoplastic transformation and progression. The molecular alterations relate to punctiform mutations, amplifications or deletions, genie translocations between chromosomes. Such molecular anomalies determine the neoplastic outcome by means of one single mechanism, or, more often, through the successive contributions by a plurality of a Iterations.
In a large number of tumoral situations, in particular of hematologic origin the main tumorigenic alterations were identified. In. such cases sequences of genetic code were identified, which may often be very short or constituted by one single replaced base, which not only result to be cancer-specific, but also, frequently, tumor-specific.
The inhibition of the functions of the genes affected by the alterations, with the normal gene being kept untouched, would make it possible remedies to be found for the damages caused by the altered gene, with the normal gene being kept unaltered. On considering that the altered gene is exclusively expressed in tumoral cells, the effect of such an inhibition would only be exerted onto the neoplastic cell, with a cancer-specific therapy being actually realized.
The possibility of inhibiting the genie functions by means of a very specific interaction with a short portion of nucleotidic sequence of the same gene is offered by the antisense oligonucleotides. The antisense oligonucleotides are constituted by short chains of single-filament synthetic DNA. The order of sequence of the single bases is so designed as to result complementary with the sequence of messenger RNA which one wants to inhibit.
The strict dependence on a perfect complementarity with the target molecule determines the genie expression inhibiting effect displayed by the antisense compounds. Therefore, the antisense compounds offer themselves as potential antitumoral compounds in those cases in which the molecular alterations are known.
On considering the above, the subject-matter of the present invention are antisense oligonucleotides (aODN's) characterized in that they comprise nucleotidic sequences hybridizing to a sequence of the hybrid gene and its messenger at the splicing region which is formed following a translocation of bcl-2 gene from chromosome 18 to chromosome 14 in a section of the latter which contains Ig-H gene (the gene for immunoglobulin H), and which block the synthesis of human BCL-2 protein.
Therefore, the present invention relates to the use of antisense oligonucleotides (aODN's) for the study and therapy of tumors in which BCL 2 protein is produced in excessive amounts owing to an alteration in the genetic code. In particular, the alteration consists in the translocation of a portion of chromosome 18 containing bcp-2 gene into a portion of chromosome 14 containing Ig-H gene. The splicing point between said portions forms a nucleotidic sequence only present in tumoral cells.
Such a mechanism is schematically displayed in Figure 1 of the accompanying drawings.
According to the present invention, antisense oligonucleotides complementary to the sequence formed owing to the translocation are capable of inhibiting the expression of BCL 2 protein in tumoral cells only, actually causing a growth inhibition and a cellular death of said cells with simultaneously respecting the integrity of the healthy cells.
A further subject-matter of the present invention is also a phramaceutical composition containing at least one antisense oligonucleotide as defined hereinabove, or a chemically modified form thereof without that the nucleotidic sequence is altered, and a therapeuticatly acceptable carrier.
A last subject-matter of the present invention is the use of such antisense compounds or of modified forms thereof in order to prepare a pharmaceutical composition to be used for the treatment of some pathological forms, in particular some tumor types.
The aODN's of the present invention can be designed on the basis of the sequence of mRNA in the splicing region between bcl-2 gene and the gene for the heavy chain of immunoglobulin Ig-H. Said sequence may be either known or determined by sequentiating samples of tumoral lymphocytes according to standardized methodologies as disclosed in a large number of manuals (Molecular Cloning, A Laboratory Manual, 1989, Sambrook, Maniatis, etc.).
For the purposes of the present invention, only those aODN's are understood as being suitable, which have a specific length suitable for hybridizing in optimal way to mRNA and target DNA. Such a length may also vary as a function of the chemical modifications introduced into the molecule constituted by natural nucleotides in order to enhance the pharmacological properties thereof. The minimal length preferably is 10 nucleotides, and the maximal length is of 100 nucleotides, preferably 50, still more preferably 30 nucleotides. The above, also on considering several mechanisms which may determine the biological effect.
Antisense compounds may act in various moments of the metabolic history of mRNA, both at nuclear and cytoplasmatic levels. It is furthermore possible that one from the antisense compounds action points is at ribosome level, or they may directly act through a direct interaction with nuclear or mitochondrial DNA.
The sequence length can also be determined on the basis of considerations known to those skilled in the art, which relate to a sufficient effectiveness of penetration through the cellular membranes. (Locke et al. PNAS 86:3474, 1989, Yakubov et al. PNAS 86: 6454, 1989).
A specific sequence defined according to such criteria as cited hereinabove can be synthetized in solid phase by means of well-known methods in the art and described, for example, by Narang (Tetrahedron 39:3, 1983), or by Itakuran (Ann. Rev. Biochem. 53:323, 1984), or in "Oligonucleotide Synthesis; A Practical Approach", Gait M. J. Ed. IRL Press, Oxford, U.K., 1984).
If so desired, such aODN's can be purified by means of known methods in the art, such as, e.g., polyacrylamide gel electrophoresis under denaturing conditions, or by high resolution chromatography, reverse phase chromatography or ion exchange column chromatography, or, finally, by capillary electrophoresis.
The aODN's according to the present invention are characterized by the possibility of inhibiting the synthesis of BCL 2 protein in the tumoral cells which display the t(14;18) translocation, by means of the use of specific antibodies, or by taking advantage from the capability of inhibiting the relevant mRNA by means of molecular probes. Also the inhibition of proteinic translation can be caused by means of a translation in cell-free system using lysates of red cells or wheat germ extracts (Sambrook, etc.).
The inhibition of synthesis of BCL 2 protein can also be determined according to a dose-effect relationship, by evaluating the growth inhibition of human tumor cells bearing the t(14;18) translocation, by evaluating the number of cells under the optical microscope or by means of the tritiated thymidine incorporation test.
The activity of the compounds according to the present invention can also be determined by analysing the induction of orderly cellular deletion in the already cited tumoral cells. The orderly cellular deletion, or apoptosis, is determined by analysing the cellular nucleus, or carrying out an electrophoresis in agarose, of the nuclear extract which appears to be fragmented into units of approximately 200 bases.
The aODN's according to the present invention can be modified for the purpose of causing the desired characteristics thereof to be improved (for a general survey also as regards general considerations relating to designing antisense oligonucleotides for the purpose of blocking the synthesis of a specific protein, reference is made to Uhlmann E. and Peymann A. Chem. Rev. 90:5543, 1990 or Englisch U. and Gauss D.H. Angewandte Chemie 30:613, 1991).
For example, such aODN's can be chemically modified at the phosphodiester linkage, or at t h e terminal 3' ends in order to render them more stable against a cleavage by nuclease enzymes, or in order to improve their penetration or hybridization characteristics, as disclosed in European Patent Application published with Publication No. 386 563. Furthermore, the aODN's can be covalently bound to a lipid, as disclosed, e.g., in International Patent Application published with Publication No. WO/9010448. In such a way, the concerned aODN can be transported more effectively through the cellular membranes and can be cleft, for example, with cellular cytopl a s ma t i c e n z yme s i n o rd e r t o l i be r a t e a c t i v e a ODN .
In order to obtain a higher absorption rate of aODN's into the cells or in order to improve the oral absorption thereof, the aODN's may also be phosphorylated, or they may be linked to cholesterin or to on olesterol derivatives, at their ends (Letsinger et. al. PNAS 86:6553, 1989).
When the aODN's are modified in order to increase their stability against an enzymatic cleavage or in order to improve the absorption thereof, care should be taken in order to make sure that such modified aODN's will still hybridize well to the specific mRNA or DNA, in order that they retain their biological and pharmacological effect. Furthermore, the aODN's of the present invention may be modified in order to secure that the drug is specifically delivered to tumoral tissues and consequently the dispersion of administered molecule is limited. According to a further aspect of the present invention, the aODN's according to the present invention may be administered in acceptable pharmaceutical forms, in particular in a suitable form for an oral administration. Dosage forms and rhythms can be parallel to those as adopted in clinical administrations of known compounds. The pharmaceutical compositions may contain at least one aODN according to the present invention in association with active substances and with either solid or liquid, pharmaceutically acceptable carriers. Any suitable carriers may be used. Furthermore, the pharmaceutical compositions may contain further pharmaceutically active substances and can be prepared according to known methods in the art.
Furthermore, the use of aODN's according to the present invention or modified forms thereof in order to prepare such pharmaceutical compositions and for diagnostic or medical purposes, for example for the treatment of some tumoral forms or of other diseases which may benefit from an inhibition of the synthesis of BCL 2 protein, are also a subject-matter of the present invention.
Furthermore, the aODN's of the present invention can be used, after being fixed onto a solid carrier according to well known methods in ther art, in order to detect genetic alterations responsible for various pathological forms, including tumors. For such diagnostic purposes, the aODN's according to the present invention may be used with a signal portion, for example a radioactive isotope, an enzyme, or a fluorescer according to well known methods in the art.
The present invention having thus being generally disclosed, it will be better understood by referring to the following specific examples, which are reported hereinunder for merely illustrative purposes, and which in no way shall be construed as being Limitative of the same invention.
E x a m p l e s
Example 1
Synthesis of an aODN
An aODN according to the present invention, with the following sequence, was synthetized by using a Beckman DNA synthetizer:
5' GGT-CCG-AGC-TTG-ACT-ACT 3'
Such a sequence is displayed in greater detail in Figure 2 of the accompanying drawings.
Example 2
Activity of an aODN
On day 1, human DHL-4 cells were grown in HB 101 media with a density of 104 cells per each well, to which an aODN according to the present invention was added in an amount comprised within the range of from 1 to 10 micromoles (microM).
On the following days, the cultures were admixed with half aODN dose and the activity was tested on from 2 to 7 days by cell count by microscope examination followed by a count of radioactivity incorporated into DNA following the addition of tritiated thymidine. The aODN's demonstrated a clear capability of reducing both the number of cells in the wells and the amount of radioactivity incorporated to cell nuclei. The activity results to be dose-dependent and specific to cells bearing the genetic damage to which aODN was directed.
In this regard, reference is made to Figures from 3 to 5 of the accompanying drawings.
Figure 3 shows the effect of an aODN on the in vitro growth of a human tumor cell line DHL-4. The growth curves respectively relate to the untreated DHL-4 line, to the same line treated with an aODN constituted by a sense sequence, a casual sequence, or the same line DHL-4 treated with an a n t i s e n s e sequence. The reported data relates to the dose of 1 mi croM.
On the ordinate, the number of cells × 103 is reported, which represents the total cells count. On the abscissa, the time is reported in days. The four curves relate to: I: reversed control, i.e., a polynucleotide having a reversed nucleotidic sequence relatively to the antisense nucleotide used in the test; U: untreated cells; S: sense nucleotide (control); AS: antisense nucleotide according to the present invention.
From the trend of the lines shown in Figure 3, it can be understood that the inhibition effect is practically inexistent for the control and untreated curves, whilst is of considerable extent for the antisense oligonucleotideo according to the present invention. Figure 4 displays the dose-dependent effect of aODN on the growth of DHL-4 tumor. The doses are indicated as concentrations (from 10 microM down to 0.001 microM) of aODN used. From the chart of Figure 4, one may understand how a decrease in the activity of inhibition of DHL-4 tumor cell growth is displayed with decreasing antisense oligonucleotide dose administered.
Figure 5 shows the specific antiproliferative effect for tumor cells bearing the genetic damage against which aODN is directed. No growth inhibiting effects are exerted against human tumor cells not bearing the molecular alteration which is the target of the present aODN.
In the chart of Figure 5, displayed on the ordinate is the number of cells × 103 which represents the total cell count. On the abscissa, the cases for as many tumor types are reported: DHL-4 is the tumor of leukemia B with 14-18 translocation, which is relevant for the purposes of the present invention; Raji is a leukemia B tumor without 14-18 translocation. LCL represents the tumor of leukemia B without translocation, YAC is the Leukemia T tumor without translocation, and, finally, K562 represents the tumor of mieloid leukemia without translocation.
For each tumor type, reported are four different cases of administration of doses of 10 microM and 1 microM for antisense (AS) and sense (S) oligonucleotides, as explained above.
From such a chart, one may see how an absolute selectivity is obtained in the effect of growth inhibition on tumor DHL-4 only, so that the use of antisense oligonucleotides according to the present invention makes it possible only such a kind of growth to be inhibited, without influencing, on the contrary, the growth of the other tumor types, which depart from the scope of the present invention.
Example 3
Induction of apoptosis
The above said cells, after being treated with the aODN's according to the present invention, result (a) to modify their morphological appearance, (b) to demonstrate DNA fragmentation and (c) to display an at all abnormal distribution of their DNA content.
Figure 6 of the accompanying drawings represents, in that regard, a cytofluorimetric analysis of cellular DNA, relevant to the effect of DNA fragmentation caused by the compounds. In this regard, the chart "A" relates to the control, in the case of untreated cells. The chart "B" relates to cells treated with an antisense oligonucleotide according to the present invention. The chart "C" relates to the overlapping of the histograms relevant to the cases of untreated cells and of cells treated with the antisense oligonucleotide. Finally, the chart "D" is obtained by overlapping the histogram "B" to the histogram of cells treated with sense oligonucleotide.
From the trend of such charts, it is evident that the effect of DNA fragmentation is only obtained with the case of treatment by means of antisense oligonucleotide according to the invention. As known, an effect of DNA fragmentation means that a mechanism of apoptosis has occurred. We wish to remind that in this kind of charts on the ordinate the number of cells is reported, and on the abscissa the intensity of fluorescence is expressed.
After classic staining with May Grundwall Giemsa, the cells display a fragmented nuclear appearance, and at all recognizable when untreated cells or cells treated with an ODN without antisense activity, or without an activity correlated with the genetic alteration taken into consideration for the purposes of the present invention, are tested for nuclear morphology. The tumor treated as already mentioned is submitted to nuclear DNA extraction according to well known methods in the art and is submitted to electrophoresis in agarose by following a standardized methodology (also described by Maniatis).
On electrophoretic examination, the DNA of the tumor treated with an aODN according to the present invention appears to have been fragmented into bands which are regularly ditributed in agarose gel and correspond to 200-base segments and are detected by U.V. light examination.
Also on flux cytofluorimetric examination, the already mentioned cells, after treatment with an aODN according to the present invention appear to be of reduced volume as compared to control cells and, after their DNA being stained with ethidium bromide, when submitted to the same cytofluorimetric examination appear to contain a reduced DNA amount, so that most cells result to emit a lower fluorescence than as corresponding to normal DNA content.
All these evidences demonstrate that the aODN's according to the present invention are capable of inducing an orderly cell deletion (apoptosis), and that said Orderly cell deletion results to be important for the pharmacological activity disclosed hereinabove.

Claims

C l a i m s
1. Antisense oligonucleotides (aODN's) characterized in that they comprise nucleotidic sequences hybridizing to a sequence of the hybrid gene and its messenger at the splicing zone which is formed following a translocation of bcl-2 gene from chromosome 18 to chromosome 14 in a section of the latter which contains Ig-H gene (the gene for immunoglobulin H), and which block the synthesis of human BCL-2 protein.
2. Antisense oligonucleotides according to claim 1, characterized in that they comprise a nucleotidic sequence including at Least one portion of bcl-2 gene and at least one portion of the gene of immunoglobulin H.
3. Antisense oligonucleotides according to claim 1, characterized in that they have a length of not more than 100 nucleotides.
4. Antisense oligonucleotides according to claim 2, characterized in that they have a length comprised within the range of from 10 to 50 nucleotides.
5. Oligonucleotides according to claim 1, characterized in that they comprise the following nucleotidic sequence:
5' GGT-CCG-AGC-TTG-ACT-ACT 3'
6. Pharmaceutical composition containing at least one antisense oligonucleotide according to claim 1 and a therapeutically acceptable carrier substance.
7. Use of an antisense oligonucleotide according to claim 1 for preparing a pharmaceutical composition.
8. Use of a sequence totally or partially corresponding to the hybrid gene resulting from the translocation of human bcl-2 gene to chromosome 14, inside the immunoglobulin gene, to be stably inserted in "antisense" direction inside the genoma of human cells, for therapeutical purpose.
9. Use of an antisense oligonucleotide probe, as claimed according to any of the preceding claims, as a diagnostic and therapeutical agent.
PCT/EP1993/001287 1992-05-22 1993-05-21 Antisense oligonucleotides with antitumoral activity, pharmaceutical compositions which comprise them, and their uses WO1993024653A1 (en)

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ITMI921235A IT1255770B (en) 1992-05-22 1992-05-22 ANTISENSE OLIGONUCLEOTIDES FOR ANTI-CANCER ACTIVITIES, PHARMACEUTICAL COMPOSITIONS THAT INCLUDE THEM, AND THEIR USE
ITMI92A001235 1992-05-22

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WO1996012017A1 (en) * 1994-10-14 1996-04-25 Otsuka Pharmaceutical Co., Ltd. Gene participating in apoptosis
WO1996027663A2 (en) * 1995-03-03 1996-09-12 Consiglio Nazionale Delle Ricerche An antisense transcript associated to tumor cells having a t(14;18) translocation and oligodeoxynucleotides useful in the diagnosis and treatment of said tumor cells
WO1996027664A2 (en) * 1995-03-03 1996-09-12 Consiglio Nazionale Delle Ricerche Antisense transcript expressed in b lymphocytes and synthetic oligonucleotides useful to inhibit the activity thereof
GB2311132A (en) * 1996-03-15 1997-09-17 Univ Heidelberg Labelled nucleic acid sequences which are specific for translocation breaking points
WO1998056905A1 (en) * 1997-06-09 1998-12-17 Novartis Ag Oligonucleotide derivatives
US6977244B2 (en) 1996-10-04 2005-12-20 Board Of Regents, The University Of Texas Systems Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US7285288B1 (en) 1997-10-03 2007-10-23 Board Of Regents, The University Of Texas System Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US7704962B1 (en) 1997-10-03 2010-04-27 Board Of Regents, The University Of Texas System Small oligonucleotides with anti-tumor activity

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WO1996012017A1 (en) * 1994-10-14 1996-04-25 Otsuka Pharmaceutical Co., Ltd. Gene participating in apoptosis
US6140492A (en) * 1995-03-03 2000-10-31 Consiglio Nazionale Delle Richerche Antisense transcript expressed in B lymphocytes and synthetic oligonucleotides useful to inhibit the activity thereof
WO1996027664A2 (en) * 1995-03-03 1996-09-12 Consiglio Nazionale Delle Ricerche Antisense transcript expressed in b lymphocytes and synthetic oligonucleotides useful to inhibit the activity thereof
WO1996027663A3 (en) * 1995-03-03 1996-10-31 Consiglio Nazionale Ricerche An antisense transcript associated to tumor cells having a t(14;18) translocation and oligodeoxynucleotides useful in the diagnosis and treatment of said tumor cells
WO1996027664A3 (en) * 1995-03-03 1996-11-07 Consiglio Nazionale Ricerche Antisense transcript expressed in b lymphocytes and synthetic oligonucleotides useful to inhibit the activity thereof
US6005095A (en) * 1995-03-03 1999-12-21 Consiglio Nazionale Delle Ricerche Antisense transcript associated to tumor cells having a T(14;18) translocation and oligodeoxynucleotides useful in the diagnosis and treatment of said tumor cells
WO1996027663A2 (en) * 1995-03-03 1996-09-12 Consiglio Nazionale Delle Ricerche An antisense transcript associated to tumor cells having a t(14;18) translocation and oligodeoxynucleotides useful in the diagnosis and treatment of said tumor cells
GB2311132A (en) * 1996-03-15 1997-09-17 Univ Heidelberg Labelled nucleic acid sequences which are specific for translocation breaking points
GB2311132B (en) * 1996-03-15 2000-05-24 Univ Heidelberg Nucleic acid sequences and methods of demonstrating translocations between chromosomes
US6977244B2 (en) 1996-10-04 2005-12-20 Board Of Regents, The University Of Texas Systems Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
WO1998056905A1 (en) * 1997-06-09 1998-12-17 Novartis Ag Oligonucleotide derivatives
US6291668B1 (en) 1997-06-09 2001-09-18 Novartis Ag Oligonucleotide derivatives
US7285288B1 (en) 1997-10-03 2007-10-23 Board Of Regents, The University Of Texas System Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides
US7704962B1 (en) 1997-10-03 2010-04-27 Board Of Regents, The University Of Texas System Small oligonucleotides with anti-tumor activity

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