Antisense modulation of bcl-x expression

#The present invention provides compositions and methods for modulating the expression of bcl-x and for inducing apoptosis. In particular, this invention relates to antisense compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding human bcl-x. Such oligonucleotides have been shown to modulate the expression of bcl-x.

#Programmed cell death, or apoptosis, is an essential feature of growth and development, as the control of cell number is a balance between cell proliferation and cell death. Apoptosis is an active rather than a passive process, resulting in cell suicide as a result of any of a number of external or internal signals. Apoptotic cell death is characterized by nuclear condensation, endonucleolytic degradation of DNA at nucleosomal intervals (“laddering”) and plasma membrane blebbing. Programmed cell death plays an essential role in, for example, immune system development and nervous system development. In the former, T cells displaying autoreactive antigen receptors are removed by apoptosis. In the latter, a significant reshaping of neural structures occurs, partly through apoptosis.

#An increasing number of genes and gene products have been implicated in apoptosis. One of these is bcl-2, which is an intracellular membrane protein shown to block or delay apoptosis. Overexpression of bcl-2 has been shown to be related to hyperplasia, autoimmunity and resistance to apoptosis, including that induced by chemotherapy (Fang et al., J. Immunol. 1994, 153, 4388-4398). A family of bcl-2-related genes has been described. All bcl-2 family members share two highly conserved domains, BH1 and BH2. These family members include, but are not limited to, A-1, mcl-1, bax and bcl-x. Bcl-x was isolated using a bcl-2 cDNA probe at low stringency due to its sequence homology with bcl-2. Bcl-x was found to function as a bcl-2-independent regulator of apoptosis (Boise et al., Cell, 1993, 74, 597-608). Two isoforms of bcl-x were reported in humans. Bcl-xl (long) contains the highly conserved BH1 and BH2 domains. When transfected into an IL-3 dependent cell line, bcl-xl inhibited apoptosis during growth factor withdrawal in a manner similar to bcl-2. In contrast, the bcl-x short isoform, bcl-xs, which is produced by alternative splicing and lacks a 63-amino acid region of exon 1 containing the BH1 and BH2 domains, antagonizes the anti-apoptotic effect of either bcl-2 or bcl-xl. As numbered in Boise et al., Cell, 1993 74:, 597-608, the bcl-x transcript can be categorized into regions described by those of skill in the art as follows: nucleotides 1-134, 5′ untranslated region (5′-UTR); nucleotides 135-137, translation initiation codon (AUG); nucleotides 135-836, coding region, of which 135-509 are the shorter exon 1 of the bcl-xs transcript and 135-698 are the longer exon 1 of the bcl-xl transcript; nucleotides 699-836, exon 2; nucleotides 834-836, stop codon; and nucleotides 837-926, 3′ untranslated region (3′-UTR). Between exons 1 and 2 (between nucleotide 698 and 699) an intron is spliced out of the pre-mRNA when the mature bcl-xl (long) mRNA transcript is produced. An alternative splice from position 509 to position 699 produces the bcl-xs (short) mRNA transcript which is 189 nucleotides shorter than the long transcript, encoding a protein product (bcl-xs) which is 63 amino acids shorter than bcl-xl. Thus nucleotide position 698 is sometimes referred to in the art as the “5′ splice site” and position 509 as the “cryptic 5′ splice site,” with nucleotide 699 sometimes referred to as the “3′ splice site.”

#FIG. 1 is a graph which shows bcl-xl mRNA levels in the Shionogi mouse tumor model. Shionogi tumors were harvested before and at several times after castration. Poly A+ RNA was extracted from each tumor tissue and then analyzed for bcl-xl and G3PDH levels by Northern blotting followed by quantitation with a laser densitometer. Each column represents the mean value with standard deviation.

#FIG. 2 is a graph which shows bcl-xl mRNA levels after normalization to G3PDH mRNA levels in Shionogi tumor cells following treatment with various concentrations of antisense bcl-xl or mismatch control oligodeoxynucleotide. Each point represents the mean of triplicate analysis with standard deviation. *: significantly different from mismatch control ODN treatment: p<0.01 (Student's t-test).

#FIG. 3 is a graph which shows the effects of antisense bcl-xl oligodeoxynucleotide ISIS 16009 administration on Shionogi tumor growth. Beginning 1 day postcastration, 12.5 mg/kg ISIS 16009 or control ODN#1 was injected i.p. once daily for 40 days into each mouse bearing Shionogi tumors. Tumor volume was measured twice weekly and calculated by the formula length×width×depth×0.5236. Each point represents the mean tumor volume in each experimental group containing 7 mice with standard deviation. * and **: significantly different from control ODN#1 treatment; p<0.05 and 0.01, respectively (Student's t-test).

#The present invention comprehends antisense compounds capable of modulating expression of human bcl-x and of its isoforms, bcl-xl and bcl-xs. Bcl-xl inhibits apoptosis and therefore inhibitors of bcl-xl, particularly specific inhibitors of bcl-xl such as the antisense compounds of the present invention, are desired as promoters of apoptosis. In contrast, the bcl-x short isoform, bcl-xs, antagonizes the anti-apoptotic effect and therefore promotes apoptosis. Inhibitors of bcl-xs are desired as inhibitors of apoptosis. Antisense compounds which specifically inhibit the expression of a particular isoform, either bcl-xl or bcl-xs, of bcl-x, or which alter the expression ratio of these two isoforms, are particularly useful for both research and therapeutic, including prophylactic, uses.

#The present invention employs oligomeric antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding bcl-x, ultimately modulating the amount of bcl-x produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding bcl-x. As used herein, the terms “target nucleic acid” and “nucleic acid encoding bcl-x” encompass DNA encoding bcl-x, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of bcl-x. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene product. In the context of the present invention, inhibition is a preferred form of modulation of gene expression and mRNA is a preferred target. Further, since many genes (including bcl-x) have multiple transcripts, “modulation” also includes an alteration in the ratio between gene products, such as alteration of mRNA splice products.

#It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding bcl-x. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding bcl-x, regardless of the sequence(s) of such codons.