Newly synthesized eukaryotic mRNA molecules, known as primary transcripts or pre-mRNA, made in the nucleus, are processed before or during transport to the cytoplasm for translation. A methylated cap structure, consisting of a terminal nucleotide, 7-methylguanylate, is added to the 5' end of the mRNA in a 5'--5' linkage with the first nucleotide of the mRNA sequence.
An approximately 200-250-base sequence of adenylate residues, referred to as poly(A), is added posttranscriptionally to a site that will become the 3' terminus of the mRNA, before entry of the mRNA into the cytoplasm. This is a multistep process which involves assembly of a processing complex, then site-specific endonucleolytic cleavage of the precursor transcript, and addition of a poly(A) "tail." In most mRNAs the polyadenylation signal sequence is a hexamer, AAUAAA, located 10 to 30 nucleotides in the 5' direction (upstream) from the site of cleavage (5'-CA-3') in combination with a U or G-U rich element 3' to the cleavage site. Multiple poly(A) sites may be present on a given transcript, of which only one is used per transcript, but more than one species of mature mRNA transcript can be produced from a given pre-mRNA via use of different poly(A) sites. It has recently been shown that stable mRNA secondary structure can affect the site of polyadenylation of an RNA construct in transfected cells. Klasens et al., Nuc. Acids Res., 1998, 26, 1870-1876. It has also been found that which of multiple polyadenylation sites is used can affect transcript stability. Chu et al., J. Immunol., 1994, 153, 4179-4189. Antisense modulation of mRNA polyadenylation has not previously been reported.
The next step in mRNA processing is splicing of the mRNA, which occurs in the maturation of 90-95% of mammalian mRNAs. Introns (or intervening sequences) are regions of a primary transcript (or the DNA encoding it) that are not included in a finished mRNA. Exons are regions of a primary transcript that remain in the mature mRNA when it reaches the cytoplasm. The exons are "spliced" together to form the mature mRNA sequence. Intron-exon junctions are also referred to as "splice sites" with the 5' side of the junction often called the "5' splice site," or "splice donor site" and the 3' side the "3' splice site" or "splice acceptor site." "Cryptic" splice sites are those which are less often used but may be used when the "usual" splice site is blocked or unavailable." Alternative splicing, i.e., the use of various combinations of exons, often results in multiple mRNA transcripts from a single gene.
A final step in RNA processing is turnover or degradation of the mRNA. Differential mRNA stabilization is one of several factors in the rate of synthesis of any protein. mRNA degradation rates seem to be related to presence or absence of poly(A) tails and also to the presence of certain sequences in the 3' end of the mRNA. For example, many mRNAs with short half-lives contain several A(U).sub.n A sequences in their 3'-untranslated regions. When a series of AUUUA sequences was inserted into a gene not normally containing them, the half life of the resulting mRNA decreased by 80%. Shaw and Kamen, Cell, 1986, 46, 659. This may be related to an increase of nucleolytic attack in sequences containing these A(U).sub.n A sequences. Other mediators of mRNA stability are also known, such as hormones, translation products (autoregulation/feedback), and low-molecular weight ligands.
Antisense compounds have generally been used to interfere with protein expression, either by interfering directly with translation of the target molecule or, more often, by RNAse-H-mediated degradation of the target mRNA. Antisense interference with 5' capping of mRNA and prevention of translation factor binding to the mRNA by oligonucleotide masking of the 5' cap have been disclosed by Baker et al. (WO 91/17755).
Antisense oligonucleotides have been used to modulate splicing, particularly aberrant splicing or splicing of mutant transcripts, often in cell-free reporter systems. A luciferase reporter plasmid system has been used to test the ability of antisense oligonucleotides targeted to the 5' splice site, 3' splice site or branchpoint to inhibit splicing of mutated or wild-type adenovirus pre-mRNA sequences in a reporter plasmid. Phosphorothioate oligodeoxynucleotides that can support RNAse H cleavage were found to be better inhibitors of expression of the wild-type adenovirus construct than the 2'-methoxy phosphorothioates that cannot support RNase H, although the reverse was true for oligonucleotides targeted to an adenovirus construct containing human .beta.-globin splice site sequences. Hodges and Crooke, Mol. Pharmacol., 1995, 48, 905-918.
Antisense oligonucleotides have been used to target mutations that lead to aberrant splicing in several genetic diseases. Use of antisense compounds to correct aberrant processing of mutated mRNA sequences is not comprehended by the present invention. Altering, i.e., controlling, the behavior of a cell, particularly the response of a cell to a stimulus, by antisense modulation of "wild-type" or native mRNA processing, the subject of the present invention, has not been described previously.
Phosphorothioate 2'-O-methyl oligoribonucleotides, have been used to target the aberrant 5' splice site of the mutant .beta.-globin gene found in patients with .beta.-thalassemia, a genetic blood disorder. Aberrant splicing of mutant .beta.-globin mRNA was blocked in vitro in vector constructs containing thalassemic human .beta.-globin pre-mRNAs using 2'-O-methyl-ribo-oligonucleotides targeted to the branch point sequence in the first intron of the mutant human .beta.-globin pre mRNAs. 2'-O-methyl oligonucleotides are used because they are stable to RNAses and form stable hybrids with RNA that are not degraded by RNAse H. Dominski and Kole, Proc. Natl. Acad. Sci. USA, 1993, 90, 8673-8677. A review article by Kole discusses use of antisense oligonucleotides targeted to aberrant splice sites created by genetic mutations such as .beta.-thalassemia or cystic fibrosis. It was hypothesized that blocking a splice site with an antisense oligonucleotide will have similar effect to mutation of the splice site, i.e., redirection of splicing. Kole, Acta Biochimica Polonica, 1997, 44, 231-238. Oligonucleotides targeted to the aberrant .beta.-globin splice site suppressed aberrant splicing and at least partially restored correct splicing in HeLa cells expressing the mutant transcript. Sierakowska et al., Nucleosides & Nucleotides, 1997, 16,1173-1182; Sierakowska et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 12840-44. U.S. Pat. No. 5,627,274 discloses and WO 94/26887 discloses and claims compositions and methods for combating aberrant splicing in a pre-mRNA molecule containing a mutation, using antisense oligonucleotides which do not activate RNAse H.
Modulation of mutant dystrophin splicing with 2'-O-methyl oligoribonucleotides has been reported both in vitro and in vivo. In dystrophin Kobe, a 52-base pair deletion mutation causes exon 19 to be skipped during splicing. An in vitro minigene splicing system was used to show that a 31-mer 2'-O-methyl oligoribonucleotide complementary to the 5' half of the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mRNA. Takeshima et al., J. Clin. Invest., 1995, 95, 515-520. The same oligonucleotide was used to induce exon skipping from the native dystrophin gene transcript in human cultured lymphoblastoid cells.
Dunckley et al., (Nucleosides & Nucleotides, 1997, 16, 1665-1668) describes in vitro constructs for analysis of splicing around exon 23 of mutated dystrophin in the mdx mouse mutant, a model for Duchenne muscular dystrophy. Plans to analyze these constructs in vitro using 2' modified oligos targeted to splice sites within and adjacent to mouse dystrophin exon 23 are discussed, though no target sites or sequences are given.
2'-O-methyl oligoribonucleotides were subsequently used to correct dystrophin deficiency in myoblasts from the mdx mouse. An antisense oligonucleotide targeted to the 3' splice site of murine dystrophin intron 22 caused skipping of the mutant exon and created a novel in-frame dystrophin transcript with a novel internal deletion. This mutated dystrophin was expressed in 1-2% of antisense treated mdx myotubes. Use of other oligonucleotide modifications such as 2'-O-methoxyethyl phosphodiesters are disclosed. Dunckley et al. (Human Mol. Genetics, 1998, 5, 1083-90).
Phosphorothioate oligodeoxynucleotides have been used to selectively suppress the expression of a mutant .alpha.2(I) collagen allele in fibroblasts from a patient with osteogenesis imperfecta, in which a point mutation in the splice donor site produces mRNA with exon 16 deleted. The oligonucleotides were targeted either to the point mutation in the pre-mRNA or to the defectively spliced transcript. In both cases mutant mRNA was decreased by half but the normal transcript is also decreased by 20%. This was concluded to be fully accounted for by an RNAse H-dependent mechanism. Wang and Marini, J. Clin Invest., 1996, 97, 448-454.
A microinjection assay was used to test the antisense effects on SV40 large T antigen (TAg) expression of oligonucleotides containing C-5 propynylpyrimidines, either as 2'-O-allyl phosphodiester oligonucleotides, which do not elicit RNAse H cleavage of the target, or as 2'-deoxy phosphorothioates, which do elicit RNAse H cleavage. Oligonucleotides targeted to the 5' untranslated region, translation initiation site, 5' splice junction or polyadenylation signal of the TAg transcript were injected into the nucleus or cytoplasm of cultured cells. The only 2'-O-allyl (non-RNAse H) oligonucleotides which were effective at inhibiting T-antigen were those targeted to the 5' untranslated region and the 5' splice junction. The 2'-O-allyl phosphodiester/C-5 propynylpyrimidine oligonucleotides, which do not elicit RNAse H, were 20 fold less potent than the oligodeoxynucleotides which had the ability to recruit RNAse H. The authors concluded that the duplexes formed between the RNA target and the 2'-O-allyl phosphodiester/C-5 propynylpyrimidine oligonucleotides dissociate rapidly in cells. Moulds et al., 1995, Biochem., 34, 5044-53. Biotinylated 2'-O-allyloligoribonucleotides incorporating 2-aminoadenine bases were targeted to the U2 small nuclear RNA (snRNA), a component of the spliceosome, in HeLa nuclear extracts. These inhibited mRNA production with a concomitant accumulation of splicing intermediates. The present invention is directed to antisense compounds targeted to mRNA.
Use of antisense compounds to block or regulate mRNA polyadenylation has not previously been described. Regulation of mRNA stability using antisense oligonucleotides targeted to RNA sequences involved in RNA turnover or degradation has also not been previously described.
There is, therefore a continued need for compositions and methods for altering the behavior of a cell, particularly the response of a cell to a stimulus, by modulation of normal mRNA processing. The present invention provides antisense compounds for such modulation. The compositions and methods of the invention can be used in therapeutics, including prophylaxis, and as research tools.