WO2009071681A2

WO2009071681A2 - Rna antagonist compounds for the modulation of bcl-2

Info

Publication number: WO2009071681A2
Application number: PCT/EP2008/066921
Authority: WO
Inventors: Anja Mølhart HØG; Jens Bo Hansen
Original assignee: Santaris Pharma A/S
Priority date: 2007-12-07
Filing date: 2008-12-05
Publication date: 2009-06-11
Also published as: WO2009071681A3

Abstract

The present invention relates to oligomer compounds (oligomers), which target BCL-2 mRNA in a cell, leading to reduced expression of Bcl-2. Reduction of BCL-2 expression is beneficial for the treatment of certain medical disorders, such as such as hyperproliferative diseases such as cancer.

Description

RNA ANTAGONIST COMPOUNDS FOR THE MODULATION OF BCL-2.

The present invention relates to oligomer compounds (oligomers), which target the Bcl-2 mRNA in a cell, leading to reduced expression of the Bcl-2. Reduction of the Bcl-2 expression is beneficial for a range of medical disorders, such as hyperproliferative diseases such as cancer. The present invention provides therapeutic compositions comprising oligomers and methods for modulating the expression of the Bcl-2 using said oligomers, including methods of treatment.

RELATED CASES

This application claims the benefit under 35 U. S. C. § 1 19(e) of U.S. Provisional Applications Serial No. US 61/012185, filed 7^th December 2007, and US 61/106261 , filed 17^th October 2008, the disclosures of which are both incorporated herein by reference in their entirety.

BACKGROUND

Human Bcl-2 is a protein which is 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 hyperproliferative diseases like restenosis, fibrosis, psoriasis or certain types of allergic diseases, in particular in tumour progression and, importantly, appears to contribute to the clinical problem of resistance to anti-neoplastic regimens, in particular standard chemotherapeutic compounds. In contrast to most normal tissues, in malignant tumours, such as a small cell lung cancer (SCLC) and non-small lung cancer (NSCLC), Bcl-2 is often over-expressed in cancer cells.

LNA containing oligonucleotides targeting the 6 first codons of the human Bcl-2 mRNA were studied in a Ph.D. thesis defended by Jan Stenvang Jepsen (May 2003, University of Copenhagen). Fluiter et al., Nucleic Acid Research, 2003, Vol. 3, 953-962, discloses in vivo tumour growth inhibition and biodistribution studies of LNA antisense oligonucleotides.

WO 2005/061710 discloses 16 nucleobase phosphorothioate LNA gapmers which comprises a target binding domain that is specifically hybridizable to a region ranging from base position No. 1459 (5') to No. 1476 (3') of the human Bcl-2 mRNA (a region corresponding to the first six codons).

An antisense oligonucleotide drug Genasense (G3139), which also targets the first six codons of the Bcl-2 mRNA has been developed to target Bcl-2. However, after disappointing results in a melanoma trial Genasense did not receive FDA approval. In an exemplary aspect, the present invention provides highly efficient LNA oligomers which target specific regions of the Bcl-2 mRNA, designed by the selection of particularly effective oligonucleotide design and target sites on the Bcl-2 mRNA.

SUMMARY OF INVENTION

The invention provides an oligomer of between 10 - 30 nucleotides in length which comprises a contiguous nucleotide sequence of a total of between 10 - 30 nucleotides, wherein said contiguous nucleotide sequence is at least 80% (e.g., 85%, 90%, 95%, 98%, or 99%) homologous to a region corresponding to the reverse complement of a mammalian BCL-2 gene or mRNA, such as SEQ ID NO: 1 or naturally occurring variant thereof. Thus, for example, the oligomer hybridizes to a single stranded nucleic acid molecule having the sequence of a portion of SEQ ID NO: 1.

The invention provides oligomer of between 10-50, such as 10 - 30 nucleotides in length which comprises a contiguous nucleobase sequence of a total of between 10-50, such as 10 - 30, nucleotides, wherein said contiguous nucleotide sequence is at least 80% homologous to a corresponding region of a nucleic acid sequence selected from the group consisting of SEQ ID No 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40.

The invention provides oligomer of between 10-50 such as 10 - 30 nucleotides in length which comprises a contiguous nucleotides sequence of a total of between 10-50 such as 10 - 30 nucleotides, wherein said contiguous nucleotides sequence is at least 80% homologous to a corresponding region of a nucleic acid sequence selected from the group consisting of SEQ ID No 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21.

In some exemplary embodiments, the oligomer comprises at least one LNA unit. The invention provides for a conjugate comprising the oligomer according to the invention, and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said oligomer.

The invention provides for a pharmaceutical composition comprising the oligomer or the conjugate according to the invention, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

The invention further provides for an oligomer according to the invention, for use in medicine.

The invention provides for the oligomer or the conjugate according to invention, for use as a medicament, such as for the treatment of one or more of the diseases referred to herein, such as hyperproliferative diseases such as cancer. The invention provides for the use of an oligomer or the conjugate according to the invention, for the manufacture of a medicament for the treatment of one or more of the diseases referred to herein, such as hyperproliferative diseases such as cancer.

The invention provides for a method of treating such as hyperproliferative diseases such as cancer, said method comprising administering an (e.g. an effective amount of) an oligomer, a conjugate or a pharmaceutical composition according to the invention, to a patient suffering from, or likely to suffer from such as hyperproliferative diseases such as cancer.

The invention provides for a method for the inhibition of BCL-2 in a cell which is expressing Bcl-2, said method comprising administering (e.g. an effective amount of) an oligomer, or a conjugate according to the invention to said cell so as to effect the inhibition of Bcl-2 in said cell.

Pharmaceutical and other compositions comprising the oligomer of the invention are also provided. Further provided are methods of down-regulating the expression of Bcl-2 in cells or tissues comprising contacting said cells or tissues, in vitro or in vivo, with one or more of the oligomers, conjugates or compositions of the invention.

Also disclosed are methods of treating an animal or a human, suspected of having or being prone to a disease or condition, associated with expression, or over-expression, of Bcl-2, by administering to said animal or human a therapeutically or prophylactically effective amount of one or more of the oligomers, conjugates or compositions of the invention.

Further, methods of using oligomers for the inhibition of expression of Bcl-2, and for treatment of diseases associated with activity of Bcl-2 are provided.

The invention provides for a method for treating an a disease or disorder, such as those referred to herein, such as a disease or disorder selected from the group consisiting of hyperproliferative diseases such as those referred to herein, such as cancer, said method comprising administering an (e.g. an effective amount of) oligomer, a conjugate, or a pharmaceutical composition according to the invention to a patient in need thereof.

The invention provides for a method of inhibiting or reducing the expression of Bcl-2 in a cell or a tissue, the method comprising the step of contacting said cell or tissue with (e.g. an effective amount of) an oligomer, a conjugate, or a pharmaceutical composition according to the invention so that expression of Bcl-2, such as Bcl-2, is inhibited or reduced.

The invention provides for a method of triggering apoptosis in a cell, such as a cancer cell, said method comprising the step of contacting said cell or tissue with (e.g. an effective amount of) an oligomer, a conjugate, or a pharmaceutical composition according to the invention so that either expression of Bcl-2 is inhibited or reduced and/or apoptosis is triggered.

BRIEF DESCRIPTION OF FIGURES

Figure 1. Oligonucleotides presented in Table 3 were evaluated for their potential to knockdown Bcl-2 at concentrations of 1 , 5 and 25 nM in 518A2 cells using lipid transfection.

Figure 2. selected oligonucleotides were evaluated for their potential to knockdown Bcl-2 in

15PC3 cells at concentration of 0.04, 0.2, 1 , 5, 12.5, and 25 nM using lipid transfection.

Figure 3. Six oligonucleotides (SEQ ID NO: 64, 69, 72, 75, 79, 88) were selected and evaluated for their potential to induce apoptosis in 15PC3 cells at concentration of 0.04, 0.2, 1 , 5, 12.5, and 25 nM using lipid transfection.

Figure 4. Homo sapiens B-cell CLL/lymphoma 2 (Bcl-2), nuclear gene encoding mitochondrial protein, transcript variant alpha, mRNA (SEQ ID NO 1 ) - Accession number

NM_000633.

Figure 5. SEQ ID NO: 2 Homo sapiens B-cell CLL/lymphoma 2 (Bcl-2), nuclear gene encoding mitochondrial protein, transcript variant beta, mRNA. Accession number

NM_000657.

Figure 6. B-cell lymphoma protein 2 alpha isoforms - SEQ ID No 94 and 95.

Figure 7. Selected oligonucleotides presented in Table 4 were evaluated for their potential to knockdown Bcl-2 using natural uptake at 5μM in 15PC3 cells. Figure 8. SEQ ID NO: 61 , 62, 63, 67, 68, 85, 91 and 93 were evaluated for their potential to down-regulate Bcl-2 in vivo in mouse liver tissue. Animals were dosed thrice weekly with

10mg/kg of antisense oligonucleotide or saline for a total of 7 doses. Liver tissue was harvested for RNA analysis 48 hours after the last dosing.

Figure 9. SEQ ID NO: 64, 69, 72, and 75, were evaluated for their potential to down- regulate Bcl-2 in vivo in mouse liver tissue. Animals were dosed daily for three consecutive days with 25mg/kg of antisense oligonucleotide or saline. Liver tissue was harvested for

RNA analysis 24 hours after the last dosing.

Figure 10. Cell viability when combining Bcl-2 targeting oligo SEQ ID NO: 63 with scrambled control oligo or with Mcl-1 targeting oligos SEQ ID NOs: 96 and 97 in 15PC3 cell line using natural uptake.

Figure 11. Caspase 3/7 induction when combining Bcl-2 targeting oligo SEQ ID NO: 63 with scrambled control oligo or with Mcl-1 targeting oligos SEQ ID NOs: 96 and 97 in 15PC3 cell line using natural uptake. DETAILED DESCRIPTION OF INVENTION

The Oligomer

The present invention employs oligomeric compounds (referred herein as oligomers), for use in modulating the function of nucleic acid molecules encoding mammalian Bcl-2, such as the Bcl-2 nucleic acid shown in SEQ ID 1 , and naturally occurring variants of such nucleic acid molecules encoding mammalian Bcl-2. The term "oligomer" in the context of the present invention, refers to a molecule formed by covalent linkage of two or more nucleotides (i.e. an oligonucleotide). The oligomer consists or comprises of a contiguous nucleotide sequence of between 10 - 50, such as 10 - 30 nucleotides in length. The term "oligomer" in the context of the present invention, refers to a molecule formed by covalent linkage of two or more nucleotides (i.e. an oligonucleotide). Herein, each single nucleotide, such as the nucleotides present in the oligomer of the invention, may also be referred to as a "monomer" or "unit". In some embodiments, the oligomer consists or comprises of a contiguous nucleotide sequence of between 10 - 30 nucleotides in length (i.e. comprises or consists of from 10 - 30 covalently linked monomers).

In various embodiments, the compound of the invention does not comprise RNA (units). It is preferred that the compound according to the invention is a linear molecule or is synthesised as a linear molecule. The oligomer is a single stranded molecule, and preferably does not comprise short regions of, for example, at least 3, 4 or 5 contiguous nucleotides, which are complementary to equivalent regions within the same oligomer (i.e. duplexes) - in this regards, the oligomer is not (essentially) double stranded. In some embodiments, the oligomer is essentially not double stranded, such as is not a siRNA. In various embodiments, the oligomer of the invention may consist entirely of the contiguous nucleotide region. Thus, the oligomer is not substantially self-complementary. siRNAs comprise of 2 complementary short RNA (or equivalent nucleobase units) sequences, such as between 21 and 23nts long, with, typically a 2nt 3' overhang on either end. In order to enhance in vivo uptake, the siRNAs may be conjugated, such as conjugated to a sterol, such as a cholesterol group (typically at the 3' or 5' termini of one or both of the strands).

The invention further provides target sequences in the Bcl2 mRNA or gene, or an allelic variant thereof, in particular those corresponding to (reverse complement of) a sequence selected from the group consisting of SEQ ID NOS: 3 - 40, wherein antisense oligonucleotides corresponding to said target sequences are capable of down-regulating McH . A variant sequence may have at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95% sequence homology to a target sequence in McM . Typically, an oligomer of the invention corresponding to said variant sequences is still capable of down-regulating Mcl1.

Specific designs of LNA oligonucleotides are also disclosed, for example those shown in SEQ ID NOS 41 - 59, and 60 - 93.. The oligomers of the invention are considered to be potent inhibitors of McM mRNA and protein expression.

In some embodiments the oligomers of the invention is able to induce apoptosis in a cell into which it is introduced, such as a cancer cell, such as the cell lines herein refered to (such as in the caspase assay herein disclosed). However, in some embodiments embodiment the oligomers of the invention may not induce apoptosis, for example as determined by the caspase assay. In this regards, whilst apoptosis may be desirable for the efficient killing of cells, e.g. cancer cells, it may, in some embodiments, be negatively associated to a more toxic effect on non-target cells and tissues. As such, depending upon the medical indication to be treated, it may be beneficial to select oligonucleotides which are very effective at triggering apoptosis, whilst others, where perhaps the medical indication is not immediately life threatening, an oligomer which is not as potent in triggering apoptosis may be appropriate.

The Target

Suitably the oligomer of the invention is capable of down-regulating expression of the Bcl-2 gene. In this regards, the oligomer of the invention can affect the inhibition of Bcl-2, typically in a mammalian such as a human cell. In some embodiments, the oligomers of the invention bind to the target nucleic acid and effect inhibition of expression of at least 10% or 20% compared to the normal expression level, more preferably at least a 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition compared to the normal expression level. In some embodiments, such modulation is seen when using between 0.04 and 25nM, such as between 0.8 and 2OnM concentration of the compound of the invention. In the same or a different embodiment, the inhibition of expression is less than 100%, such as less than 98% inhibition, less than 95% inhibition, less than 90% inhibition, less than 80% inhibition, such as less than 70% inhibition. Modulation of expression level may be determined by measuring protein levels, e.g. by the methods such as SDS-PAGE followed by western blotting using suitable antibodies raised against the target protein. Alternatively, modulation of expression levels can be determined by measuring levels of mRNA, e.g. by northern blotting or quantitative RT-PCR. When measuring via mRNA levels, the level of down-regulation when using an appropriate dosage, such as between 0.04 and 25nM, such as between 0.8 and 2OnM concentration, is, in some embodiments, typically to a level of between 10-20% the normal levels in the absence of the compound of the invention. The invention therefore provides a method of down-regulating or inhibiting the expression of Bcl-2 protein and/or mRNA in a cell which is expressing Bcl-2 protein and/or mRNA, said method comprising administering the oligomer or conjugate according to the invention to said cell to down-regulating or inhibiting the expression of Bcl-2 protein and/or mRNA in said cell. The administration is typically performed as an effective amount of said oligomer. Suitably the cell is a mammalian cell such as a human cell and/or a cancer cell. The administration may occur, in some embodiments, in vitro. The administration may occur, in some embodiments, in vivo.

The term "target nucleic acid", as used herein refers to the DNA or RNA encoding mammalian Bcl-2 polypeptide, such as human Bcl-2, such as SEQ ID NO: 1. Bcl-2 encoding nucleic acids or naturally occurring variants thereof, and RNA nucleic acids derived therefrom, preferably mRNA, such as pre-mRNA, although preferably mature mRNA. In some embodiments, for example when used in research or diagnostics the "target nucleic acid" may be a cDNA or a synthetic oligonucleotide derived from the above DNA or RNA nucleic acid targets. The oligomer according to the invention is preferably capable of hybridising to the target nucleic acid. It will be recognised that SEQ ID NO: 1 is a cDNA sequences, and as such, corresponds to the mature mRNA target sequence, although uracil is replaced with thymidine in the cDNA sequences.

The term "naturally occurring variant thereof refers to variants of the Bcl-2 polypeptide of nucleic acid sequence which exist naturally within the defined taxonomic group, such as mammalian, such as mouse, monkey, and preferably human. Typically, when referring to "naturally occurring variants" of a polynucleotide the term also may encompass any allelic variant of the Bcl-2encoding genomic DNA which are found at the Chromosome Chr 18: 58.94 - 59.14 Mb by chromosomal translocation or duplication, and the RNA, such as mRNA derived therefrom. "Naturally occurring variants" may also include variants derived from alternative splicing of the Bcl-2 mRNA. A variant sequence may have at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% sequence homology to a target sequence in Bcl-2. Typically, an oligomer of the invention corresponding to said variant sequences is still capable of down-regulating Bcl-2.

When referenced to a specific polypeptide sequence, e.g., the term also includes naturally occurring forms of the protein which may therefore be processed, e.g. by co- or post-translational modifications, such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc. Oligomer Sequences

The oligomers comprise or consist of a contiguous nucleotide sequence which corresponds to the reverse complement of a nucleotide sequence present in SEQ ID NO: 1. Thus, the oligomer can comprise or consist of, or a sequence selected from the group consisting of SEQ ID NOS: 22 - 40 and 3 - 21 , wherein said oligomer (or contiguous nucleotide portion thereof) may optionally have one or two, mismatches against said selected sequence.

The oligomer may comprise or consist of a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to the equivalent region of a nucleic acid which encodes a mammalian Bcl-2 (e.g., SEQ ID NO: 1 ). Thus, the oligomer can comprise or consist of a fully complementary antisense nucleotide sequence.

However, in some embodiments, the oligomer may tolerate 1 or 2 mismatches, when hybridising to the target sequence and still sufficiently bind to the target to show the desired effect, i.e. down-regulation of the target. Mismatches may, for example, be compensated by increased length of the oligomer nucleotide sequence and/or an increased number of nucleotide analogues, such as LNA, present within the nucleotide sequence.

In some embodiments, the contiguous nucleotide sequence comprises no more than 2 mismatches when hybridizing to the (complementary) target sequence, such as to the corresponding region of a nucleic acid which encodes a mammalian Bcl-2, such as SEQ ID NO 1.

In some embodiments, the contiguous nucleotide sequence comprises no more than a single mismatch when hybridizing to the target sequence, such as the corresponding region of a nucleic acid which encodes a mammalian Bcl-2.

The nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to a corresponding sequence selected from the group consisting of SEQ ID NOS: 22 - 40 and 3 - 21 , such as at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%, at least 98%, at least 99%, such as 100% homologous (identical). The nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% homologous to the reverse complement of a corresponding sequence present in SEQ ID NO: 1 , such as at least 85%, at least 90%, at least 91 %, at least 92%at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%, at least 98%, at least 99%, such as 100% homologous (identical). The nucleotide sequence of the oligomers of the invention or the contiguous nucleotide sequence is preferably at least 80% complementary to a sub-sequence present in SEQ ID NO: 1 , such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% complementary, at least 97%, at least 98%, at least 99%, such as 100% complementary (perfectly complementary).

In some embodiments the oligomer (or contiguous nucleotide portion thereof) is selected from, or comprises, one of the sequences selected from the group consisting of SEQ ID NO 23, 23, 26, 27, 28, 29, 30, 31 , 33, 37, 39 and 40, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 1 1 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof, wherein said oligomer (or contiguous nucleotide portion thereof) may optionally comprise one, or two mismatches against said selected sequence.

In some embodiments the oligomer (or contiguous nucleotide portion thereof) is selected from, or comprises, one of the sequences selected from the group consisting of SEQ ID NOS: 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 , or a sub- sequence of at least 10 contiguous nucleotides thereof, wherein said oligomer (or contiguous nucleotide portion thereof) may optionally comprise one or two mismatches when compared to the sequence.

In some embodiments the sub-sequence may consist of 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29 contiguous nucleotides, such as between 12 -22, such as between 12-18 nucleotides. Suitably, in some embodiments, the sub-sequence is of the same length as the contiguous nucleotide sequence of the oligomer of the invention.

Other exemplary oligomers include a (contiguous) nucleotide sequence, such as a sequence of 12, 13, 14, 15 or 16 contiguous nucleotides in length, which have a nucleotide sequence selected from a sequence from the group consisting of SEQ ID NO 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 , wherein said oligomer (or contiguous nucleotide portion thereof) may optionally comprise one, or two mismatches against said selected sequence.

However, it is recognised that, in some embodiments the nucleotide sequence of the oligomer may comprise additional 5' or 3' nucleotides, such as, independently, 1 , 2, 3, 4 or 5 additional nucleotides 5' and/or 3', which are non-complementary to the target sequence. In this respect the oligomer of the invention, may, in some embodiments, comprise a contiguous nucleotide sequence which is flanked 5' and or 3' by additional nucleotides. In some embodiments the additional 5' or 3' nucleotides are naturally occurring nucleotides, such as DNA or RNA. In some embodiments, the additional 5' or 3' nucleotides may represent region D as referred to in the context of gapmer oligomers herein. In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 3 or SEQ ID 22, such as SEQ ID 60, or a subsequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof. In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 4, or SEQ ID 23, such as SEQ ID NO 61 , or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 5, or SEQ ID 24, such as SEQ ID NO 62, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 6, or SEQ ID 25, such as SEQ ID NO 63, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 7, or SEQ ID 26, such as SEQ ID NO 91 , 92, 93, 64, 65 or 66, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 1 1 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 8, or SEQ ID 27, such as SEQ ID 67, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 1 1 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof. In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 9, or SEQ ID 28, such as SEQ ID 68, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 10, or SEQ ID 29, such as SEQ ID 69, 70 or 71 or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 1 1 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 1 1 , or SEQ ID 30, such as SEQ ID 72, 73 or 74 or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 1 1 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 12, or SEQ ID 31 , such as SEQ ID 75, 76 and 77, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 13, or SEQ ID 32, such as SEQ ID 78 or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 1 1 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 14, or SEQ ID 33, such as SEQ ID 79, 80, or 81 , or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof. In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 15, or SEQ ID 34, such as SEQ ID 82, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 16, or SEQ ID 35, such as SEQ ID 83, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 17, or SEQ ID 36, such as SEQ ID 84, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 18, or SEQ ID 37, such as SEQ ID 85, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 11 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 19, or SEQ ID 38, such as SEQ ID 86, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 1 1 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof. In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 20, or SEQ ID 39, such as SEQ ID 87, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 1 1 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof. In some embodiments the oligomer according to the invention consists or comprises of a nucleobase sequence according to SEQ ID 21 , or SEQ ID 40, such as SEQ ID 88, 89 or 90, or a sub-sequence of at least 10 contiguous nucleotides thereof, such as 1 1 , 12, 13, 14, 15 or 16 contiguous nucleotides thereof.

When determining "homology" between the oligomers of the invention (or contiguous nucleotide sequence) and the nucleic acid which encodes the mammalian Bcl-2 or the reverse complement thereof, such as those disclosed herein, the determination of homology may be made by a simple alignment with the corresponding nucleotide sequence of the compound of the invention and the corresponding region of the nucleic acid which encodes the mammalian Bcl-2 (or target nucleic acid), or the reverse complement thereof, and the homology is determined by counting the number of bases which align and dividing by the total number of contiguous nucleotides in the compound of the invention, and multiplying by 100. In such a comparison, if gaps exist, it is preferable that such gaps are merely mismatches rather than areas where the number of nucleotides within the gap differ between the nucleotide sequence of the invention and the target nucleic acid. The terms "corresponding to" and "corresponds to" refer to the comparison between the nucleotide sequence of the oligomer or contiguous nucleotide sequence (a first sequence) and the equivalent contiguous nucleotide sequence of a further sequence selected from either i) a sub-sequence of the reverse complement of the nucleic acid target, such as the mRNA which encodes the Bcl-2 protein, such as SEQ ID NO: 1 , and/or ii) the sequence of nucleotides provided herein such as the group consisting of SEQ ID NOS: 22 - 40 and 3 - 21 , or sub-sequence thereof. Nucleotide analogues are compared directly to their equivalent or corresponding nucleotides. A first sequence which corresponds to a further sequence under i) or ii) typically is identical to that sequence over the length of the first sequence (such as the contiguous nucleotide sequence) or, as described herein may, in some embodiments, is at least 80% homologous to a corresponding sequence, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% homologous, at least 97%, at least 98%, at least 99% such as 100% homologous (identical).

The terms "corresponding nucleotide analogue" and "corresponding nucleotide" are intended to indicate that the nucleotide in the nucleotide analogue and the naturally occurring nucleotide are identical. For example, when the 2-deoxyribose unit of the nucleotide is linked to an adenine, the "corresponding nucleotide analogue" contains a pentose unit (different from 2-deoxyribose) linked to an adenine.

Length The oligomers comprise or consist of a contiguous nucleotide sequence of a total of between 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides in length.

In some embodiments, the oligomers comprise or consist of a contiguous nucleotide sequence of a total of between 10 - 22, such as 12 - 18, such as 13 - 17 or 12 - 16, such as 13, 14, 15, 16 contiguous nucleotides in length.

In some embodiments, the oligomers comprise or consist of a contiguous nucleotide sequence of a total of 10, 11 , 12, 13, or 14 contiguous nucleotides in length.

In some embodiments, the oligomer according to the invention consists of no more than 22 nucleotides, such as no more than 20 nucleotides, such as no more than 18 nucleotides, such as 15, 16 or 17 nucleotides. In some embodiments the oligomer of the invention comprises less than 20 nucleotides.

Nucleotide analogues

The term "nucleotide" as used herein, refers to a glycoside comprising a sugar moiety, a base moiety and a covalently linked phosphate group and covers both naturally occurring nucleotides, such as DNA or RNA, preferably DNA, and non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to as "nucleotide analogues" herein.

Non-naturally occurring nucleotides include nucleotides which have modified sugar moieties, such as bicyclic nucleotides or 2' modified nucleotides, such as 2' substituted nucleotides.

"Nucleotide analogues" are variants of natural nucleotides, such as DNA or RNA nucleotides, by virtue of modifications in the sugar and/or base moieties. Analogues could in principle be merely "silent" or "equivalent" to the natural nucleotides in the context of the oligonucleotide, i.e. have no functional effect on the way the oligonucleotide works to inhibit target gene expression. Such "equivalent" analogues may nevertheless be useful if, for example, they are easier or cheaper to manufacture, or are more stable to storage or manufacturing conditions, or represent a tag or label. Preferably, however, the analogues will have a functional effect on the way in which the oligomer works to inhibit expression; for example by producing increased binding affinity to the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into the cell. Specific examples of nucleoside analogues are described by e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and in Scheme 1 :

Phosphorthioate 2'-O-Methyl 2'-MOE 2'-Fluoro

2

Morpholino

2'-F-ANA

2 '- (3-hydroxy )propy 1

Boranophosphates

Scheme 1

The oligomer may thus comprise or consist of a simple sequence of natural occurring nucleotides - preferably 2'-deoxynucleotides (referred to here generally as "DNA"), but also possibly ribonucleotides (referred to here generally as "RNA"), or a combination of such naturally occurring nucleotides and one or more non-naturally occurring nucleotides, i.e. nucleotide analogues. Such nucleotide analogues may suitably enhance the affinity of the oligomer for the target sequence.

Examples of suitable and preferred nucleotide analogues are provided by PCT/DK2006/000512 or are referenced therein.

Incorporation of affinity-enhancing nucleotide analogues in the oligomer, such as LNA or 2'-substituted sugars, can allow the size of the specifically binding oligomer to be reduced, and may also reduce the upper limit to the size of the oligomer before non-specific or aberrant binding takes place.

In some embodiments the oligomer comprises at least 2 nucleotide analogues. In some embodiments, the oligomer comprises from 3-8 nucleotide analogues, e.g. 6 or 7 nucleotide analogues. In the by far most preferred embodiments, at least one of said nucleotide analogues is a locked nucleic acid (LNA); for example at least 3 or at least 4, or at least 5, or at least 6, or at least 7, or 8, of the nucleotide analogues may be LNA. In some embodiments all the nucleotides analogues may be LNA.

It will be recognised that when referring to a preferred nucleotide sequence motif or nucleotide sequence, which consists of only nucleotides, the oligomers of the invention which are defined by that sequence may comprise a corresponding nucleotide analogue in place of one or more of the nucleotides present in said sequence, such as LNA units or other nucleotide analogues, which raise the duplex stability/T_m of the oligomer/target duplex (i.e. affinity enhancing nucleotide analogues). In some embodiments, any mismatches between the nucleotide sequence of the oligomer and the target sequence are preferably found in regions outside the affinity enhancing nucleotide analogues, such as region B as referred to herein, and/or region D as referred to herein, and/or at the site of non modified such as DNA nucleotides in the oligonucleotide, and/or in regions which are 5' or 3' to the contiguous nucleotide sequence. Examples of such modification of the nucleotide include modifying the sugar moiety to provide a 2'-substituent group or to produce a bridged (locked nucleic acid) structure which enhances binding affinity and may also provide increased nuclease resistance.

A preferred nucleotide analogue is LNA, such as oxy-LNA (such as beta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such as beta-D-amino-LNA and alpha-L-amino- LNA) and/or thio-LNA (such as beta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA and alpha-L-ENA). Most preferred is beta-D-oxy-LNA.

In some embodiments the nucleotide analogues present within the oligomer of the invention (such as in regions A and C mentioned herein) are independently selected from, for example: 2'-O-alkyl-RNA units, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabino nucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (intercalating nucleic acid -Christensen, 2002. Nucl. Acids. Res. 2002 30: 4918-4925, hereby incorporated by reference) units and 2'MOE units. In some embodiments there is only one of the above types of nucleotide analogues present in the oligomer of the invention, or contiguous nucleotide sequence thereof. In some embodiments the nucleotide analogues are 2'-O-methoxyethyl-RNA (2'MOE), 2'-fluoro-DNA monomers or LNA nucleotide analogues, and as such the oligonucleotide of the invention may comprise nucleotide analogues which are independently selected from these three types of analogue, or may comprise only one type of analogue selected from the three types. In some embodiments at least one of said nucleotide analogues is 2'-MOE- RNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-MOE-RNA nucleotide units. In some embodiments at least one of said nucleotide analogues is 2'-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-fluoro-DNA nucleotide units.

In some embodiments, the oligomer according to the invention comprises at least one Locked Nucleic Acid (LNA) unit, such as 1 , 2, 3, 4, 5, 6, 7, or 8 LNA units, such as between 3 - 7 or 4 to 8 LNA units, or 3, 4, 5, 6 or 7 LNA units. In some embodiments, all the nucleotide analogues are LNA. In some embodiments, the oligomer may comprise both beta-D-oxy-LNA, and one or more of the following LNA units: thio-LNA, amino-LNA, oxy- LNA, and/or ENA in either the beta-D or alpha-L configurations or combinations thereof. In some embodiments all LNA cytosine units are 5'methyl-Cytosine. In some embodiments of the invention, the oligomer may comprise both LNA and DNA units. Preferably the combined total of LNA and DNA units is 10-25, preferably 10-20, even more preferably 12-16. In some embodiments of the invention, the nucleotide sequence of the oligomer, such as the contiguous nucleotide sequence consists of at least one LNA and the remaining nucleotide units are DNA units. In some embodiments the oligomer comprises only LNA nucleotide analogues and naturally occurring nucleotides (such as RNA or DNA, most preferably DNA nucleotides), optionally with modified internucleotide linkages such as phosphorothioate.

The term "nucleobase" refers to the base moiety of a nucleotide and covers both naturally occuring a well as non-naturally occurring variants. Thus, "nucleobase" covers not only the known purine and pyrimidine heterocycles but also heterocyclic analogues and tautomeres thereof.

Examples of nucleobases include, but are not limited to adenine, guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine, 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.

In some embodiments, at least one of the nucleobases present in the oligomer is a modified nucleobase selected from the group consisting of 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine. LNA

The term "LNA" refers to a bicyclic nucleotide analogue, known as "Locked Nucleic Acid". It may refer to an LNA monomer, or, when used in the context of an "LNA oligonucleotide" refers to an oligonucleotide containing one or more such bicyclic nucleotide analogues.

The LNA used in the oligonucleotide compounds of the invention preferably has the structure of the general formula I

Formula 1 wherein X is selected from -O-, -S-, -N(R^N>, -C(R⁶R^6*)-; B is selected from hydrogen, optionally substituted Ci_-4-alkoxy, optionally substituted d-₄-alkyl, optionally substituted Ci_-4-acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;

P designates the radical position for an internucleotide linkage to a succeeding monomer, or a 5'-terminal group, such internucleotide linkage or 5'-terminal group optionally including the substituent R⁵ or equally applicable the substituent R^5*;

P^* designates an internucleotide linkage to a preceding monomer, or a 3'-terminal group;

R^4* and R^2* together designate a biradical consisting of 1-4 groups/atoms selected from -C(R^aR^b)-, -C(R^a)=C(R^b)-, -C(R^a)=N-, -O-, -Si(R^a)₂-, -S-, -SO₂-, -N(R^a)-, and >C=Z, wherein Z is selected from -O-, -S-, and -N(R^a)-, and R^a and R^b each is independently selected from hydrogen, optionally substituted Ci.-i₂-alkyl, optionally substituted C_2-i₂-alkenyl, optionally substituted C_2-i₂-alkynyl, hydroxy,

C_2-i₂-alkoxyalkyl, C_2-i₂-alkenyloxy, carboxy, Ci_i₂-alkoxycarbonyl, Ci_i₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_6-alkyl)amino, carbamoyl, mono- and di(Ci_6-alkyl)-amino-carbonyl, amino- d-₆-alkyl-aminocarbonyl, mono- and di(Ci.₆-alkyl)amino-Ci.₆-alkyl-aminocarbonyl, Ci_-6-alkyl- carbonylamino, carbamido, Ci_-6-alkanoyloxy, sulphono, Ci_-6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci_-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents R^a and R^b together may designate optionally substituted methylene (=CH₂), and each of the substituents R^1*, R², R³, R⁵, R^5*, R⁶ and R^6*, which are present is independently selected from hydrogen, optionally substituted Ci.-ι₂-alkyl, optionally substituted C₂-i₂-alkenyl, optionally substituted C₂-i₂-alkynyl, hydroxy,

C_2-i₂-alkoxyalkyl, C_2-i₂-alkenyloxy, carboxy, Ci.-i₂-alkoxycarbonyl, Ci.-i₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_6-alkyl)amino, carbamoyl, mono- and di(Ci_6-alkyl)-amino-carbonyl, amino- d-₆-alkyl-aminocarbonyl, mono- and di(Ci.₆-alkyl)amino-Ci.₆-alkyl-aminocarbonyl, Ci_-6-alkyl- carbonylamino, carbamido, Ci_-6-alkanoyloxy, sulphono, Ci_-6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci_-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from -O-, -S-, and -(NR^N)- where R^N is selected from hydrogen and Ci_-4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and R^N*, when present and not involved in a biradical, is selected from hydrogen and Ci_-4-alkyl; and basic salts and acid addition salts thereof; In some embodiments R^5* is selected from H, -CH₃, -CH₂-CH₃,- CH₂-O-CH₃, and -

CH=CH₂.

In some embodiments, R^4* and R^2* together designate a biradical selected from - C(R^aR^b)-O-, -C(R^aR^b)-C(R^cR^d)-O-, -C(R^aR^b)-C(R^cR^d)-C(R^eR^f)-O-, -C(R^aR^b)-O-C(R^cR^d)-, - C(R^aR^b)-O-C(R^cR^d)-O-, -C(R^aR^b)-C(R^cR^d)-, -C(R^aR^b)-C(R^cR^d)-C(R^eR^f)-, - C(R^a)=C(R^b)-C(R^cR^d)-, -C(R^aR^b)-N(R^c)-, -C(R^aR^b)-C(R^cR^d)- N(R⁶)-, -C(R^aR^b)-N(R^c)-O-, and - C(R^aR^b)-S-, -C(R^aR^b)-C(R^cR^d)-S-, wherein R^a, R^b, R^c, R^d, R^e, and R^f each is independently selected from hydrogen, optionally substituted Ci_i₂-alkyl, optionally substituted C_2-i₂-alkenyl, optionally substituted C_2-i₂-alkynyl, hydroxy, Ci_i₂-alkoxy, C_2-i₂-alkoxyalkyl, C_2-i₂-alkenyloxy, carboxy, Ci_i₂-alkoxycarbonyl, Ci_i₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_-6-alkyl)amino, carbamoyl, mono- and di(Ci_-6-alkyl)-amino-carbonyl, amino- d-₆-alkyl-aminocarbonyl, mono- and di(Ci.₆-alkyl)amino-Ci.₆-alkyl-aminocarbonyl, Ci_-6-alkyl- carbonylamino, carbamido, Ci_-6-alkanoyloxy, sulphono, Ci_-6-alkylsulphonyloxy, nitro, azido, sulphanyl, d-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents R^a and R^b together may designate optionally substituted methylene (=CH₂),

In a further embodiment R^4* and R^2* together designate a biradical (bivalent group) selected from -CH₂-O-, -CH₂-S-, -CH₂-NH-, -CH₂-N(CH₃)-, -CH₂-CH₂-O-, -CH₂-CH(CH₃)-, - CH₂-CH₂-S-, -CH₂-CH₂-NH-, -CH₂-CH₂-CH₂-, -CH₂-CH₂-CH₂-O-, -CH₂-CH₂-CH(CH₃)-, - CH=CH-CH₂-, -CH₂-O-CH₂-O-, -CH₂-NH-O-, -CH₂-N(CH₃J-O-, -CH₂-O-CH₂-, -CH(CH₃J-O-, - CH(CH₂-O-CH₃)-O-.

For all chiral centers, asymmetric groups may be found in either R or S orientation.

Preferably, the LNA used in the oligomer of the invention comprises at least one LNA unit according to any of the formulas

wherein Y is -0-, -0-CH₂- ,-S-, -NH-, or N(R^H); Z and Z^* are independently selected among an internucleotide linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety, and R^H is selected from hydrogen and Ci_-4- alkyl.

Specifically preferred LNA units are shown in scheme 2:

β-D-amino-LNA Scheme 2

The term "thio-LNA" comprises a locked nucleotide in which Y in the general formula above is selected from S or -CH₂-S-. Thio-LNA can be in both beta-D and alpha-L- configuration. The term "amino-LNA" comprises a locked nucleotide in which Y in the general formula above is selected from -N(H)-, N(R)-, CH₂-N(H)-, and -CH₂-N(R)- where R is selected from hydrogen and C-_M-alkyl. Amino-LNA can be in both beta-D and alpha-L- configuration.

The term "oxy-LNA" comprises a locked nucleotide in which Y in the general formula above represents -O- or -CH₂-O-. Oxy-LNA can be in both beta-D and alpha-L- configuration.

The term "ENA" comprises a locked nucleotide in which Y in the general formula above is -CH₂-O- (where the oxygen atom of -CH₂-O- is attached to the 2'-position relative to the base B). In a preferred embodiment LNA is selected from beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particular beta-D-oxy-LNA.

RNAse recruitment

It is recognised that an oligomeric compound may function via non RNase mediated degradation of target mRNA, such as by steric hindrance of translation, or other methods, however, the preferred oligomers of the invention are capable of recruiting an endoribonuclease (RNase), such as RNase H.

It is preferable that the oligomer, or contiguous nucleotide sequence, comprises of a region of at least 6, such as at least 7 consecutive nucleotide units, such as at least 8 or at least 9 consecutive nucleotide units (residues), including 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 consecutive nucleotides, which, when formed in a duplex with the complementary target RNA is capable of recruiting RNase. The contiguous sequence which is capable of recruiting RNAse may be region B as referred to in the context of a gapmer as described herein. In some embodiments the size of the contiguous sequence which is capable of recruiting RNAse, such as region B, may be higher, such as 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units.

EP 1 222 309 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. A oligomer is deemed capable of recruiting RNase H if, when provided with the complementary RNA target, it has an initial rate, as measured in pmol/l/min, of at least 1 %, such as at least 5%, such as at least 10% or less than 20% of the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309. In some embodiments, an oligomer is deemed essentially incapable of recruiting

RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is less than 1 %, such as less than 5%, such as less than 10% or less than 20% of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309.

In other embodiments, an oligomer is deemed capable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is at least 20%, such as at least 40 %, such as at least 60 %, such as at least 80 % of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2' substitutions, with phosphorothioate linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91 - 95 of EP 1 222 309. Typically the region of the oligomer which forms the consecutive nucleotide units which, when formed in a duplex with the complementary target RNA is capable of recruiting RNase consists of nucleotide units which form a DNA/RNA like duplex with the RNA target - and include both DNA units and LNA units which are in the alpha-L configuration, particularly preferred being alpha-L-oxy LNA.

The oligomer of the invention may comprise a nucleotide sequence which comprises both nucleotides and nucleotide analogues, and may be in the form of a gapmer, a headmer or a mixmer.

A headmer is defined by a contiguous stretch of non-RNase recruiting nucleotide analogues at the 5'-end followed by a contiguous stretch of DNA or modified nucleotide units recognizable and cleavable by the RNase towards the 3'-end (such as at least 7 such nucleotides), and a tailmer is defined by a contiguous stretch of DNA or modified nucleotides recognizable and cleavable by the RNase at the 5'-end (such as at least 7 such nucleotides), followed by a contiguous stretch of non-RNase recruiting nucleotide analogues towards the 3'-end. Other chimeras according to the invention, called mixmers consisting of an alternate composition of DNA or modified nucleotides recognizable and cleavable by RNase and non-RNase recruiting nucleotide analogues. Some nucleotide analogues may also be able to mediate RNaseH binding and cleavage. Since α-L-LNA recruits RNaseH activity to a certain extent, smaller gaps of DNA or modified nucleotides recognizable and cleavable by the RNaseH for the gapmer construct might be required, and more flexibility in the mixmer construction might be introduced.

Gapmer Design Preferably, the oligomer of the invention is a gapmer. A gapmer oligomer is an oligomer which comprises a contiguous stretch of nucleotides which is capable of recruiting an RNAse, such as RNAseH, such as a region of at least 6 or 7 DNA nucleotides, referred to herein in as region B, wherein region B is flanked both 5' and 3' by regions of affinity enhancing nucleotide analogues, such as between 1 - 6 nucleotide analogues 5' and 3' to the contiguous stretch of nucleotides which is capable of recruiting RNAse - these regions are referred to as regions A and C respectively.

Preferably the gapmer comprises a (poly)nucleotide sequence of formula (5' to 3'), A- B-C, or optionally A-B-C-D or D-A-B-C, wherein; region A (5' region) consists or comprises of at least one nucleotide analogue, such as at least one LNA unit, such as between 1-6 nucleotide analogues, such as LNA units, and; region B consists or comprises of at least five consecutive nucleotides which are capable of recruiting RNAse (when formed in a duplex with a complementary RNA molecule, such as the mRNA target), such as DNA nucleotides, and; region C (3'region) consists or comprises of at least one nucleotide analogue, such as at least one LNA unit, such as between 1-6 nucleotide analogues, such as LNA units, and; region D, when present consists or comprises of 1 , 2 or 3 nucleotide units, such as DNA nucleotides.

In some embodiments, region A consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as between 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units; and/or region C consists of 1 , 2, 3, 4, 5 or 6 nucleotide analogues, such as LNA units, such as between 2-5 nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 3 or 4 LNA units.

In some embodiments B consists or comprises of 5, 6, 7, 8, 9, 10, 11 or 12 consecutive nucleotides which are capable of recruiting RNAse, or between 6-10, or between 7-9, such as 8 consecutive nucleotides which are capable of recruiting RNAse. In some embodiments region B consists or comprises at least one DNA nucleotide unit, such as 1-12 DNA units, preferably between 4-12 DNA units, more preferably between 6-10 DNA units, such as between 7-10 DNA units, most preferably 8, 9 or 10 DNA units.

In some embodiments region A consist of 3 or 4 nucleotide analogues, such as LNA, region B consists of 7, 8, 9 or 10 DNA units, and region C consists of 3 or 4 nucleotide analogues, such as LNA. Such designs include (A-B-C) 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3, and may further include region D, which may have one or 2 nucleotide units, such as DNA units.

Further gapmer designs are disclosed in WO2004/046160 and are hereby incorporated by reference.

US provisional application, 60/977409, hereby incorporated by reference, refers to 'shortmer' gapmer oligomers, which, in some embodiments may be the gapmer oligomer according to the present invention.

In some embodiments the oligomer is consisting of a contiguous nucleotide sequence of a total of 10, 11 , 12, 13 or 14 nucleotide units, wherein the contiguous nucleotide sequence is of formula (5' - 3'), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein; A consists of 1 , 2 or 3 nucleotide analogue units, such as LNA units; B consists of 7, 8 or 9 contiguous nucleotide units which are capable of recruiting RNAse when formed in a duplex with a complementary RNA molecule (such as a mRNA target); and C consists of 1 , 2 or 3 nucleotide analogue units, such as LNA units. When present, D consists of a single DNA unit.

In some embodiments A consists of 1 LNA unit. In some embodiments A consists of 2 LNA units. In some embodiments A consists of 3 LNA units. In some embodiments C consists of 1 LNA unit. In some embodiments C consists of 2 LNA units. In some embodiments C consists of 3 LNA units. In some embodiments B consists of 7 nucleotide units. In some embodiments B consists of 8 nucleotide units. In some embodiments B consists of 9 nucleotide units. In some embodiments B comprises of between 1 - 9 DNA units, such as 2, 3, 4, 5, 6, 7 or 8 DNA units. In some embodiments B consists of DNA units. In some embodiments B comprises of at least one LNA unit which is in the alpha-L configuration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA units in the alpha-L-configuration. In some embodiments B comprises of at least one alpha-L-oxy LNA unit or wherein all the LNA units in the alpha-L- configuration are alpha-L-oxy LNA units. In some embodiments the number of nucleotides present in A-B-C are selected from the group consisting of (nucleotide analogue units - region B - nucleotide analogue units): 1 -8-1 , 1 -8-2, 2-8-1 , 2-8-2, 3-8-3, 2-8- 3, 3-8-2, 4-8-1 , 4-8-2, 1-8-4, 2-8-4, or; 1-9-1 , 1-9-2, 2-9-1 , 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1 , 4- 9-1 , 1-9-4, or; 1-10-1 , 1-10-2, 2-10-1 , 2-10-2, 1-10-3, 3-10-1. In some embodiments the number of nucleotides in A-B-C are selected from the group consisting of: 2-7-1 , 1-7-2, 2-7- 2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3. In some embodiments both A and C consists of two LNA units each, and B consists of 8 or 9 nucleotide units, preferably DNA units. lnternucleotide Linkages

The terms "linkage group" or "internucleotide linkage" are intended to mean a group capable of covalently coupling together two nucleotides, two nucleotide analogues, and a nucleotide and a nucleotide analogue, etc. Specific and preferred examples include phosphate groups and phosphorothioate groups. The nucleotides of the oligomer of the invention or contiguous nucleotides sequence thereof are coupled together via linkage groups. Suitably each nucleotide is linked to the 3' adjacent nucleotide via a linkage group.

Suitable internucleotide linkages include those listed within PCT/DK2006/000512, for example the internucleotide linkages listed on the first paragraph of page 34 of PCT/DK2006/000512 (hereby incorporated by reference).

It is, in some embodiments, preferred to modify the internucleotide linkage from its normal phosphodiester to one that is more resistant to nuclease attack, such as phosphorothioate or boranophosphate - these two, being cleavable by RNase H, also allow that route of antisense inhibition in reducing the expression of the target gene. Suitable sulphur (S) containing internucleotide linkages as provided herein may be preferred. Phosphorothioate internucleotide linkages are also preferred, particularly for the gap region (B) of gapmers. Phosphorothioate linkages may also be used for the flanking regions (A and C, and for linking A or C to D, and within region D, as appropriate).

Regions A, B and C, may however comprise internucleotide linkages other than phosphorothioate, such as phosphodiester linkages, particularly, for instance when the use of nucleotide analogues protects the internucleotide linkages within regions A and C from endo-nuclease degradation - such as when regions A and C comprise LNA nucleotides.

The internucleotide linkages in the oligomer may be phosphodiester, phosphorothioate or boranophosphate so as to allow RNase H cleavage of targeted RNA. Phosphorothioate is preferred, for improved nuclease resistance and other reasons, such as ease of manufacture.

In one aspect of the oligomer of the invention, the nucleotides and/or nucleotide analogues are linked to each other by means of phosphorothioate groups.

It is recognised that the inclusion of phosphodiester linkages, such as one or two linkages, into an otherwise phosphorothioate oligomer, particularly between or adjacent to nucleotide analogue units (typically in region A and or C) can modify the bioavailability and/or bio-distribution of an oligomer - see WO2008/053314, hereby incorporated by reference.

In some embodiments, such as the embodiments referred to above, where suitable and not specifically indicated, all remaining linkage groups are either phosphodiester or phosphorothioate, or a mixture thereof.

In some embodiments all the internucleotide linkage groups are phosphorothioate.

When referring to specific gapmer oligonucleotide sequences, such as those provided herein it will be understood that, in various embodiments, when the linkages are phosphorothioate linkages, alternative linkages, such as those disclosed herein may be used, for example phosphate (phosphodiester) linkages may be used, particularly for linkages between nucleotide analogues, such as LNA, units. Likewise, when referring to specific gapmer oligonucleotide sequences, such as those provided herein, when the C residues are annotated as 5'methyl modified cytosine, in various embodiments, one or more of the Cs present in the oligomer may be unmodified C residues. in some embodimentsin some embodiments

Oligomeric Compounds

The oligomers of the invention may, for example, be selected from the group consisting of: 40 - 59 or 60 - 92. Some preferred oligomers, include oligomers which comprise or consist of the sequence of bases present in SEQ ID NOs 68, 61 and 62.

Conjugates

In the context the term "conjugate" is intended to indicate a heterogenous molecule formed by the covalent attachment ("conjugation") of the oligomer as described herein to one or more non-nucleotide, or non-polynucleotide moieties. Examples of non-nucleotide or non- polynucleotide moieties include macromolecular agents such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof. Typically proteins may be antibodies for a target protein. Typical polymers may be polyethylene glycol.

Therefore, in various embodiments, the oligomer of the invention may comprise both a polynucleotide region which typically consists of a contiguous sequence of nucleotides, and a further non-nucleotide region. When referring to the oligomer of the invention consisting of a contiguous nucleotide sequence, the compound may comprise non-nucleotide components, such as a conjugate component.

In various embodiments of the invention the oligomeric compound is linked to ligands/conjugates, which may be used, e.g. to increase the cellular uptake of oligomeric compounds. WO2007/031091 provides suitable ligands and conjugates, which are hereby incorporated by reference.

The invention also provides for a conjugate comprising the compound according to the invention as herein described, and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said compound. Therefore, in various embodiments where the compound of the invention consists of a specified nucleic acid or nucleotide sequence, as herein disclosed, the compound may also comprise at least one non-nucleotide or non- polynucleotide moiety (e.g. not comprising one or more nucleotides or nucleotide analogues) covalently attached to said compound. Conjugation (to a conjugate moiety) may enhance the activity, cellular distribution or cellular uptake of the oligomer of the invention. Such moieties include, but are not limited to, antibodies, polypeptides, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g. Hexyl-s-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-o- hexadecyl-rac-glycero-S-h-phosphonate, a polyamine or a polyethylene glycol chain, an adamantane acetic acid, a palmityl moiety, an octadecylamine or hexylamino-carbonyl- oxycholesterol moiety.

The oligomers of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. In certain embodiments the conjugated moiety is a sterol, such as cholesterol.

In various embodiments, the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptides of, for example between 1 -50, such as 2 - 20 such as 3 - 10 amino acid residues in length, and/or polyalkylene oxide such as polyethylglycol(PEG) or polypropylene glycol - see WO 2008/034123, hereby incorporated by reference. Suitably the positively charged polymer, such as a polyalkylene oxide may be attached to the oligomer of the invention via a linker such as the releasable inker described in WO 2008/034123.

By way of example, the following conjugate moieties may be used in the conjugates of the invention:

S^<" OUGOM ER -3^'

5'- OLIGOMER -3^*

Activated oligomers

The term "activated oligomer," as used herein, refers to an oligomer of the invention that is covalently linked (i.e., functionalized) to at least one functional moiety that permits covalent linkage of the oligomer to one or more conjugated moieties, i.e., moieties that are not themselves nucleic acids or monomers, to form the conjugates herein described. Typically, a functional moiety will comprise a chemical group that is capable of covalently bonding to the oligomer via, e.g., a 3'-hydroxyl group or the exocyclic NH₂ group of the adenine base, a spacer that is preferably hydrophilic and a terminal group that is capable of binding to a conjugated moiety (e.g., an amino, sulfhydryl or hydroxyl group). In some embodiments, this terminal group is not protected, e.g., is an NH₂ group. In other embodiments, the terminal group is protected, for example, by any suitable protecting group such as those described in "Protective Groups in Organic Synthesis" by Theodora W Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons, 1999). Examples of suitable hydroxyl protecting groups include esters such as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, or triphenylmethyl, and tetrahydropyranyl. Examples of suitable amino protecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl, triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groups such as trichloroacetyl or trifluoroacetyl. In some embodiments, the functional moiety is self- cleaving. In other embodiments, the functional moiety is biodegradable. See e.g., U.S. Patent No. 7,087,229, which is incorporated by reference herein in its entirety.

In some embodiments, oligomers of the invention are functionalized at the 5' end in order to allow covalent attachment of the conjugated moiety to the 5' end of the oligomer. In other embodiments, oligomers of the invention can be functionalized at the 3' end. In still other embodiments, oligomers of the invention can be functionalized along the backbone or on the heterocyclic base moiety. In yet other embodiments, oligomers of the invention can be functionalized at more than one position independently selected from the 5' end, the 3' end, the backbone and the base.

In some embodiments, activated oligomers of the invention are synthesized by incorporating during the synthesis one or more monomers that is covalently attached to a functional moiety. In other embodiments, activated oligomers of the invention are synthesized with monomers that have not been functionalized, and the oligomer is functionalized upon completion of synthesis. In some embodiments, the oligomers are functionalized with a hindered ester containing an aminoalkyl linker, wherein the alkyl portion has the formula (CH₂)_W, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group is attached to the oligomer via an ester group (-0-C(O)- (CH₂)_WNH).

In other embodiments, the oligomers are functionalized with a hindered ester containing a (CH₂)_w-sulfhydryl (SH) linker, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (-O-C(O)-(CH₂)_WSH)

In some embodiments, sulfhydryl-activated oligonucleotides are conjugated with polymer moieties such as polyethylene glycol or peptides (via formation of a disulfide bond). Activated oligomers containing hindered esters as described above can be synthesized by any method known in the art, and in particular by methods disclosed in PCT Publication No. WO 2008/034122 and the examples therein, which is incorporated herein by reference in its entirety.

In still other embodiments, the oligomers of the invention are functionalized by introducing sulfhydryl, amino or hydroxyl groups into the oligomer by means of a functionalizing reagent substantially as described in U.S. Patent Nos. 4,962,029 and 4,914,210, i.e., a substantially linear reagent having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposing end which comprises a protected or unprotected sulfhydryl, amino or hydroxyl group. Such reagents primarily react with hydroxyl groups of the oligomer. In some embodiments, such activated oligomers have a functionalizing reagent coupled to a 5'-hydroxyl group of the oligomer. In other embodiments, the activated oligomers have a functionalizing reagent coupled to a 3'- hydroxyl group. In still other embodiments, the activated oligomers of the invention have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer. In yet further embodiments, the oligomer of the invention is functionalized with more than one of the functionalizing reagents as described in U.S. Patent Nos. 4,962,029 and 4,914,210, incorporated herein by reference in their entirety. Methods of synthesizing such functionalizing reagents and incorporating them into monomers or oligomers are disclosed in U.S. Patent Nos. 4,962,029 and 4,914,210. In some embodiments, the 5'-terminus of a solid-phase bound oligomer is functionalized with a dienyl phosphoramidite derivative, followed by conjugation of the deprotected oligomer with, e.g., an amino acid or peptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of monomers containing 2'-sugar modifications, such as a 2'-carbamate substituted sugar or a 2'-(O-pentyl-N-phthalimido)- deoxyribose sugar into the oligomer facilitates covalent attachment of conjugated moieties to the sugars of the oligomer. In other embodiments, an oligomer with an amino-containing linker at the 2'-position of one or more monomers is prepared using a reagent such as, for example, 5'-dimethoxytrityl-2'-O-(e-phthalimidylaminopentyl)-2'-deoxyadenosine-3'- N, N- diisopropyl-cyanoethoxy phosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters, 1991 , 34, 7171.

In still further embodiments, the oligomers of the invention may have amine-containing functional moieties on the nucleobase, including on the N6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or 5 positions of cytosine. In various embodiments, such functionalization may be achieved by using a commercial reagent that is already functionalized in the oligomer synthesis.

Some functional moieties are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from the Pierce Co. (Rockford, III.). Other commercially available linking groups are 5'-Amino-Modifier C6 and 3'-Amino-Modifier reagents, both available from Glen Research Corporation (Sterling, Va.). 5'-Amino-Modifier C6 is also available from ABI (Applied Biosystems Inc., Foster City, Calif.) as Aminolink-2, and 3'-Amino-Modifier is also available from Clontech Laboratories Inc. (Palo Alto, Calif.). In some embodimentsin some embodiments

Compositions The oligomer of the invention may be used in pharmaceutical formulations and compositions. Suitably, such compositions comprise a pharmaceutically acceptable diluent, carrier, salt or adjuvant. PCT/DK2006/000512 provides suitable and preferred pharmaceutically acceptable diluent, carrier and adjuvants - which are hereby incorporated by reference. Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in PCT/DK2006/000512 - which are also hereby incorporated by reference.

Applications

The oligomers of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis. In research, such oligomers may be used to specifically inhibit the synthesis of BCL-2 protein (typically by degrading or inhibiting the mRNA and thereby prevent protein formation) in cells and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention.

In diagnostics the oligomers may be used to detect and quantitate BCL-2 expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques. For therapeutics, an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of BCL-2 is treated by administering oligomeric compounds in accordance with this invention. Further provided are methods of treating a mammal, such as treating a human, suspected of having or being prone to a disease or condition, associated with expression of BCL-2 by administering a therapeutically or prophylactically effective amount of one or more of the oligomers or compositions of the invention. The oligomer, a conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.

The invention also provides for the use of the compound or conjugate of the invention as described for the manufacture of a medicament for the treatment of a disorder as referred to herein, or for a method of the treatment of as a disorder as referred to herein.

The invention also provides for a method for treating a disorder as referred to herein said method comprising administering a compound according to the invention as herein described, and/or a conjugate according to the invention, and/or a pharmaceutical composition according to the invention to a patient in need thereof.

Medical Indications

The oligomers and other compositions according to the invention can be used for the treatment of conditions associated with over expression or expression of mutated version of the Bcl-2. The invention further provides use of a compound of the invention in the manufacture of a medicament for the treatment of a disease, disorder or condition as referred to herein.

Generally stated, one aspect of the invention is directed to a method of treating a mammal suffering from or susceptible to conditions associated with abnormal levels of Bcl-2, comprising administering to the mammal and therapeutically effective amount of an oligomer targeted to Bcl-2 that comprises one or more LNA units. The oligomer, a conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.

For therapeutics, an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of Bcl-2 is treated by administering antisense compounds in accordance with this invention. Further provided are methods of treating a mammal, such as treating a human, suspected of having or being prone to a disease or condition, associated with expression of Bcl-2 by administering a therapeutically or prophylactically effective amount of one or more of the oligomers or compositions of the invention. The cancer, as referred to herein, is in some embodiments selected from the group consisting of melanoma, leukemia, myeloma, lymphoma, glioma, and carcinoma.

In some embodiments the cancer is selected from the group consisting of leukemia, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL) and acute lymphoid leukemia (ALL), malignant glioma, melanoma, multiple myeloma, and hepatocellular carcinoma.

In some embodiments the cancer selected from the group consisting of leukemia, melanoma, myeloma, and melanoma. In some embodiments the cancer is leukemia, such as chronic lymphocytic leukemia (CLL), acute lymphoid leukemia (ALL), and chronic myeloid leukemia. In some embodiments the cancer is lymphoma, such as non-hodgkin's lymphomas, follicular lymphoma and diffuse large B-cell lymphoma. In some embodiments the cancer is myleoma such as multiple myeloma. In some embodiments the cancer is melanoma, such as malignant melanoma. In one embociment the cancer is malignant glioma. In some embodiments the cancer is a carcinoma such as a hepatocellular carcinoma. The oligomers, conjugates, and compositions may, in one preferable embodiment, may be for the use in the treatment of the cancers. In some embodiments, the cancer is liver or kidney cancer. In some embodiments the cancer is lung cancer, or a breast cancer or a prostate cancer. In some embodiments the cancer is a malignant tumour.

In some embodiments, for example for the treatment of brain cancer, it is preferred that phosphorothioate linkages are not used in the compound according to the invention.

In some embodiments, the term 'treatment' as used herein refers to both treatment of an exisiting disease (e.g. a cancer as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognised that treatment as referred to herein may, in some embodiments, be prophylactic. The invention also provides for the use of the compound or conjugate of the invention as described for the manufacture of a medicament for the treatment of cancer, or for a method of the treatment of cancer.

The invention also provides for a method for treating cancer, said method comprising administering a compound according to the invention as herein described, and/or a conjugate according to the invention, and/or a pharmaceutical composition according to the invention to a patient in need thereof.

Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in PCT/DK2006/000512 - which are hereby incorporated by reference. The invention also provides for a pharmaceutical composition comprising a compound or a conjugate as herein described or a conjugate, and a pharmaceutically acceptable diluent, carrier or adjuvant. PCT/DK2006/000512 provides suitable and preferred pharmaceutically acceptable diluent, carrier and adjuvants - which are hereby incorporated by reference.

Pharmaceutical compositions comprising more than one active ingredient (i.e. comprise a further therapeutic agent or treatment),

Bcl-2 antisense agents have been used in combination therapies, particularly anticancer therapies, such as radiotherapy or chemotherapy. Indeed Bcl-2 antisense agents have been considered as chemosensitisation agents, for example in the treatment of malignant melanoma {Lancet. 2000;356(9243):1728-1732).

Suitably both the oligomer targeting Bcl2 and the further active ingredient are administered in effective amounts. In this respect, it is considered that for some further active ingredients, the down-regulation of Bcl2 is beneficial to the treatment with the further active ingredient and may alleviate a non-responsiveness or low-responsiveness to the further active ingredient.

In some embodiments the method of treatment according to the invention is a method of treating a hyperproliferative disorder, such as cancer, wherein said treatment comprises both the administration of the pharmaceutical composition according to the invention and radiation therapy (see Invest New Drugs. 2007 Oct;25(5):411-6 and Clinical Cancer Research Vol. 1 1 , 8131-8144, November 15, 2005, which are hereby incorporated by reference).

In some embodiments the pharmaceutical composition according to the invention may comprise both the oligomer or conjugate according to the invention and a further therapeutic agent, such as Fludara® (fludarabine) and/or Cytoxan® (cyclophosphamide). It is known that the combination of Bcl2 targeting antisense agents and/or Fludara® (fludarabine) and/or Cytoxan® (cyclophosphamide) improves the response rate and duration of response in relapsed or refractory chronic lymphocytic leukemia (CLL).

The pharmaceutical composition according to the invention may further comprise other active ingredients, including those which are indicated as being useful for the treatment of hyperproliferative diseases and cancer, such as those referred to herein.

The further active ingredients may for instance be selected from one or more of the following (the references referred to which indicate the benefit of combination of the further active ingredient with Bcl-2 antisense agents are hereby incorporated by reference): • BH3 mimetic small molecule inhibitors, such as ABT-737 (Oltersdorf T, et al. Nature. 435:677-681. PMID: 15902208, hereby incorporated by reference).

• Gimatecan (Eur J Cancer. 2005 May;41 (8):1213-22)

• Paclitaxel, paclitaxel albumin nanoparticles, imatinib, sorafenib, sunitinib, and erlotinib (Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part

I. VoI 25, No. 18S (June 20 Supplement), 2007: 14061 ) - human melanoma.

• Pacltaxel - small-lung cell cancer (Annals of Ocology 13: 539-545, 2002).

• Cisplatin - breast cancer (Journal of Biomedical Science, VoI 12/6 999-101 1 )

• Rituximab, fludarabine, cyclo-phosphamid, dacarbazine, temozolomide docetaxel (Drugs R D. 2007;8(5):321-34.)

• Doxorubicin and dexamethasone and thalidomide (Hematology Am Soc Hematol Educ Program. 2003;:248-78)

The invention provides for use of an oligomer targeting Bcl2, such as one or more of the oligomers described herein, for the preparation of a medicament, wherein said medicament is for the use in the treatment of cancer in combination with a further active ingredient, such a a further active ingredient selected from those listed above or an inhibitor of McH , such as an oligomer targeting McH .

The invention provides for a medicament comprising an oligomer targeting Bcl2, such as one or more of the oligomers described herein, wherein said medicament is for the use in the treatment of cancer in combination with with a further active ingredient, such as a further active ingredient selected from those listed above or an inhibitor of McM , such as an oligomer targeting McM .

The invention further relates to methods of treating a disease, such as those referred to herein, such as cancer, comprising administering to a patient in need there of an effective amount of an oligomer that targets Bcl2 mRNA in a cell and an effective amount of a a further active ingredient, such as a further active ingredient selected from those listed above or an inhibitor of Mcl1 , such as an oligomer targeting Mcl1.

The further active ingredient is typically administered in the form of a pharmaceutical composition which further comprises a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

Administration

The oligomer targeting Bcl2 may be administered at regular intervals (Dose intervals, Dl) of between 3 days and two weeks, such as 4, 5, 6, 7, 8, 9, 0, 1 1 , 12, 13 days, such as about 1 week, such as 6, 7 or 8 days. Suitably at least two doses are provides with a Dl period between the two dosages, such as 3, 4, 5, 6, 7, 8, 9 or 10 dosages, each with a dose interval (Dl) between each dose of LNA oligomer. The Dl period between each dosage may the same, such as between 3 days and two weeks, such as 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 days, such as about 1 week, such as 6, 7 or 8 days.

In some embodiments, each dose of the oligomer targeting Bcl2 may be between about 0.25mg/kg - about 10mg/kg, such as about 0.5mg/kg, about 1 mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg. In some embodiments, each dose of the LNA oligomer targeting Bcl2 may be between about 2 mg/kg - about 8mg/kg, or about 4 to about 6 mg/kg or about 4mg/kg to about 5mg/kg. In some embodiments, each dose of the LNA oligomer targeting Bcl2 is at least 2mg/kg, such as 2, 3_S 4, 5, 6, 7 or 8 mg/kg, such as 6 mg/kg.

Administration of the oligomer is typically performed by parenteral administration, such as subcutaneous, intramuscular, intravenous or intra peritoneal administration. Intravenously administration is preferred.

In some embodiments the dosage regime for the oligomer may be repeated after an initial dosage regime, indeed the dosage regime may be repeated as necessary in order to treat or prevent the progression of the disease.

One advantage of the oligomers targeting Bcl2 according to the invention is that they may be administered over a relatively short time period rather than continuously. This provides a marked improvement in the quality of life for the patient as they are not required to be hospital bound for long periods of time. Therefore in a preferred embodiment, the LNA oligomer targeting Bcl2 is not administered by continuous infusion. Each dose of the oligomer may therefore be administered to the patient in a time period of less than 12 hours, such as less than about 8 hours, less than about 4 hours, such as less than about 3 hours. Each dose of the LNA oligomer may therefore be administered to the patient in a time period of between about 1 and about 4 hours, such as between about 2 and about 3 hours, or about 2 hours. The oligomer may be administered to the patient in a time period of at least 30 minutes such as at least 1 hour. Such administrations may be given intravenously, for example.

A pharmaceutically effective dose of the further active ingredient may, in some embodiments be administered prior to, during or subsequently to the administration of one or more pharmaceutically effective doses of the LNA oligomer targeting Bcl2. Typically, one or more effective doses of the further active ingredient is administered so that the both the LNA oligomer and the further active ingredient provide their therapeutic benefit concurrently within the patient or subject. Combination with Antisense oligomers targeting Mcl-1 In some embodiments the pharmaceutical composition according to the invention may comprise a further therapeutic agent (active ingredient) such active ingredients which target Mcl-1 such as antisense oligomers which target Mcl-1 mRNA. (Further) therapeutic agents which target Mcl-1 are described in Mandelin and Pope et al., Expert Opinion Ther. Targets (2007) 1 1 (3):363-373, hereby incorporated by reference.

One further active ingredient may be an antisense oligomer which targets Mcl-1. The Bcl-2 oligomers according to the invention may, in some embodiments be used in combination with compounds which target Mcl-1 , such as antisense oligomers which are complementary to the Mcl-1 mRNA. WO2007/109174 and US 6,001 ,992, hereby incorporated by reference, disclose 2'MOE antisense agents which target Mcl-1 , which may therefore be considered for use in combination with oligomers which target Bcl-2.

In some embodiments, the oligomers which target Mcl-1 , which may be used in combination with the oligomers which target Bcl-2, such as those disclosed herein, are LNA oligomers, such as gapmers and shortmers, which comprises a contiguous nucleobase sequence of a total of between 10-50 nucleobases, wherein said contiguous nucleobase sequence is at least 80%, such as at least homologous to a corresponding region of a nucleic acid which encodes a mammalian Mcl-1.

With respect to the structure of the oligomers, such as gapmers and shortmers, and conjugates thereof, which target Mcl-1 , with the exception of the fact that such oligomers will be complementary to a corresponding region of the Mcl-1 mRNA, or in one case, comprise 1 , 2, 3, or 4 mismatches to the corresponding region of the target Mcl-1 , may, in some embodiments may have the same structure as the oligomers according to the present invention (i.e. apart from the specific sequence of nucleobases).

The administration of a pharmaceutical composition comprising an oligomer which targets McH may be performed as per the oligomers which target Bcl-2 as disclosed herein, and may be performed prior to, during or after the administration of the pharmaceutical composition comprising the oligomer of the invention, are as part of the same pharmaceutical composition.

The invention therefore provides for methods for the simultaneous (concurrent) inhibition of expression of both Bcl-2 and Mcl-1 in a cell, such as a cancer cell, which is expressing Bcl-2 and Mcl-1 , said method comprising a. administering an effective amount of a Bcl2 inhibitor, such as a oligomer which targets Bcl2, such as oligomer according to the invention, or a conjugate or pharmaceutical composition thereof, to said cell so as to inhibit Bcl2 in said cell, b. administering an effective amount of a Mcl1 inhibitor, such as a oligomer which targets Mc11 , such as SEQ ID NO 96 or 97, or a conjugate or pharmaceutical composition thereof, to said cell so as to inhibit McH in said cell, wherein steps a) and b) may be performed in any order or simultaneously and lead to the simultaneous inhibition (down-regulation) of both Mcl-1 and Bcl-2 inhibition in said cell; wherein said method is performed either in vivo or in vitro. Suitably the cell may be a cancer cell in a subject, such as a human subject suffering from cancer. In some embodiments, the method may result in cell death such as apoptosis of the cell.

Suitable McH inhibitors to be used in conjunction with the Bcl2 inhibitor are referenced above and include also include those disclosed in U.S. Provisional Applications Serial No. US 61/012191 , filed 7^th December 2007, and US 61/095955, filed 1 1^th September 2008, the contents of which are hereby incorporated in their entirity. Kits of parts

The invention also provides a kit of parts wherein a first part comprises the oligomer, the conjugate and/or the pharmaceutical composition according to the invention and a further part comprises an further active ingredient (i.e. further therapeutic agent), such as those referred to herein. It is therefore envisaged that the kit of parts may be used in a method of treatment, as referred to herein, where the method comprises administering both the first part and the further part, either simultaneously or one after the other. The invention also provides a kit of parts wherein a first part comprises the oligomer, the conjugate and/or the pharmaceutical composition according to the invention and a further part comprises an antisense oligonucleotide capable of lowering the expression of McI 1. It is therefore envisaged that the kit of parts may be used in a method of treatment, as referred to herein, where the method comprises administering both the first part and the further part, either simultaneously or one after the other. Medical methods and use

The oligomers and other compositions according to the invention can be used for the treatment of conditions associated with over expression or expression of mutate version of the Bcl-2. It has been suggested by leading scientists in the field that pharmaceutical intervention with Bcl-2 will result in therapeutic options against cancer, such as those referred to herein.

Further conditions which may be associated with abnormal levels of Bcl-2, and which, therefore may be treated using the compositions, conjugates and compounds according to the invention include disorders selected form the group consisting of hyperproliferative diseases, such as cancer. Hyperprol iterative diseases refer to disorders which are characterised by the uncontrolled and detrimental proliferation of cells within the body, such as cancer such as those referred to herein.

The invention further provides use of a compound of the invention in the manufacture of a medicament for the treatment of a disease, disorder or condition as referred to herein.

Generally stated, one aspect of the invention is directed to a method of treating a mammal suffering from or susceptible to conditions associated with abnormal levels of Bcl-2, comprising administering to the mammal and therapeutically effective amount of an oligomer targeted to Bcl-2 that comprises one or more LNA units. An interesting aspect of the invention is directed to the use of an oligomer (compound) as defined herein or as conjugate as defined herein for the preparation of a medicament for the treatment of a disease, disorder or condition as referred to herein.

The methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels of Bcl-2. Furthermore, the invention described herein encompasses a method of preventing or treating a disease comprising a therapeutically effective amount of a Bcl-2 modulating oligomer to a human in need of such therapy. The invention further encompasses the use of a short period of administration of a Bcl-2 modulating oligonucleotide compound.

The oligomers of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

Alternatively stated, the invention is furthermore directed to a method for treating abnormal levels of Bcl-2, said method comprising administering a oligomer of the invention, or a conjugate of the invention or a pharmaceutical composition of the invention to a patient in need thereof and further comprising the administration of a further chemotherapeutic agent or further active ingredient. Said further administration may be such that the further chemotherapeutic agent or further active ingredient is conjugated to the compound of the invention, is present in the pharmaceutical composition, or is administered in a separate formulation.

The invention also relates to an oligomer, a composition or a conjugate as defined herein for use as a medicament.

The invention further relates to use of a compound, composition, or a conjugate as defined herein for the manufacture of a medicament for the treatment of abnormal levels of Bcl-2 or expression of mutant forms of Bcl-2 (such as allelic variants, such as those associated with one of the diseases referred to herein). Moreover, the invention relates to a method of treating a subject suffering from a disease or condition selected from hyperproliferative diseases and cancer, such as those referred to herein.

A patient who is in need of treatment is a patient suffering from or likely to suffer from the disease or disorder.

EMBODIMENTS

The following embodiments of the present invention may be used in combination with the other embodiments described herein. 1. An oligomer of between 10-50 nucleobases in length which comprises a contiguous nucleobase sequence of a total of between 10-50 nucleobases, wherein said contiguous nucleobase sequence is at least 80% homologous to a corresponding region of a nucleic acid sequence selected from the group consiting of SEQ ID No 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40. 2. The oligomer according to embodiment 1 , wherein the contiguous nucleobase sequence comprises no more than 3, such as no more than 2 mismatches to the corresponding region of the nucleic acid, selected from the group consiting of SEQ ID No 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40

3. The oligomer according to embodiment 1 , wherein said contiguous nucleobase sequence comprises no more than a single mismatch to the corresponding region of the nucleic acid.selected from the group consiting of SEQ ID No 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40.

4. The oligomer according to embodiment 1 , wherein said contiguous nucleobase sequence comprises no mismatches, when compared to the corresponding region of the nucleic acid.selected from the group consiting of SEQ ID No 22, 23, 24, 25, 26, 27,

28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 and 40.

5. The oligomer according to any one of embodiments 1 - 4, wherein the nucleobase sequence of the oligomer consists of the contiguous nucleobase sequence.

6. .The oligomer according to any one of embodiments 1 - 5, wherein the contiguous nucleobase sequence comprises a contiguous subsequence of at least 7 nucleobase residues which, when formed in a duplex with the complementary Bcl-2 target RNA is capable of recruiting RNaseH.

7. The oligomer according to embodiment 6, wherein the contiguous nucleobase sequence comprises of a contiguous subsequence of at least 8, at least 9 or at least 10 nucleobase residues which, when formed in a duplex with the complementary Bcl-2 target RNA is capable of recruiting RNaseH.

8. The oligomer according to any one of embodiments 6 or 7 wherein said contiguous subsequence is at least 9 or at least 10 nucleobases in length, such as at least 12 nucleobases or at least 14 nucleobases in length, such as 14, 15 or 16 nucleobases residues which, when formed in a duplex with the complementary Bcl-2 target RNA is capable of recruiting RNaseH.

9. The oligomer according to embodiment any one of embodiments 1 - 8 wherein said oligomer is conjugated with one or more non-nucleobase compounds. 10. The oligomer according to any one of embodiments 1 - 9, wherein said oligomer has a length of between 10 - 22 nucleobases.

1 1. The oligomer according to any one of embodiments 1 - 9, wherein said oligomer has a length of between 12 - 18 nucleobases.

12. The oligomer according to any one of embodiments 1 - 9, wherein said oligomer has a length of 14, 15 or 16 nucleobases.

13. The oligomer according to any one of embodiments 1 -12, wherein the oligomer or contiguous nucleobase sequence comprises, or is selected from a corresponding nucleobase sequence present in a nucleotide sequence selected from the group consisting of SEQ ID NO 3 - 21. 14. The oligomer according to any one of embodiments 1 - 13, wherein said contiguous nucleobase sequence comprises at least one affinity enhancing nucleotide analogue, such as LNA.

15. The oligomer according to embodiment 14, wherein said contiguous nucleobase sequence comprises a total of 2, 3, 4, 5, 6, 7, 8, 9 or 10 affinity enhancing nucleotide analogues, such as between 5 and 8 affinity enhancing nucleotide analogues.

16. The oligomer according to any one of embodiments 1 - 15 which comprises at least one affinity enhancing nucleotide analogue, wherein the remaining nucleobases are selected from the group consisting of DNA nucleotides and RNA nucleotides, preferably DNA nucleotides. 17. The oligomer according to any one of embodiments 1 - 16, wherein the oligomer comprises of a sequence of nucleobases of formula, in 5' to 3' direction, A-B-C, and optionally of formula A-B-C-D, wherein:

A consists or comprises of at least one nucleotide analogue, such as 1 , 2, 3, 4, 5 or 6 nucleotide analogues, preferably between 2-5 nucleotide analogues, preferably 2, 3 or 4 nucleotide analogues, most preferably 2, 3 or 4 consecutive nucleotide analogues and; B consists or comprises at least five consecutive nucleobases which are capable of recruiting RNAseH (when formed in a duplex with a complementary RNA molecule, such as the Bcl-2 mRNA target), such as

DNA nucleobases, such as 5, 6, 7, 8, 9, 10, 1 1 or 12 consecutive nucleobases which are capable of recruiting RNAseH, or between 6-10, or between 7-9, such as 8 consecutive nucleobases which are capable of recruiting RNAseH, and; C consists or comprises of at least one nucleotide analogue, such as 1 , 2, 3, 4,

5, or 6 nucleotide analogues, preferably between 2-5 nucleotide analogues, such as 2, 3 or 4 nucleotide analogues, most preferably 2, 3 or 4 consecutive nucleotide analogues, and;

D when present, consists or comprises, preferably consists, of one or more DNA nucleotide, such as between 1-3 or 1-2 DNA nucleotides.

18. The oligomer according to embodiment 17, wherein region A consists or comprises of 2, 3 or 4 consecutive nucleotide analogues.

19. The oligomer according to any one of embodiments 17 - 18, wherein region B consists or comprises of 7, 8, 9 or 10 consecutive DNA nucleotides or equivalent nucleobases which are capable of recruiting RNAseH when formed in a duplex with a complementary RNA ,such as the Bcl-2 mRNA target.

20. The oligomer according to any one of embodiments 17 - 19, wherein region C consists or comprises of 2, 3 or 4 consecutive nucleotide analogues.

21. The oligomer according to any one of embodiments 17 - 20, wherein region D consists, where present, of one or two DNA nucleotides.

22. The oligomer according to any one of embodiments 17 - 21 , wherein:

A Consists or comprises of 3 contiguous nucleotide analogues;

B Consists or comprises of 7, 8, 9 or 10 contiguous DNA nucleotides or equivalent nucleobases which are capable of recruiting RNAseH when formed in a duplex with a complementary RNA, such as the Bcl-2 mRNA target;

C Consists or comprises of 3 contiguous nucleotide analogues; D Consists, where present, of one or two DNA nucleotides.

23. The oligomer according to embodiment 22, wherein the contiguous nucleobase sequence consists of 10, 1 1 , 12, 13 or 14 nucleobases, and wherein; A Consists of 1 ,2 or 3 contiguous nucleotide analogues; B Consists of 7, 8, or 9 consecutive DNA nucleotides or equivalent nucleobases which are capable of recruiting RNAseH when formed in a duplex with a complementary RNA, such as the Bcl-2 mRNA target; C Consists of 1 ,2 or 3 contiguous nucleotide analogues;

D Consists, where present, of one DNA nucleotide.

24. The oligomer according to anyone of embodiments 17 - 23, wherein B comprises at least one LNA nucleobase which is in the alpha-L configuration, such as alpha-L-oxy LNA. 25. The oligomer according to any one of embodiments 1 - 24, wherein the nucleotide analogue(s) are independently or collectively selected from the group consisting of: Locked Nucleic Acid (LNA) units; 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino- DNA units, 2'-fluoro-DNA units, PNA units, HNA units, and INA units.

26. The oligomer according to embodiment 25 wherein all the nucleotide analogues(s) are LNA units.

27. The oligomer according to any one of embodiments 1 - 26, which comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA units such as between 2 and 8 nucleotide LNA units.

28. The oligomer according to any one of the embodiments 25 - 27, wherein the LNAs are independently selected from oxy-LNA, thio-LNA, and amino-LNA, in either of the beta-D and alpha-L configurations or combinations thereof.

29. The oligomer according to embodiment 28, wherein the LNAs are all beta-D-oxy-LNA.

30. The oligomer according to any one of embodiments 17 - 29, wherein the nucleotide analogues or nucleobases of regions A and C are beta-D-oxy-LNA.

31. The oligomer according to any one of embodiments 1 - 30, wherein at least one of the nucleobases present in the oligomeris a modified nucleobase selected from the group consisting of 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5- propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6- aminopurine.

32. The oligomer according to any one of embodiments 1 - 31 , wherein said oligomer hybridises with a corresponding mammalian Bcl-2 mRNA with a T_m of at least 50⁰C.

33. The oligomer according to any one of embodiments 1 - 32, wherein said oligomer hybridises with a corresponding mammalian Bcl-2 mRNA with a T_m of no greater than 80⁰C. 34. The oligomer according to any one of embodiments 1 - 33, wherein the internucleoside linkages are independently selected from the group consisting of: phosphodiester, phosphorothioate and boranophosphate.

35. The oligomer according to embodiment 34, wherein the oligomer comprises at least one phosphorothioate internucleoside linkage.

36. The oligomer according to embodiment 35, wherein the internucleoside linkages adjacent to or between DNA or RNA units, or within region B are phosphorothioate linkages.

37. The oligomer according to embodiment 35 or 36, wherein the linkages between at least one pair of consecutive nucleotide analogues is a phosphodiester linkage.

38. The oligomer according to embodiment 34 or 36, wherein all the linkages between consecutive nucleotide analogues are phosphodiester linkages.

39. The oligomer according to embodiment 38 wherein all the internucleoside linkages are phosphorothioate linkages. 40. A conjugate comprising the oligomer according to any one of the embodiments 1-39 and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said compound.

41. A pharmaceutical composition comprising an oligomer as defined in any of embodiments 1-39 or a conjugate as defined in embodiment 40, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

42. A pharmaceutical composition according to 41 , wherein the oligomer is constituted as a pro-drug.

43. A pharmaceutical composition according to embodiment 41 or 42, which further comprises a further therapeutic agent such as an active ingredient which targets Mcl-1 such as an antisense oligomer which targets Mcl-1 mRNA.

44. Use of an oligomer as defined in any one of the embodiments 1 -39, or a conjugate as defined in embodiment 40, for the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of hyperproliferative diseases such as cancer, such as those referred to herein, such as a cancer selected from the group consisting of acute myeloid leukemia, lymphoma, chronic lymphocytic leukemia and non-hodgkin's lymphomas particularly follicular lymphoma and diffuse large B-cell lymphoma.

45. A method for treating a hyperproliferative disease such as cancer, said method comprising administering an oligomer as defined in one of the embodiments 1-39, or a conjugate as defined in embodiment 40, or a pharmaceutical composition as defined in any one of the embodiments 41 - 43, to a patient in need thereof.

46. A method of reducing or inhibiting the expression of Bcl-2 in a cell or a tissue (which is expressing Bcl-2), the method comprising the step of contacting said cell or tissue with a compound as defined in one of the embodiments 1-39, or a conjugate as defined in embodiment 40, or a pharmaceutical composition as defined in any one of the embodiments 41 - 43, so that expression of Bcl-2 is reduced or inhibited.

47. A method of triggering apoptosis in a cell, such as a cancer cell, said method comprising the step of contacting said cell or tissue with an oligomer as defined in one of the embodiments 1-39, or a conjugate as defined in embodiment 40, or a pharmaceutical composition as defined in any one of the embodiments 41 - 43, so that so that either expression of Bcl-2 is inhibited or reduced and/or apoptosis is triggered.

EXAMPLES

Example 1: Monomer synthesis The LNA monomer building blocks and derivatives thereof were prepared following published procedures and references cited therein, (WO 03/095467 A1 ; D. S. Pedersen, C.

Rosenbohm, T. Koch (2002) Preparation of LNA Phosphoramidites, Synthesis 6, 802-808;

M. D. Sørensen, L. Kvaernø, T. Bryld, A. E. Hakansson, B. Verbeure, G. Gaubert, P.

Herdewijn, J. Wengel (2002) α-L-r/ιbo-configured Locked Nucleic Acid (α-l-LNA): Synthesis and Properties, J. Am. Chem. Soc, 124, 2164-2176; S. K. Singh, R. Kumar, J. Wengel

(1998) Synthesis of Novel Bicyclo[2.2.1] Ribonucleosides: 2'-Amino- and 2'-TNo-LNA

Monomeric Nucleosides, J. Org. Chem. 1998, 63, 6078-6079; C. Rosenbohm, S. M.

Christensen, M. D. Sørensen, D. S. Pedersen, L. E. Larsen, J. Wengel, T. Koch (2003)

Synthesis of 2'-amino-LNA: a new strategy, Org. Biomol. Chem. 1 , 655-663; D. S. Pedersen, T. Koch (2003) Analogues of LNA (Locked Nucleic Acid). Synthesis of the 2'-TNo-LNA

Thymine and 5-Methyl Cytosine Phosphoramidites, Synthesis 4, 578-582.

Example 2: Oligonucleotide synthesis

Oligonucleotides were synthesized according to the method described in WO07/031081.

Table 1 shows examples of antisense oligonucleotide sequences of the invention. Tables 2 and 3 show examples of antisense oligonucleotides (oligos) of the invention.

Example 3: Design of the oligonucleotides

In accordance with the present invention, a series of different oligonucleotides were designed to target different regions of human Bcl-2 (GenBank accession number

NM_000657 and NM_000633, SEQ ID NO: 1 and 2). Table 1 Antisense oligonucleotide sequences of the invention SEQ ID NOS:3-40 are oligo sequences designed to target human Bcl-2.

Table 2: Oligonucleotide designs of the invention

In SEQ ID NOs: 41-59, upper case letters indicates nucleotide analogue units, such as LNA, such as Beta-D-oxy LNA, and subscript "s" represents phosphorothiote linkage. Lower case letters represent nucleotide units, such as DNA units. Absence of "s" (if any) indicates phosphodiester linkage. Nucleotide analogue cytosines may be 5-methylcytosine.

Example 4: In vitro model: Cell culture

The effect of antisense oligonucleotides on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. The target can be expressed endogenously or by transient or stable transfection of a nucleic acid encoding said target. The expression level of target nucleic acid can be routinely determined using, for example, Northern blot analysis, Real-Time PCR, Ribonuclease protection assays. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. Cells were cultured in the appropriate medium as described below and maintained at 37⁰C at 95-98% humidity and 5% CO₂. Cells were routinely passaged 2-3 times weekly. 15PC3: The human prostate cancer cell line 15PC3 was cultured in DMEM (Sigma) + 10% fetal bovine serum (FBS) + 2 mM Glutamax I + gentamicin (25μg/ml). 518A2: The human melanoma cell line was cultured in DMEM (Sigma) + 10% fetal bovine serum (FBS) + 2 mM Glutamax I + gentamicin (25μg/ml).

Example 5: In vitro model: Treatment with antisense oligonucleotide using lipid transfection

The cell lines listed in example 4 were treated with oligonucleotide using the cationic liposome formulation LipofectAMINE 2000 (Gibco) as transfection vehicle. Cells were seeded in 6-well cell culture plates (NUNC) and treated when 80-90% confluent. Oligo concentrations used ranged from 0.04 nM to 25 nM final concentration. Formulation of oligo- lipid complexes were carried out essentially as described by the manufacturer using serum- free OptiMEM (Gibco) and a final lipid concentration of 5 μg/mL LipofectAMINE 2000 for 15PC3 cells and 2.5 μg/ml for 518A2 cells. Cells were incubated at 37⁰C for 4 hours and treatment was stopped by removal of oligo-containing culture medium. Cells were washed and serum-containing media was added. After oligo treatment cells were allowed to recover for 20 hours before they were harvested for RNA analysis. Example 6: In vitro model: Extraction of RNA and cDNA synthesis Total RNA Isolation and First strand synthesis Total RNA was extracted from cells transfected as described above and using the Qiagen RNeasy kit (Qiagen cat. no. 74104) according to the manufacturer's instructions. First strand synthesis was performed using Reverse Transcriptase reagents from Ambion according to the manufacturer's instructions.

For each sample 0.5 μg total RNA was adjusted to (10.8 μl) with RNase free H₂O and mixed with 2 μl random decamers (50 μM) and 4 μl dNTP mix (2.5 mM each dNTP) and heated to 70 ⁰C for 3 min after which the samples were rapidly cooled on ice. After cooling the samples on ice, 2 μl 1 Ox Buffer RT, 1 μl MMLV Reverse Transcriptase (100 U/μl) and 0.25 μl

RNase inhibitor (10 U/μl) was added to each sample, followed by incubation at 42 ⁰C for 60 min, heat inactivation of the enzyme at 95°C for 10 min and then the sample was cooled to 4

⁰C. Example 7: In vitro model: Analysis of Oligonucleotide Inhibition of Bcl-2 Expression by Real-time PCR

Antisense modulation of Bcl-2 expression can be assayed in a variety of ways known in the art. For example, Bcl-2 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or mRNA.

Methods of RNA isolation and RNA analysis such as Northern blot analysis is routine in the art and is taught in, for example, Current Protocols in Molecular Biology, John Wiley and

Sons.

Real-time quantitative (PCR) can be conveniently accomplished using the commercially available Multi-Color Real Time PCR Detection System, available from Applied Biosystem.

Real-time Quantitative PCR Analysis of Bcl-2 mRNA Levels

The sample content of human Bcl-2 mRNA was quantified using the human Bcl-2 ABI Prism

Pre-Developed TaqMan Assay Reagents (Applied Biosystems cat. no. Hs00608023_m1 according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA quantity or beta actin (ACTB) mRNA quantity was used as an endogenous control for normalizing any variance in sample preparation.

The sample content of human GAPDH mRNA was quantified using the human GAPDH ABI

Prism Pre-Developed TaqMan Assay Reagent (Applied Biosystems cat. no. 4310884E) according to the manufacturer's instructions.

The sample content of human beta actin mRNA was quantified using the human ACTB ABI

Prism Pre-Developed TaqMan Assay Reagent (Applied Biosystems cat. no. 4310681 E) according to the manufacturer's instructions.

Real-time Quantitative PCR is a technique well known in the art and is taught in for example Heid et al. Real time quantitative PCR, Genome Research (1996), 6: 986-994. Real time PCR: The cDNA from the first strand synthesis performed as described in example 5 was diluted 2-20 times, and analyzed by real time quantitative PCR using Taqman 7500 FAST or 7900 FAST from Applied Biosystems. The primers and probe were mixed with 2 x Taqman Fast Universal PCR master mix (2x) (Applied Biosystems Cat.# 4364103) and added to 4 μl cDNA to a final volume of 10 μl. Each sample was analysed in duplicate. Assaying 2 fold dilutions of a cDNA that had been prepared on material purified from a cell line expressing the RNA of interest generated standard curves for the assays. Sterile H₂O was used instead of cDNA for the no template control. PCR program: 60° C for 2 minutes, then 95° C for 30 seconds, followed by 40 cycles of 95° C, 3 seconds, 60° C, 20-30 seconds. Relative quantities of target mRNA sequence were determined from the calculated Threshold cycle using the Applied Biosystems Fast System SDS Software Version 1.3.1.21. or SDS Software Version 2.3.

Example 8: In vitro analysis: Antisense Inhibition of Human Bcl-2 Expression by oligonucleotide compounds Oligonucleotides presented in Table 3 were evaluated in the 518A2 cell line for their potential to knockdown of Bcl-2 at concentrations of 1 , 5 and 25 nM using lipid transfection (see Figure 1 ). Six oligonucleotides (SEQ ID NO: 64, 69, 72, 75, 79, 88) were selected and evaluated in the 15PC3 cell line for their potential to knockdown Bcl-2 at a wider range of concentration of 0.04, 0.2, 1 , 5, 12.5, and 25 nM using lipid transfection (see Figure 2). Table 3: Antisense Inhibition of Human Bcl-2 expression by oligonucleotides. The data in Table 3 are presented as percentage down-regulation relative to mock transfected cells at 25 nM in 518A2 cells and at 1 nM in 15PC3 cells. Lower case letters represent DNA units, bold upper case letters represent, LNA preferably β-D-oxy-LNA units. All LNA C are preferably 5'methyl C. Subscript "s" represents phosphorothioate linkage.

As shown in Table 3, oligonucleotides of SEQ ID NOs: 60, 61 , 64, 67, 68, 69, 72, 75, 79, 85, 87, and 88 demonstrated about 84% or greater inhibition of Bcl-2 expression in these experiments and are therefore preferred.

Also preferred are oligonucleotides based on the illustrated antisense oligo sequences, for example varying the length (shorter or longer) and/or nucleobase content (e.g. the type and/or proportion of analogue units), which also provide good inhibition of Bcl-2 expression. Example 9: In vitro analysis: Apoptosis induction by LNA antisense oligomeric compounds using lipid transfection. Six oligonucleotides (SEQ ID NOs: 64, 69, 72, 75, 79, and 88) were selected and evaluated in the 15PC3 cell line for their potential to induce apoptosis. Cells were seeded to a density of 6, 000 cells per well in white 96 well plate (Nunc 136101 ) in DMEM the day prior to transfection. The next day cells were washed once in prewarmed OptiMEM followed by addition of 72 μl OptiMEM containing 5 μg/ml Lipofectamine2000 (In vitrogen). Cells were incubated for 7 min before adding 18 μl oligonucleotides diluted in OptiMEM. The final oligonucleotide concentration ranged from 0.04 nM to 25 nM. After 4 h of treatment, cells were washed in OptiMEM and 50 μl DMEM containing serum was added. Following treatment with the oligomeric compound, cells were allowed to recover for the period indicated before they were removed from the CO₂ incubator and equilibrated to room temperature for 15 min. An equal volume of highly sensitive Caspase 3/7-Glo™ Reagent (Promega) was added directly to the cells in 96 wells, and plates were incubated for 60 min before recording luminescence (luciferase activity) in Luminoskan Ascent instrument from Thermo Labsystems after further 1 min lag period. The luciferase activity is measured as Relative Light Units per seconds (RLU/s). The data were processed in the Ascent software 2.6 and graphs were drawn in excel. (See Figure 3). Example 10: In vitro model: Natural uptake of antisense oligonucleotide

The cell line, 15PC3, listed in example 4 was incubated with oligo dissolved in sterile water without any transfection vehicle. Cells were seeded in 6-well cell culture plates (NUNC) and incubated with oligo when 10-30% confluent. Oligo concentrations used ranged from 1 μM to 50μM, final concentration. Cells were incubated at 37⁰C in the oligo containing normal growth serum for 1 to 15 days before they were harvested for RNA analysis.

As shown in Table 4, oligonucleotides of SEQ ID NOs: 61 , 62, 63, 64, 67, 68, 69, 72, 75, 85, 91 and 93 demonstrated about 85% or greater inhibition of Bcl-2 expression in these experiments and are therefore preferred. Also preferred are oligonucleotides based on the illustrated antisense oligo sequences, for example varying the length (shorter or longer) and/or nucleobase content (e.g. the type and/or proportion of analogue units), which also provide good inhibition of Bcl-2 expression. See Figure 7).

Example 11: In vivo screen of antisense oligonucleotides

The antisense oligonucleotides were screened in vivo at a dose of 10mg/kg three times weekly for a total of 7 doses or 25mg/kg daily for three consecutive days. The animals were dosed with 10 ml per kg body weight i.v. of the antisense oligonucleotide compounds formulated in the vehicle or vehicle alone. Liver tissue was harvested 24 or 48 hours after the last dose for RNA analysis. The sample content of murine Bcl-2 mRNA was quantified using the murine Bcl-2 ABI Prism Pre-Developed TaqMan Assay Reagents (Applied Biosystems cat. no. Mm00477631_m1 ) according to the manufacturer's instructions. The sample content of murine b-actin mRNA was quantified using the murine ACTB ABI Prism Pre-Developed TaqMan Assay Reagents (Applied Biosystems cat. no. 4352341 E) according to the manufacturer's instructions

The sample content of murine GAPDH mRNA was quantified using the murine GAPDH ABI Prism Pre-Developed TaqMan Assay Reagents (Applied Biosystems cat. no. 435239E) according to the manufacturer's instructions

Example 12: Preparation of conjugates of oligomers with polyethylene glycol The oligomers having sequences shown as SEQ ID NO: 68, 61 and 62 are functionalized on the 5' terminus by attaching an aminoalkyl group, such as hexan-1 -amine blocked with a blocking group such as Fmoc to the 5' phosphate groups of the oligomers using routine phosphoramidite chemistry, oxidizing the resultant compounds, deprotecting them and purifying them to achieve the functionalized oligomers, respectively, having the formulas (IA) - (IC):

wherein the bold uppercase letters represent nucleoside analogue monomers, lowercase letters represent DNA monomers, the subscript "s" represents a phosphorothioate linkage, and ^MeC represents 5-methylcytosine. A solution of activated PEG, such as the one shown in formula (II):

(H) wherein the PEG moiety has an average molecular weight of 12,000, and each of the compounds of formulas (IA) and (IB) in PBS buffer are stirred in separate vessels at room temperature for 12 hours. The reaction solutions are extracted three times with methylene chloride and the combined organic layers are dried over magnesium sulphate and filtered and the solvent is evaporated under reduced pressure. The resulting residues are dissolved in double distilled water and loaded onto an anion exchange column. Unreacted PEG linker is eluted with water and the products are eluted with NH₄HCO₃ solution. Fractions containing pure products are pooled and lypophilized to yield the conjugates SEQ ID NOs: 68, 61 and 62, respectively as show in formulas (NIA) and (NIB):

wherein each of the oligomers of SEQ ID NOs: 68, 61 and 62 is attached to a PEG polymer having average molecular weight of 12,000 via a releasable linker. Chemical structures of PEG polymer conjugates that can be made with oligomers having sequences shown in SEQ ID NOs: 68, 61 and 62 using the process described above are respectively shown in formulas (IVA), (IVB) and (IVC):

Xissss/ H 6

wherein bold uppercase letters represent beta-D-oxy-LNA monomers, lowercase letters represent DNA monomers, the subscript "s" represents a phosphorothioate linkage and ^MeC represent 5-methylcytosine.

Activated oligomers that can be used in this process to respectively make the conjugates shown in formulas (IVA), (IVB) and (IVC) have the chemical structures shown in formulas (VA), (VB) and (VC):

Example 13: In vitro model: Combination of antisense oligonucleotides targeting Bcl-2 and antisense oligonucleotides targeting Mcl-1 on caspase 3/7 induction and cell viability One preferred Bcl-2 targeting oligo SEQ ID NO: 63 was combined with Mcl-1 targeting oligos SEQ ID NOs: 96 and 97 in 15PC3 cells using natural uptake for evaluation of caspase 3/7 induction and cell viability. The cells were treated with a final concentration of 5μM of each oligo giving a total final concentration of 10μM in each well. The combinations were compared to cells incubated with SEQ ID NO 63 plus scrambled control oligo, SEQ ID NO 96 plus scrambled control oligo, and SEQ ID NO 97 plus scrambled control oligo again with a final concentration of 5μM of each oligo.

SEQ ID NO 96 = G_sT_sA_sa_sg_sa_sc_sa_sa_sa_sc_sA_sG_sA,

SEQ ID NO 97 = 1^*0^^^^^^° C^

15PC3 cells were seeded at day 0 to a density of 5000 cells per well in 10Oμl of normal growth medium containing the oligonucleotide combinations without any transfection reagent in clear 96-well cell culture plates for cell viability measurement and in white luminometer 96- well cell culture plates for caspase 3/7 induction measurements. The cells were incubated at 37⁰C for 0, 1 , 2 and 3 days. At day 0, 1 , 2, and 3 cell viability was measured by adding 10 μl CellTiter 96® AQueous One Solution Reagent (CellTiter 96® AQueous One Solution Cell Proliferation, G3582, Promega) per well to the clear 96-well cell culture plates. The plates were incubated at room temperature for 1 hour before the absorbance was measured at 490nm and 650nm using a spectrophometer (Powerwave X, Bio-Tek Instruments) (see figure 10). When using a standard t-test on the results on day 3 the combination of SEQ ID NO 63 + SEQ ID NO 96 showed significantly (p < 0.05) less cell viability than SEQ ID NO 96 + scrambled control and significantly (p < 0.05) less cell viability than SEQ ID NO 63 + scrambled control giving an advantage of combining the Bcl-1 targeting oligo SEQ ID NO 63 with the Mcl-1 targeting oligo. The combination of SEQ ID NO 63 + SEQ ID NO 97 showed significantly (p < 0.05) less cell viability than SEQ ID NO 97 + scrambled control and significantly (p < 0.05) less cell viability than SEQ ID NO 63 + scrambled control giving an advantage of combining the Bcl-2 targeting oligo SEQ ID NO 63 with the Mcl-1 targeting oligo. At day 1 , 2, and 3 caspase 3/7 induction was measured by cooling of the plates to room temperature and then adding 100 μl Caspase 3/7-Glo™ Reagent (Promega) per well to the luminometer plates and the plates were incubated at room temperature for 60 minutes before recording luminescence (luciferase activity) in Luminoskan Ascent instrument from Thermo Labsystems after further 1 min lag period. The luciferase activity is measured as Relative Light Units per seconds (RLU/s). The data were processed in the Ascent software 2.6 and graphs were drawn in excel (see figure 11 ). For the caspase 3/7 activity the induction was compared to the scrambled control treated cells. When using a standard t-test on the results on day 3 the combination of SEQ ID NO 63 + SEQ ID NO 96 showed significantly (p < 0.05) higher caspase 3/7 induction than SEQ ID NO 63 + scrambled control and significantly (p < 0.05) higher caspase 3/7 induction than SEQ ID NO 96 + scrambled control giving an advantage of combining the Bcl-2 targeting oligo SEQ ID NO 63 with the Mcl-1 targeting oligo. The combination of SEQ ID NO 63 + SEQ ID NO 97 showed higher caspase 3/7 induction than SEQ ID NO 97 + scrambled control and significantly (p < 0.05) higher caspase 3/7 induction than SEQ ID NO 63 + scrambled control giving an advantage of combining the Bcl-2 targeting oligo SEQ ID NO 63 with the Mcl-1 targeting oligo.

Claims

1. An oligomer of between 10 - 30 nucleotides in length, capable of the down-regulating expression of a mammalian Bcl-2 gene, which comprises a contiguous nucleotide sequence of a total of between 10 - 30 nucleotides, wherein said contiguous nucleotide sequence is at least 90% homologous to a corresponding region of either; a. SEQ ID NOs 28 or 9; or b. SEQ ID NO 23 or SEQ ID NO 4; or c. SEQ ID NO 24 or 5, or any of SEQ ID NO 3 - 21 or 22 - 40.

2. The oligomer according to claim 1 , wherein the contiguous nucleotide sequence comprises no mismatches or no more than one mismatch with the reverse complement of a corresponding region of SEQ ID NO 1.

3. The oligomer according to any one of claims 1 - 2, wherein the nucleotide sequence of the oligomer consists of the contiguous nucleotide sequence.

4. The oligomer according to any one of claims 1 - 3, wherein the contiguous nucleotide sequence is between 10 - 18 nucleotides in length.

5. The oligomer according to any one of claims 1 - 4, wherein the contiguous nucleotide sequence comprises nucleotide analogues.

6. The oligomer according to claim 5, wherein the nucleotide analogues are sugar modified nucleotides, such as sugar modified nucleotides selected from the group consisting of: Locked Nucleic Acid (LNA) units; 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-amino-

DNA units, and 2'-fluoro-DNA units.

7. The oligomer according to claim 6, wherein the nucleotide analogues are LNA.

8. The oligomer according to any one of claims 5 - 7 which is a gapmer.

9. The oligomer according to any one of claims 1 - 8, which inhibits the expression of BCL- 2 gene or mRNA in a cell which is expressing BCL-2 gene or mRNA.

10. The olgimer according to any one of claims 1 - 9, wherein the oligomer consists or comprises an contiguous nucleotide sequence selected from τ_sA_sτ_st_sg_sg_sa_st_sg_st_sg_sc_st_sτ_sτ_sG (^{SEQ I D N0 68}); ^or

G_sA_sA_sc_sa_sc_st_st_sg_sa_st_st_sc_sT_sG_sG (SEQ ID NO 62); wherein uppercase letters denote LNA nucleotides such as beta-D-oxy-LNA nucleotides, and lowercase letters denote DNA monomers, the subscript "s" denotes a phosphorothioate linkage, and ^MeC denotes a LNA cytosine nucleotide, such as an LNA 5-methylcytosine nucleotide.

1 1. A conjugate comprising the oligomer according to any one of claims 1 - 10, and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said oligomer.

12. A pharmaceutical composition comprising the oligomer according to any one of claims 1 - 10, or the conjugate according to claim 11 , and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

13. The oligomer according to any one of claims 1 - 10, or the conjugate according to claim 1 1 , for use as a medicament, such as for the treatment of such as hyperproliferative diseases such as cancer.

14. The use of an oligomer according to any one of the claims 1 -10, or a conjugate according to claim 11 , for the manufacture of a medicament for the treatment of such as hyperproliferative diseases such as cancer.

15. A method of treating such as hyperproliferative diseases such as cancer, said method comprising administering an effective amount of an oligomer according to any one of the claims 1-10, or a conjugate according to claim 1 1 , or a pharmaceutical composition according to claim 12, to a patient suffering from, or likely to suffer from such as hyperproliferative diseases such as cancer.

16. A method for the inhibition of Bcl-2 in a cell, such as a cancer cell, which is expressing Bcl-2, said method comprising administering an oligomer according to any one of the claims 1-10, or a conjugate according to claim 1 1 to said cell so as to inhibit Bcl-2 in said cell, wherein said method is performed either in vivo or in vitro.

17. A method for the simultaneous inhibition of expression of both Bcl-2 and Mcl-1 in a cell, such as a cancer cell, which is expressing Bcl-2 and Mcl-1 , said method comprising a. administering an effective amount of a Bcl2 inhibitor, such as a oligomer which targets Bcl2, such as oligomer according to any one of the claims 1-10, or a conjugate thereof to said cell so as to inhibit Bcl-2 in said cell, b. administering an effective amount of a Mcl1 inhibitor, such as a oligomer which targets McH , or a conjugate thereof to said cell so as to inhibit McH in said cell, wherein steps a) and b) may be performed in any order or simultaneously and lead to the simultaneous inhibition (down-regulation) of both Mcl-1 and Bcl-2 inhibition in said cell; wherein said method is performed either in vivo or in vitro.