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-spec f c 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 neoplastic cells and healthy cells.

During the 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 t rans locati ons 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 origir the main tumorigenic alterations were identified. I. 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,

SUBSTITUTESHEET

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 rea li zed.

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

SUBSTITUTESHEET

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 ranslocation 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 oligonucleo ides 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 phramaceuti ca I composition containing at least one antisense oligonucleotide as defined hereinabove, or a chemically modified form thereof

SUBSTITUTESHEET

without that the nucleotidic sequence is altered, and a therapeut cat ly 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 sequent i at i ng 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

SUBSTITUTESHEET

cytoplasmati c levels. It is furthermore possible that one from the antisense compounds action points is at riboso e level, or they may directly act through a direct interaction with nuclear or itochondrial 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 "0 li gonuc leo i e 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 e lect rophoresi s under denaturing conditions, or by high resolution chromatography, reverse phase chromatography or ion exchange column chromatography, or, finally, by capillary e lectrophoresi s.

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

SUBSTITUTESHEET

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 e lec rophoresi s 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

SUBSTITUTESHEET

modified at the phosphodi ester linkage, or at the 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 cytoplas ati c enzymes in order to liberate active aODN.

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 ortocrioissteroi 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.

SUBSTITUTESHEET

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,

SUBSTITUTESHEET

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_ ^β _i_e_s l£3!DB.L£_l

SyDlt)S§i§_2 _SD_§Qδt!

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.

Examg _, le_2 Acti_yi.tγ_of_an_a0DN On day 1, human DHL-4 cells were grown in HB 101 media with a density of 10* 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 thy idine. The aODN's demonstrated a clear

SUBSTITUTESHEET

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 iD_yUI2 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 antisense sequence. The reported data relates to the dose of 1 mi croM.

On the ordinate, the number of cells x 10 ³ 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 polynuc leotide 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 oli gonucleoti de according to the present invention.

SUBSTITUTESHEET

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 oligonucleot i de dose admini stered.

Figure 5 shows the specific anti pro li ferati ve 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 x 10 ³ 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 translocat on. 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

SUBSTITUTESHEET

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.

ExamβJ.e_3 lDd £li2D_2i_SB2Bi2§i§ 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 cytof Luorimetri c 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 oli gonuc Leotide 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 o li gonuc Leoti de . Finally, the chart "D" is obtained by overlapping the histogram "B" to the histogram of cells treated with sense o I i gonuc Leo i de .

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

SUBSTITUTESHEET

oligonuc leotide 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 e lect rophoresi s in agarose by following a standardized methodology (also described by Maniatis).

On e lectrophoreti c 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 cytof luorimet ri c 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 cytof luorimetri c examination

SUBSTITUTE SHEET

U.

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.

SUBSTITUTESHEET