MODULATION OF APOLIPOPROTEIN (a) EXPRESSION

Abstract

Compounds, compositions and methods are provided for modulating the expression of apolipoprotein(a). The compositions comprise oligonucleotides, targeted to nucleic acid encoding apolipoprotein(a). Methods of using these compounds for modulation of apolipoprotein(a) expression and for diagnosis and treatment of disease associated with expression of apolipoprotein(a) are provided.

Description

SEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled ISPH0595USC6SEQ_ST25.txt, created on Mar. 11, 2015 which is 84 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] The present invention provides compositions and methods for modulating the expression of Apolipoprotein(a).

[0003] Lipoproteins are globular, micelle-like particles that consist of a non-polar core of acylglycerols and cholesteryl esters, surrounded by an amphiphilic coating consisting of protein, phospholipid and cholesterol. Lipoproteins have been classified into five broad categories on the basis of their functional and physical properties: chylomicrons (which transport dietary lipids from intestine to tissues), very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), (all of which transport triacylglycerols and cholesterol from the liver to tissues), and high density lipoproteins (HDL) (which transport endogenous cholesterol from tissues to the liver).

[0004] Lipoprotein particles undergo continuous metabolic processing and have variable properties and compositions. Lipoprotein densities increase without decreasing particle diameter because the density of their outer coatings is less than that of the inner core. The protein components of lipoproteins are known as apolipoproteins. At least nine apolipoproteins are distributed in significant amounts among the various human lipoproteins.

[0005] Lipoprotein(a) (also known as Lp(a)) is a cholesterol rich particle of the pro-atherogenic LDL class. Since Lp(a) is found only in Old World primates and European hedgehogs, it has been suggested that it does not play an essential role in lipid and lipoprotein metabolism. Most studies have shown that high concentrations of Lp(a) are strongly associated with increased risk of cardiovascular disease (Rainwater and Kammerer, J. Exp. Zool., 1998, 282, 54-61). These observations have stimulated numerous studies in humans and other primates to investigate the factors that control Lp(a) concentrations and physiological properties (Rainwater and Kammerer, J. Exp. Zool., 1998, 282, 54-61).

[0006] Lp(a) contains two disulfide-linked distinct proteins, apolipoprotein(a) (or ApoA) and apolipoprotein B (or ApoB) (Rainwater and Kammerer, J. Exp. Zool., 1998, 282, 54-61). Apolipoprotein(a) is a unique apolipoprotein encoded by the LPA gene which has been shown to exclusively control the physiological concentrations of Lp(a) (Rainwater and Kammerer, J. Exp. Zool., 1998, 282, 54-61). It varies in size due to interallelic differences in the number of tandemly repeated Kringle 4-encoding 5.5 kb sequences in the LPA gene (Rainwater and Kammerer, J. Exp. Zool., 1998, 282, 54-61).

[0007] Cloning of human apolipoprotein(a) in 1987 revealed homology to human plasminogen (McLean et al., Nature, 1987, 330, 132-137). The gene locus LPA encoding apolipoprotein(a) was localized to chromosome 6q26-27, in close proximity to the homologous gene for plasminogen (Frank et al., Hum. Genet., 1988, 79, 352-356).

[0008] Transgenic mice expressing human Apolipoprotein(a) were found to be more susceptible than control mice to the development of lipid-staining lesions in the aorta. Consequently, apolipoprotein(a) is co-localized with lipid deposition in the artery walls (Lawn et al., Nature, 1992, 360, 670-672). As an extension of these studies, it was established that the major in vivo action of apolipoprotein(a) is inhibition of conversion of plasminogen to plasmin which causes decreased activation of latent transforming growth factor-beta. Since transforming growth factor-beta is a negative regulator of smooth muscle cell migration and proliferation, inhibition of plasminogen activation indicates a possible mechanism for apolipoprotein(a) induction of atherosclerotic lesions (Grainger et al., Nature, 1994, 370, 460-462).

[0009] Elevated plasma levels of Lp(a), caused by increased expression of apolipoprotein(a), are associated with increased risk for atherosclerosis and its manifestations, which include hypercholesterolemia (Seed et al., N. Engl. J. Med., 1990, 322, 1494-1499), myocardial infarction (Sandkamp et al., Clin. Chem., 1990, 36, 20-23), and thrombosis (Nowak-Gottl et al., Pediatrics, 1997, 99, E11).

[0010] Moreover, the plasma concentration of Lp(a) is strongly influenced by heritable factors and is refractory to most drug and dietary manipulation (Katan and Beynen, Am. J. Epidemiol., 1987, 125, 387-399; Vessby et al., Atherosclerosis, 1982, 44, 61-71.). Pharmacologic therapy of elevated Lp(a) levels has been only modestly successful and apheresis remains the most effective therapeutic modality (Hajjar and Nachman, Annu. Rev. Med., 1996, 47, 423-442).

[0011] Morishita et al. have reported the use of three ribozyme oligonucleotides against apolipoprotein(a) for inhibition of apolipoprotein(a) expression in HepG2 cells (Morishita et al., Circulation, 1998, 98, 1898-1904).

[0012] U.S. Pat. No. 5,721,138 refers to nucleotide sequences encoding the human apolipoprotein(a) gene 5'-regulatory region and isolated nucleotide sequences comprising at least thirty consecutive complementary nucleotides from human apolipoprotein(a) from nucleotide position -208 to -1448 (Lawn, 1998).

[0013] To date, investigative and therapeutic strategies aimed at inhibiting apolipoprotein(a) function have involved the previously cited use of Lp(a) apheresis and ribozyme oligonucleotides. Currently no existing drugs are available to specifically lower lipoprotein(a) levels in humans, and limited models exist in which to perform drug discovery. Consequently, there remains a long-felt need for additional agents and methods capable of effectively modulating, e.g., inhibiting, apolipoprotein(a) function, and particularly a need for agents capable of safe and efficacious administration to lower alipoprotein(a) levels in patients at risk for the development of coronary artery disease.

SUMMARY OF THE INVENTION

[0014] The present invention provides compositions and methods for modulating the expression of apolipoprotein(a). Such novel compositions and methods enable research into the pathways of plasminogen and apolipoprotein(a), as well as other lipid metabolic processes. Such novel compositions and methods are useful in assessing the toxicity of chemical and pharmaceutical compounds on apolipoprotein(a) function, plasminogen or other lipid metabolic processes. Such novel compositions and methods are useful for drug discovery for the treatment of cardiovascular conditions, including myocardial infarction and atherosclerosis, among others.

[0015] In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules or sequences encoding apolipoprotein(a). Such compounds are shown herein to modulate the expression of apolipoprotein(a). Additionally disclosed are embodiments of oligonucleotide compounds that hybridize with nucleic acid molecules encoding apolipoprotein(a) in preference to nucleic acid molecules or sequences encoding plasminogen.

[0016] The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding apolipoprotein(a), and which modulate the expression of apolipoprotein(a). Pharmaceutical and other compositions comprising the compounds of the invention are also provided.

[0017] Further provided are methods of screening for modulators of apolipoprotein(a) and methods of modulating the expression of apolipoprotein(a) in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of apolipoprotein(a) are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

A. Overview of the Invention

[0018] The present invention employs compounds, preferably oligonucleotides and similar species, for use in modulating the function or effect of nucleic acid molecules encoding apolipoprotein(a). This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding apolipoprotein(a). As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding apolipoprotein(a)" have been used for convenience to encompass DNA encoding apolipoprotein(a), RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as "antisense". Antisense technology is emerging as an effective means of reducing the expression of specific gene products and is uniquely useful in a number of therapeutic, diagnostic and research applications involving modulation of Apolipoprotein(a) expression.

[0019] Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as "antisense inhibition." Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

[0020] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of apolipoprotein(a). In the context of the present invention, "modulation" and "modulation of expression" mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

[0021] In the context of this invention, "hybridization" means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0022] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired. Such conditions include, i.e., physiological conditions in the case of in vivo assays or therapeutic treatment, and conditions in which assays are performed in the case of in vitro assays.

[0023] In the present invention the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. In the context of this invention, "stringent conditions" under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.

[0024] "Complementary," as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases that can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

[0025] The sequence of an antisense compound can be, but need not necessarily be, 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event. In one embodiment of this invention, the antisense compounds of the present invention comprise at least 70%, or at least 75%, or at least 80%, or at least 85% sequence complementarity to a target region within the target nucleic acid. In other embodiments, the antisense compounds of the present invention comprise at least 90% sequence complementarity and even comprise at least 95% or at least 99% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0026] Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some preferred embodiments, homology, sequence identity or complementarity, between the oligomeric and target is between about 50% to about 60%. In some embodiments, homology, sequence identity or complementarity, is between about 60% to about 70%. In other embodiments, homology, sequence identity or complementarity, is between about 70% and about 80%. In still other embodiments, homology, sequence identity or complementarity, is between about 80% and about 90%. In yet other embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.

B. Compounds of the Invention

[0027] According to the present invention, "compounds" include antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds. Specifically excluded from the definition of "compounds" herein are ribozymes that contain internal or external "bulges" that do not hybridize to the target sequence. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.

[0028] One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

[0029] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0030] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs that are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).

[0031] The oligonucleotides of the present invention also include modified oligonucleotides in which a different base is present at one or more of the nucleotide positions in the oligonucleotide. For example, if the first nucleotide is an adenosine, modified oligonucleotides may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the oligonucleotide. These oligonucleotides are then tested using the methods described herein to determine their ability to inhibit expression of apolipoprotein(a) mRNA.

[0032] In the context of this invention, the term "oligomeric compound" refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0033] While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to, oligonucleotide analogs and mimetics such as those described herein.

[0034] The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

[0035] In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0036] In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0037] In one embodiment, compounds of this invention are oligonucleotides from about 12 to about 50 nucleobases. In another embodiment, compounds of this invention comprise from about 15 to about 30 nucleobases.

[0038] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0039] Exemplary antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5'-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5'-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly exemplary antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3'-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3'-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases).

[0040] Exemplary compounds of this invention may be found identified in the Examples and listed in Table 1. In addition to oligonucleotide compounds that bind to target sequences of apolipoprotein(a) in general, there are also exemplified oligonucleotide compounds of this invention that bind to target nucleotide sequences of apolipoprotein(a), but do not bind to, or do not bind preferentially to, sequences of plasminogen due to lack of homology between the two nucleic acid molecules or sufficient number of mismatches in the target sequences. These latter compounds are also useful in various therapeutic methods of this invention. Examples of antisense compounds to such `mismatched` target sequences as described above include SEQ ID NO: 12 and SEQ ID NO: 23 of Table I below. See, also, the discussion of target regions below.

[0041] One having skill in the art armed with the exemplary antisense compounds illustrated herein will be able, without undue experimentation, to identify further useful antisense compounds.

C. Targets of the Invention

[0042] "Targeting" an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid 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 nucleic acid encodes apolipoprotein(a).

[0043] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term "region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. "Segments" are defined as smaller or sub-portions of regions within a target nucleic acid. "Sites," as used in the present invention, are defined as positions within a target nucleic acid.

[0044] 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 has 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). 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 transcribed from a gene encoding apolipoprotein(a), regardless of the sequence(s) of such codons. A translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively).

[0045] The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon. Consequently, the "start codon region" (or "translation initiation codon region") and the "stop codon region" (or "translation termination codon region") are all regions that may be targeted effectively with the antisense compounds of the present invention.

[0046] The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0047] Another target region includes the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene). Still another target region includes the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA (or corresponding nucleotides on the gene). The 5' cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. Another target region for this invention is the 5' cap region.

[0048] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. In one embodiment, targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, is particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. An aberrant fusion junction due to rearrangement or deletion is another embodiment of a target site. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". Introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0049] Alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants". More specifically, "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

[0050] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants". If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0051] Variants can be produced through the use of alternative signals to start or stop transcription. Pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also embodiments of target nucleic acids.

[0052] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as "preferred target segments." As used herein the term "preferred target segment" is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid that are accessible for hybridization.

[0053] While the specific sequences of certain exemplary target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional target segments are readily identifiable by one having ordinary skill in the art in view of this disclosure.

[0054] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0055] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5'-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3'-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[0056] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0057] In various embodiments of this invention, the oligomeric compounds are targeted to regions of the target apollipoprotein(a) nucleobase sequence (e.g., such as those disclosed in Example 13) comprising nucleobases 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3751-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4751-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, or 4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300, 7301-7350, 7351-7400, 7401-7450, 7451-7500, 7501-7550, 7551-7600, 7601-7650, 7651-7700, 7701-7750, 7751-7800, 7801-7850, 7851-7900, 7901-7950, 7951-8000, 8001-8050, 8051-8100, 8101-8150, 8151-8200, 8201-8250, 8251-8300, 8301-8350, 8351-8400, 8401-8450, 8451-8500, 8501-8550, 8551-8600, 8601-8650, 8651-8700, 8701-8750, 8751-8800, 8801-8850, 8851-8900, 8901-8950, 8951-9000, 9001-9050, 9051-9100, 9101-9150, 9151-9200, 9201-9250, 9251-9300, 9301-9350, 9351-9400, 9401-9450, 9451-9500, 9501-9550, 9551-9600, 9601-9650, 9651-9700, 9701-9750, 9751-9800, 9801-9850, 9851-9900, 9901-9950, 9951-10000, 10001-10050, 10051-10100, 10101-10150, 10151-10200, 10201-10250, 10251-10300, 10301-10350, 10351-10400, 10401-10450, 10451-10500, 10501-10550, 10551-10600, 10601-10650, 10651-10700, 10701-10750, 10751-10800, 10801-10850, 10851-10900, 10901-10950, 10951-11000, 11001-11050, 11051-11100, 11101-11150, 11151-11200, 11201-11250, 11251-11300, 11301-11350, 11351-11400, 11401-11450, 11451-11500, 11501-11550, 11551-11600, 11601-11650, 11651-11700, 11701-11750, 11751-11800, 11801-11850, 11851-11900, 11901-11950, 11951-12000, 12001-12050, 12051-12100, 12101-12150, 12151-12200, 12201-12250, 12251-12300, 12301-12350, 12351-12400, 12401-12450, 12451-12500, 12501-12550, 12551-12600, 12601-12650, 12651-12700, 12701-12750, 12751-12800, 12801-12850, 12851-12900, 12901-12950, 12951-13000, 13001-13050, 13051-13100, 13101-13150, 13151-13200, 13201-13250, 13251-13300, 13301-13350, 13351-13400, 13401-13450, 13451-13500, 13501-13550, 13551-13600, 13601-13650, 13651-13700, 13701-13750, 13751-13800, 13801-13850, 13851-13900, 13901-13938, of apolipoprotein(a) or any combination thereof.

[0058] In one embodiment, the oligonucleotide compounds of this invention are 100% complementary to these sequences or to small sequences found within each of the above listed sequences. In another embodiment the oligonucleotide compounds have from at least 3 or 5 mismatches per 20 consecutive nucleobases in individual nucleobase positions to these target regions. Still other compounds of the invention are targeted to overlapping regions of the above-identified portions of the apolipoprotein(a) sequence.

[0059] In still another embodiment, target regions include those portions of the apolipoprotion(a) sequence that do not overlap with plasminogen sequences. For example, among such apolipoprotein(a) target sequences are included those found within the following nucleobase sequences: 10624-10702, 10963-11036, 11325-11354, 11615-11716, 11985-12038, 12319-12379, 13487-13491, and 13833-13871. As a further example, target sequences of apolipoprotein(a) that have at least 6 mismatches with the sequence of pla sminogen over at least 20 consecutive nucleotides are desirable targets for antisense compounds that bind preferentially to apolipoprotein(a) rather than to plasminogen. Such target sequences can readily be identified by a BLAST comparison of the two GenBank sequences of plasminogen (e.g., GenBank Accession No. NM 000301) and apolipoprotein(a)(e.g., GenBank Accession No. NM 005577.1).

D. Screening and Target Validation

[0060] In a further embodiment, the "preferred target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of apolipoprotein(a). "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding apolipoprotein(a) and which comprise at least an 8-nucleobase portion that is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding apolipoprotein(a) with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding apolipoprotein(a). Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding apolipoprotein(a), the modulator may then be employed in further investigative studies of the function of apolipoprotein(a), or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0061] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0062] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).

[0063] The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between apolipoprotein(a) and a disease state, phenotype, or condition. These methods include detecting or modulating apolipoprotein(a) comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of apolipoprotein(a) and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0064] The compounds of the present invention can be utilized for diagnostics, therapeutics, and prophylaxis, and as research reagents and components of kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[0065] For use in kits and diagnostics and in various biological systems, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, are useful as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0066] As used herein the term "biological system" or "system" is defined as any organism, cell, cell culture or tissue that expresses, or is made competant to express products of the LPA gene. These include, but are not limited to, humans, transgenic animals, cells, cell cultures, tissues, xenografts, transplants and combinations thereof.

[0067] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.

[0068] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000 480, 17-24; Celis, et al., FEBS Lett., 2000 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0069] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding apolipoprotein(a). For example, oligonucleotides that hybridize with such efficiency and under such conditions as disclosed herein as to be effective apolipoprotein(a) inhibitors are effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding apolipoprotein(a) and in the amplification of said nucleic acid molecules for detection or for use in further studies of apolipoprotein(a). Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding apolipoprotein(a) can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide, or any other suitable detection means. Kits using such detection means for detecting the level of apolipoprotein(a) in a sample may also be prepared.

[0070] The specificity and sensitivity of antisense are also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0071] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of apolipoprotein(a) is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a apolipoprotein(a) inhibitor. The apolipoprotein(a) inhibitors of the present invention effectively inhibit the activity of the apolipoprotein(a) protein or inhibit the expression of the apolipoprotein(a) protein. In one embodiment, the activity or expression of apolipoprotein(a) in an animal is inhibited by about 10%. Preferably, the activity or expression of apolipoprotein(a) in an animal is inhibited by about 30%. More preferably, the activity or expression of apolipoprotein(a) in an animal is inhibited by 50% or more. Thus, the oligomeric compounds modulate expression of apolipoprotein(a) mRNA by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%.

[0072] For example, the reduction of the expression of apolipoprotein(a) may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding apolipoprotein(a) protein and/or the apolipoprotein(a) protein itself. For example, apolipoprotein(a) is produced in the liver, and can be found in normal and atherosclerotic vessel walls.

[0073] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

F. Modifications

[0074] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

[0075] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0076] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0077] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0078] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts.

[0079] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

[0080] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0081] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH.sub.2--NH--O--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene (methylimino) or MMI backbone], --CH.sub.2--O--N(CH.sub.3)--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and --O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native phosphodiester backbone is represented as --O--P--O--CH.sub.2] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified Sugars

[0082] Modified oligonucleotides may also contain one or more substituted sugar moieties.

[0083] Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O--, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3].sub.2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2, also described in examples hereinbelow.

[0084] Other preferred modifications include 2'-methoxy (2'-O--CH.sub.3), 2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl (2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl (2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0085] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (--CH.sub.2--).sub.n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in published International Patent Application Nos. WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

[0086] Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (--C.ident.C--CH.sub.3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0087] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

Conjugates

[0088] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenan-thridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosures of which are

incorporated herein by reference. Conjugate moieties include, but are not limited to, 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 phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodo-benzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999), which is incorporated herein by reference in its entirety.

[0089] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

Chimeric Compounds

[0090] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0091] The present invention also includes antisense compounds that are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0092] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

G. Formulations

[0093] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0094] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.

[0095] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0096] The present invention also includes pharmaceutical compositions and formulations that include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0097] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0098] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0099] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0100] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 .mu.m in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug that may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0101] Formulations of the present invention include liposomal formulations. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0102] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0103] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0104] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0105] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0106] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoyl-phosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoyl-phosphatidyl ethanolamine DOTMA).

[0107] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

[0108] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U. S. Published Patent Application No. 2003/0040497 (Feb. 27, 2003) and its parent applications; U. S. Published Patent Application No. 2003/0027780 (Feb. 6, 2003) and its parent applications; and U.S. patent application Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0109] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0110] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents that function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethyl-nitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclo-phosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0111] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. For example, the first target may be a apolipoprotein(a) target, and the second target may be a region from another nucleotide sequence. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same apolipoprotein(a) nucleic acid target. Numerous examples of antisense compounds are illustrated herein and others may be selected from among suitable compounds known in the art. Two or more combined compounds may be used together or sequentially.

H. Dosing

[0112] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC.sub.50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 .mu.g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 .mu.g to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0113] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. Each of the references, GenBank accession numbers, as well as each application from which the present application claims priority, and the like recited in the present application is incorporated herein by reference in its entirety.

EXAMPLES

Example 1

Synthesis of Nucleoside Phosphoramidites

[0114] The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published International Patent Application No. WO 02/36743; 5'-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5'-O-Dimethoxytrityl-2'-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N.sup.4-benzoyl-5-methylcy- tidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2'-Fluorodeoxyadenosine, 2'-Fluorodeoxyguanosine, 2'-Fluorouridine, 2'-Fluorodeoxycytidine, 2'-O-(2-Methoxyethyl) modified amidites, 2'-O-(2-methoxyethyl)-5-methyluridine intermediate, 5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi- n-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5'-O-Dimethoxytrityl-2'-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5'-O-dimethoxytrityl-2'-O-(2-methoxyethyl)-N.sup.4-benzoyl-5-methyl-cytid- ine penultimate intermediate, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.4-benzo- yl-5-methylcytidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.6-benzo- yladenosin-3'-O-yl]-2-cyanoethyl-N, N-diisopropylphosphoramidite (MOE A amdite), [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.su- p.4-isobutyrylguanosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidit- e (MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and 2'-O-(dimethylaminooxyethyl) nucleoside amidites, 2'-(Dimethylaminooxyethoxy) nucleoside amidites, 5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-5-methyluridine, 5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine, 2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine, 5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluri- dine, 5'-O-tert-Butyldiphenylsilyl-2'-O--[N,N dimethylaminooxyethyl]-5-methyluridine, 2'-O-(dimethylaminooxyethyl)-5-methyluridine, 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine, 5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoe- thyl)-N,N-diisopropylphosphoramidite], 2'-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(- 4,4'-dimethoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphora- midite], 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside amidites, 2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

Oligonucleotide and Oligonucleoside Synthesis

[0115] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0116] Oligonucleotides: Unsubstituted and substituted phosphodiester (P.dbd.O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0117] Phosphorothioates (P.dbd.S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1M NH.sub.4OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0118] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0119] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0120] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or 5,366,878, herein incorporated by reference.

[0121] Alkylphosphonothioate oligonucleotides are prepared as described in published International patent application Nos. PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0122] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0123] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0124] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

[0125] Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P.dbd.O or P.dbd.S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0126] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0127] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

RNA Synthesis

[0128] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5'-hydroxyl in combination with an acid-labile orthoester protecting group on the 2'-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2' hydroxyl.

[0129] Following this procedure for the sequential protection of the 5'-hydroxyl in combination with protection of the 2'-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

[0130] RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3'- to 5'-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3'-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5'-end of the first nucleoside. The support is washed and any unreacted 5'-hydroxyl groups are capped with acetic anhydride to yield 5'-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5'-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

[0131] Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S.sub.2Na.sub.2) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55.degree. C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2'-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

[0132] The 2'-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group that has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine, which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

[0133] Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0134] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 .mu.l of each of the complementary strands of RNA oligonucleotides (50 .mu.M RNA oligonucleotide solution) and 15 .mu.l of 5.times. annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90.degree. C., then 1 hour at 37.degree. C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4

Synthesis of Chimeric Oligonucleotides

[0135] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers".

[2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0136] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3' wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at 55.degree. C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0137] [2'-O-(2-methoxyethyl)]-[2'-deoxy]-[-2'-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2'-O-methyl chimeric oligonucleotide, with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites.

[2'-O-(2-Methoxyethyl)Phosphodiester]-[2'-deoxy Phosphorothioate]-[2'-O-(2-Methoxyethyl)Phosphodiester] Chimeric Oligonucleotides

[0138] [2'-O-(2-methoxyethyl phosphodiester]-[2'-deoxy phosphorothioate]-[2'-O-(methoxyethyl)phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2'-O-methyl chimeric oligonucleotide with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0139] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

Design and Screening of Duplexed Antisense Compounds Targeting Apolipoprotein(a)

[0140] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target apolipoprotein(a). The nucleobase sequence of the antisense strand of the duplex comprises at least an 8-nucleobase portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.

[0141] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 74) and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:

##STR00001##

[0142] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 .mu.M. Once diluted, 30 .mu.L of each strand is combined with 15 .mu.L of a 5.times. solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 pt. This solution is incubated for 1 minute at 90.degree. C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37.degree. C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 .mu.M. This solution can be stored frozen (-20.degree. C.) and freeze-thawed up to 5 times.

[0143] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate apolipoprotein(a) expression.

[0144] When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 .mu.L OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 .mu.L of OPTI-MEM-1 containing 12 .mu.g/mL LIPOFECTIN reagent (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6

Oligonucleotide Isolation

[0145] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55.degree. C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH.sub.4OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full-length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis were determined by the ratio of correct molecular weight relative to the -16 amu product (+/-32+/-48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

Oligonucleotide Synthesis

96 Well Plate Format

[0146] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0147] Oligonucleotides were cleaved from support and deprotected with concentrated NH.sub.4OH at elevated temperature (55-60.degree. C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

Oligonucleotide Analysis

96-Well Plate Format

[0148] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE.TM. MDQ instrument) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000 instrument, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

Cell Culture and Oligonucleotide Treatment

[0149] The effects of antisense compounds on target nucleic acid expression are tested in any of a variety of cell types, provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. 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. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

T-24 Cells:

[0150] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 .mu.g/mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0151] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

A549 Cells:

[0152] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 .mu.g/mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

NHDF Cells:

[0153] Human neonatal dermal fibroblasts (NHDFs) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

HEK Cells:

[0154] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

Treatment with Antisense Compounds:

[0155] When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 .mu.L OPTI-MEM.TM.-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 .mu.L of OPTI-MEM.TM.-1 medium containing 3.75 .mu.g/mL LIPOFECTIN.TM. reagent (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37.degree. C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0156] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2'-O-methoxyethyl gapmers (2'-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

Analysis of Oligonucleotide Inhibition of Apolipoprotein(a) Expression

[0157] Antisense modulation of apolipoprotein(a) expression can be assayed in a variety of ways known in the art. For example, apolipoprotein(a) mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM.TM. 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0158] Protein levels of apolipoprotein(a) can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to apolipoprotein(a) can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11

Design of Phenotypic Assays and In Vivo Studies for the Use of Apolipoprotein(a) Inhibitors

Phenotypic Assays

[0159] Once apolipoprotein(a) inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of apolipoprotein(a) in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0160] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with apolipoprotein(a) inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0161] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status, which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

[0162] Analysis of the genotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the apolipoprotein(a) inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

[0163] The cells subjected to the phenotypic assays described herein derive from in vitro cultures or from tissues or fluids isolated from living organisms, both human and non-human. In certain embodiments, a tissue and its constituent cells comprise, but are not limited to, blood (e.g., hematopoietic cells, such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34.sup.+ cells CD4.sup.+ cells), lymphocytes and other blood lineage cells, bone marrow, brain, stem cells, blood vessel, liver, lung, bone, breast, cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial, epithelial, esophagus, facia, fibroblast, follicular, ganglion cells, glial cells, goblet cells, kidney, lymph node, muscle, neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, small intestine, spleen, stomach, testes and fetal tissue. In other embodiments, a fluid and its constituent cells comprise, but is not limited to, blood, urine, synovial fluid, lymphatic fluid and cerebro-spinal fluid. The phenotypic assays may also be performed on tissues treated with apolipoprotein(a) inhibitors ex vivo.

In Vivo Studies

[0164] The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, including humans.

[0165] The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study.

[0166] To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or apolipoprotein(a) inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a apolipoprotein(a) inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.

[0167] Volunteers receive either the apolipoprotein(a) inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding apolipoprotein(a) or apolipoprotein(a) protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.

[0168] Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.

[0169] Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and apolipoprotein(a) inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the apolipoprotein(a) inhibitor show positive trends in their disease state or condition index at the conclusion of the study.

Example 12

RNA Isolation

[0170] Poly(A)+mRNA Isolation

[0171] Poly(A)+mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 .mu.L of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 .mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70.degree. C., was added to each well, the plate was incubated on a 90.degree. C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0172] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Total RNA Isolation

[0173] Total RNA was isolated using an RNEASY 96.TM. kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 .mu.L cold PBS. 150 .mu.L Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 .mu.L of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96.TM. well plate attached to a QIAVAC.TM. manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 .mu.L of Buffer RW1 was added to each well of the RNEASY 96.TM. plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 .mu.L of Buffer RW1 was added to each well of the RNEASY 96.TM. plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96.TM. plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC.TM. manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC.TM. manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 .mu.L of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0174] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 instrument (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

Real-Time Quantitative PCR Analysis of Apolipoprotein(a) mRNA Levels

[0175] Quantitation of apolipoprotein(a) mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM.TM. 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM.TM. Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0176] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ("single-plexing"), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0177] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 .mu.L PCR cocktail (2.5.times.PCR buffer minus MgCl.sub.2, 6.6 mM MgCl.sub.2, 375 .mu.M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units of PLATINUM.RTM. Taq reagent, 5 Units MuLV reverse transcriptase, and 2.5.times.ROX dye) to 96-well plates containing 30 .mu.L total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48.degree. C. Following a 10 minute incubation at 95.degree. C. to activate the PLATINUM.RTM. Taq reagent, 40 cycles of a two-step PCR protocol were carried out: 95.degree. C. for 15 seconds (denaturation) followed by 60.degree. C. for 1.5 minutes (annealing/extension).

[0178] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen.TM. reagent (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen.TM. RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen.TM. reagent are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0179] In this assay, 170 .mu.L of RiboGreen.TM. working reagent (RiboGreen.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L purified, cellular RNA. The plate is read in a CytoFluor.TM. 4000 apparatus (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0180] Probes and primers to human apolipoprotein(a) were designed to hybridize to a human apolipoprotein(a) sequence, using published sequence information (GenBank accession number NM.sub.--005577.1, incorporated herein as SEQ ID NO: 4). For human apolipoprotein(a) the PCR primers were:

forward primer: CAGCTCCTTATTGTTATACGAGGGA (SEQ ID NO: 5) reverse primer: TGCGTCTGAGCATTGCGT (SEQ ID NO: 6) and the PCR probe was: FAM-CCCGGTGTCAGGTGGGAGTACTGC-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

Northern Blot Analysis of Apolipoprotein(a) mRNA Levels

[0181] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM. reagent(TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER.TM. UV Crosslinker 2400 apparatus (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB.TM. hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0182] To detect human apolipoprotein(a), a human apolipoprotein(a) specific probe was prepared by PCR using the forward primer CAGCTCCTTATTGTTATACGAGGGA (SEQ ID NO: 5) and the reverse primer TGCGTCTGAGCATTGCGT (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0183] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER.TM. apparatus and IMAGEQUANT.TM. Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

Antisense Inhibition of Human Apolipoprotein(a) Expression by Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap

[0184] In accordance with the present invention, a series of antisense compounds was designed to target different regions of the human apolipoprotein(a) RNA, using published sequences (GenBank accession number NM.sub.--005577.1, incorporated herein as SEQ ID NO: 4). The compounds are shown in Table 1. "Target site" indicates the first (5'-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P.dbd.S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.

[0185] Apolipoprotein(a) is found in humans, nonhuman primates and the European hedgehog, but not in common laboratory animals such as rats and mice. Transgenic mice which express human apolipoprotein(a) have been engineered (Chiesa et al., J. Biol. Chem., 1992, 267, 24369-24374). The use of primary hepatocytes prepared from human apolipoprotein(a) transgenic mice circumvents the issue of variability when testing antisense oligonucleotide activity in primary human hepatocytes. Accordingly, primary mouse hepatocytes prepared from the human apolipoprotein(a) transgenic mice were used to investigate the effects of antisense oligonucleotides on human apolipoprotein(a) expression. The human apolipoprotein(a) transgenic mice were obtained from Dr. Robert Pitas and Dr. Matthias Schneider in the Gladstone Institute at the University of California, San Francisco. Primary hepatocytes were isolated from these mice and were cultured in DMEM, high glucose (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum, (Invitrogen Corporation, Carlsbad, Calif.), 100 units per mL penicillin/100 .mu.g/mL streptomycin (Invitrogen Corporation, Carlsbad, Calif.). For treatment with oligonucleotide, cells were washed once with serum-free DMEM and subsequently transfected with a dose of 150 nM of antisense oligonucleotide using LIPOFECTIN reagent (Invitrogen Corporation, Carlsbad, Calif.) as described in other examples herein. The compounds were analyzed for their effect on human apolipoprotein(a) mRNA levels by quantitative real-time PCR as described in other examples herein. Gene target quantities obtained by real time RT-PCR were normalized using mouse GAPDH. For mouse GAPDH the PCR primers were:

forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO: 71) reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO: 72) and the PCR probe was: 5' JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3' (SEQ ID NO: 73) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

[0186] Data are averages from three experiments in which primary transgenic mouse hepatocytes were treated with the antisense oligonucleotides of the present invention.

TABLE-US-00001 TABLE 1 Inhibition of human apolipoprotein(a) mRNA levels by chimeric phosphorothioate oligonucleo- tides having 2'-MOE wings and a deoxy gap TARGET SEQ SEQ ID TARGET % ID ISIS # REGION NO SITE SEQUENCE INHIB NO 144367 Coding 4 174 ggcaggtcct 53 11 tcctgtgaca 144368 Coding 4 352 tctgcgtctg 87 12 agcattgcgt 144369 Coding 4 522 aagcttggca 0 13 ggttcttcct 144370 Coding 4 1743 tcggaggcgc 40 14 gacggcagtc 144371 Coding 4 2768 cggaggcgcg 0 15 acggcagtcc 144372 Coding 4 2910 ggcaggttct 65 16 tcctgtgaca 144373 Coding 4 3371 ataacaataa 50 17 ggagctgcca 144374 Coding 4 4972 gaccaagctt 62 18 ggcaggttct 144375 Coding 4 5080 taacaataag 36 19 gagctgccac 144376 Coding 4 5315 tgaccaagct 25 20 tggcaggttc 144377 Coding 4 5825 ttctgcgtct 38 21 gagcattgcg 144378 Coding 4 6447 aacaataagg 29 22 agctgccaca 144379 Coding 4 7155 acctgacacc 79 23 gggatccctc 144380 Coding 4 7185 ctgagcattg 16 24 cgtcaggttg 144381 Coding 4 8463 agtagttcat 71 25 gatcaagcca 144382 Coding 4 8915 gacggcagtc 34 26 ccttctgcgt 144383 Coding 4 9066 ggcaggttct 5 27 tccagtgaca 144384 Coding 4 10787 tgaccaagct 31 28 tggcaagttc 144385 Coding 4 11238 tataacacca 9 29 aggactaatc 144386 Coding 4 11261 ccatctgaca 66 30 ttgggatcca 144387 Coding 4 11461 tgtggtgtca 36 31 tagaggacca 144388 Coding 4 11823 atgggatcct 55 32 ccgatgccaa 144389 Coding 4 11894 acaccaaggg 58 33 cgaatctcag 144390 Coding 4 11957 ttctgtcact 59 34 ggacatcgtg 144391 Coding 4 12255 cacacggatc 58 35 ggttgtgtaa 144392 Coding 4 12461 acatgtcctt 51 36 cctgtgacag 144393 Coding 4 12699 cagaaggagg 33 37 ccctaggctt 144394 Coding 4 13354 ctggcggtga 52 38 ccatgtagtc 144395 3' UTR 4 13711 tctaagtagg 68 39 ttgatgcttc 144396 3' UTR 4 13731 tccttaccca 70 40 cgtttcagct 144397 3' UTR 4 13780 ggaacagtgt 63 41 cttcgtttga 144398 3' UTR 4 13801 gtttggcata 44 42 gctggtagct 144399 3' UTR 4 13841 accttaaaag 57 43 cttatacaca 144400 3' UTR 4 13861 atacagaatt 21 44 tgtcagtcag 144401 3' UTR 4 13881 gtcatagcta 46 45 tgacacctta

[0187] As shown in Table 1, SEQ ID NOs 11, 12, 14, 16, 17, 18, 19, 21, 23, 25, 30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 42, 43 and 45 demonstrated at least 35% inhibition of human apolipoprotein(a) expression in this assay and are therefore preferred. More preferred are SEQ ID NOs 23, 12 and 40. The target regions to which these preferred sequences are complementary are herein referred to as "preferred target segments" and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 2. These sequences are shown to contain thymine (T) but one of skill in the art will appreciate that thymine (T) is generally replaced by uracil (U) in RNA sequences. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 2 is the species in which each of the preferred target segments was found.

TABLE-US-00002 TABLE 2 Sequence and position of preferred target segments identified in apolipoprotein(a). TAR- REV GET COMP SEQ OF SEQ SITE ID TARGET SEQ ACTIVE ID ID NO SITE SEQUENCE ID IN NO 57364 4 174 tgtcacaggaag 11 H. 46 gacctgcc sapiens 57365 4 352 acgcaatgctca 12 H. 47 gacgcaga sapiens 57367 4 1743 gactgccgtcgc 14 H. 48 gcctccga sapiens 57369 4 2910 tgtcacaggaag 16 H. 49 aacctgcc sapiens 57370 4 3371 tggcagctcctt 17 H. 50 attgttat sapiens 57371 4 4972 agaacctgccaa 18 H. 51 gcttggtc sapiens 57372 4 5080 gtggcagctcct 19 H. 52 tattgtta sapiens 57374 4 5825 cgcaatgctcag 21 H. 53 acgcagaa sapiens 57376 4 7155 gagggatcccgg 23 H. 54 tgtcaggt sapiens 57378 4 8463 tggcttgatcat 25 H. 55 gaactact sapiens 57383 4 11261 tggatcccaatg 30 H. 56 gtcagatg sapiens 57384 4 11461 tggtcctctatg 31 H. 57 acaccaca sapiens 57385 4 11823 ttggcatcggag 32 H. 58 gatcccat sapiens 57386 4 11894 ctgagattcgcc 33 H. 59 cttggtgt sapiens 57387 4 11957 cacgatgtccag 34 H. 60 tgacagaa sapiens 57388 4 12255 ttacacaaccga 35 H. 61 tccgtgtg sapiens 57389 4 12461 ctgtcacaggaa 36 H. 62 ggacatgt sapiens 57391 4 13354 gactacatggtc 38 H. 63 accgccag sapiens 57392 4 13711 gaagcatcaacc 39 H. 64 tacttaga sapiens 57393 4 13731 agctgaaacgtg 40 H. 65 ggtaagga sapiens 57394 4 13780 tcaaacgaagac 41 H. 66 actgttcc sapiens 57395 4 13801 agctaccagcta 42 H. 67 tgccaaac sapiens 57396 4 13841 tgtgtataagct 43 H. 68 tttaaggt sapiens 57398 4 13881 taaggtgtcata 45 H. 69 gctatgac sapiens

[0188] As these "preferred target segments" have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of apolipoprotein(a).

[0189] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds that hybridize to at least a portion of the target nucleic acid.

Example 16

Western Blot Analysis of Apolipoprotein(a) Protein Levels

[0190] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 .mu.l/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to apolipoprotein(a) is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER.TM. apparatus (Molecular Dynamics, Sunnyvale Calif.).

Example 17

Antisense Inhibition of Human Apolipoprotein(a) in Transgenic Primary Mouse Hepatocytes: Dose Response

[0191] In accordance with the present invention, antisense oligonucleotides identified as having good activity based on the results in Example 15 were further investigated in dose-response studies. Primary hepatocytes from human apolipoprotein(a) transgenic mice were treated with 10, 50, 150 or 300 nM of ISIS 144396 (SEQ ID NO: 40), ISIS 144368 (SEQ ID NO: 12), ISIS 144379 (SEQ ID NO: 23) or ISIS 113529 (CTCTTACTGTGCTGTGGACA, SEQ ID NO: 70). ISIS 113529 was used as a scrambled control oligonucleotide and is a chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap" region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings". The wings are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P.dbd.S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.

[0192] Following 24 hours of exposure to antisense oligonucleotides, target mRNA expression levels were evaluated by quantitative real-time PCR as described in other examples herein. The results are the average of 4 experiments for apolipoprotein(a) antisense oligonucleotides and the average of 12 experiments for the control oligonucleotide. The data are expressed as percent inhibition of apolipoprotein(a) expression relative to untreated controls and are shown in Table 3.

TABLE-US-00003 TABLE 3 Antisense inhibition of human apolipoprotein(a) in transgenic primary mouse hepatocytes: dose response % Inhibition of transgenic human lipoprotein(a) ISIS # Oligonucleotide dose 144396 144368 144379 113529 10 nM 0 11 55 N.D. 50 nM 0 26 73 N.D. 150 nM 0 58 85 N.D. 300 nM 9 62 89 0

[0193] These data demonstrate that the oligonucleotides of the present invention are able to inhibit the expression of human apolipoprotein(a) in a dose-dependent fashion.

Example 18

Oil Red O Stain

[0194] Hepatic steatosis, or clearing of lipids from the liver, is assessed by routine histological analysis of frozen liver tissue sections stained with oil red O stain, which is commonly used to visualize lipid deposits, and counterstained with hematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.

Example 19

Animal Models

[0195] In addition to human systems, which express apolipoprotein (a), biological systems of other mammals are also available for studies of expression products of the LPA gene as well as for studies of the Lp(a) particles and their role in physiologic processes.

[0196] Transgenic mice which express human apolipoprotein(a) have been engineered (Chiesa et al., J. Biol. Chem., 1992, 267, 24369-24374) and are used as an animal model for the investigation of the in vivo activity of the oligonucleotides of this invention. Although transgenic mice expressing human apolipoprotein(a) exist, they fail to assemble Lp(a) particles because of the inability of human apolipoprotein(a) to associate with mouse apolipoprotein B. When mice expressing human apolipoprotein(a) are bred to mice expressing human apolipoprotein B, the Lp(a) particle is efficiently assembled (Callow et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 2130-2134). Accordingly mice expressing both human apolipoprotein(a) and human apolipoprotein B transgenes are used for animal model studies in which the secretion of the Lp(a) particle is evaluated.

[0197] Where additional genetic alterations are necessary, mice with either a single human transgene (human apolipoprotein(a) or human apolipoprotein B) or both human transgenes (human apolipoprotein(a) and human apolipoprotein B) are bred to mice with a desired genetic mutation. The offspring with the desired combination of transgene(s) and genetic mutation(s) is selected for use as an animal model. In one nonlimiting example, mice expressing both human apolipoprotein(a) and human apolipoprotein B are bred to mice with a mutation in the leptin gene, yielding offspring producing human Lp(a) particles in an ob/ob model of obesity and diabetes.

ob/ob Mice

[0198] Leptin is a hormone produced by fat that regulates appetite. Deficiencies in this hormone in both humans and non-human animals leads to obesity. ob/ob mice have a mutation in the leptin gene which results in obesity and hyperglycemia. As such, these mice are a useful model for the investigation of obesity and treatments designed to reduce obesity.

[0199] Seven-week old male C57B1/6J-Lep ob/ob mice (Jackson Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of 10-15% and are subcutaneously injected with oligonucleotides of the present invention or a control oligonucleotide at a dose of 5, 10 or 25 mg/kg two times per week for 4 weeks. Saline-injected animals and leptin wildtype littermates (i.e. lean littermates) serve as controls. After the treatment period, mice are sacrificed and target levels are evaluated in liver, brown adipose tissue (BAT) and white adipose tissue (WAT). RNA isolation and target mRNA expression level quantitation are performed as described by other examples herein.

[0200] To assess the physiological effects resulting from antisense inhibition of target apolipoprotein(a) mRNA, the ob/ob mice that receive antisense oligonucleotide treatment are further evaluated at the end of the treatment period for serum lipids, serum apolipoproteins, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, and fat tissue triglycerides. Serum components are measured on routine clinical diagnostic instruments. Tissue triglycerides are extracted using an acetone extraction technique known in the art, and subsequently measured by ELISA. The presence of the Lp(a) particle in the serum is measured using a commercially available ELISA kit (ALerCHEK Inc., Portland, Me.). Hepatic steatosis, or clearing of lipids from the liver, is assessed by measuring the liver triglyceride content. Hepatic steatosis is also assessed by routine histological analysis of frozen liver tissue sections stained with oil red O stain, which is commonly used to visualize lipid deposits, and counterstained with hematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.

[0201] The effects of apolipoprotein(a) inhibition on glucose and insulin metabolism are also evaluated in the ob/ob mice treated with antisense oligonucleotides of this invention. Plasma glucose is measured at the start of the antisense oligonucleotide treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly at the beginning to of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose and insulin levels are measured before the insulin or glucose challenge and at 15, 20 or 30 minute intervals for up to 3 hours.

[0202] To assess the metabolic rate of ob/ob mice treated with antisense oligonucleotides of this invention, the respiratory quotient and oxygen consumption of the mice are also measured.

[0203] The ob/ob mice that received antisense oligonucleotide treatment are further evaluated at the end of the treatment period for the effects of apolipoprotein(a) inhibition on the expression of genes that participate in lipid metabolism, cholesoterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism. These genes include, but are not limited to, HMG-CoA reductase, acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2, carnitine palmitoyltransferase I and glycogen phosphorylase, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1, lipoprotein lipase and hormone sensitive lipase. mRNA levels in liver and white and brown adipose tissue are quantitated by real-time PCR as described in other examples herein, employing primer-probe sets that were generated using published sequences of each gene of interest.

db/db Mice

[0204] A deficiency in the leptin hormone receptor mice also results in obesity and hyperglycemia. These mice are referred to as db/db mice and, like the ob/ob mice, are used as a mouse model of obesity.

[0205] Seven-week old male C57B1/6J-Lepr db/db mice (Jackson Laboratory, Bar Harbor, Me.) are fed a diet with a fat content of 15-20% and are subcutaneously injected with oligonucleotides of this invention or a control oligonucleotide at a dose of 5, 10 or 25 mg/kg two times per week for 4 weeks. Saline-injected animals and leptin receptor wildtype littermates (i.e. lean littermates) serve as controls. After the treatment period, mice are sacrificed and apolipoprotein(a) levels are evaluated in liver, brown adipose tissue (BAT) and white adipose tissue (WAT). RNA isolation and apolipoprotein(a) mRNA expression level quantitation are performed as described by other examples herein.

[0206] After the treatment period, mice are sacrificed and apolipoprotein(a) levels are evaluated in liver, brown adipose tissue (BAT) and white adipose tissue (WAT). RNA isolation and apolipoprotein(a) mRNA expression level quantitation are performed as described by other examples herein.

[0207] To assess the physiological effects resulting from antisense inhibition of apolipoprotein(a) mRNA, the db/db mice that receive antisense oligonucleotide treatment are further evaluated at the end of the treatment period for serum lipids, serum apolipoproeins, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, and fat tissue triglycerides. Serum components are measured on routine clinical diagnostic instruments. Tissue triglycerides are extracted using an acetone extraction technique known in the art, and subsequently measured by ELISA. The presence of the Lp(a) particle in the serum is measured using a commercially available ELISA kit (ALerCHEK Inc., Portland, Me.). Hepatic steatosis, or clearing of lipids from the liver, are assessed by measuring the liver triglyceride content. Hepatic steatosis is also assessed by routine histological analysis of frozen liver tissue sections stained with oil red O stain, which is commonly used to visualize lipid deposits, and counterstained with hematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.

[0208] The effects of apolipoprotein(a) inhibition on glucose and insulin metabolism are also evaluated in the db/db mice treated with antisense oligonucleotides. Plasma glucose is measured at the start of the antisense oligonucleotide treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly at the beginning to of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose levels are measured before the insulin or glucose challenge and 15, 30, 60, 90 and 120 minutes following the injection.

[0209] To assess the metabolic rate of db/db mice treated with antisense oligonucleotides, the respiratory quotient and oxygen consumption of the mice are also measured.

[0210] The db/db mice that received antisense oligonucleotide treatment are further evaluated at the end of the treatment period for the effects of apolipoprotein(a) inhibition on the expression of genes that participate in lipid metabolism, cholesoterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism. These genes include, but are not limited to, HMG-CoA reductase, acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2, carnitine palmitoyltransferase I and glycogen phosphorylase, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1, lipoprotein lipase and hormone sensitive lipase. mRNA levels in liver and white and brown adipose tissue are quantitated by real-time PCR as described in other examples herein, employing primer-probe sets that were generated using published sequences of each gene of interest.

Lean Mice

[0211] C57B1/6 mice are maintained on a standard rodent diet and are used as control (lean) animals. Seven-week old male C57B1/6 mice are fed a diet with a fat content of 4% and are subcutaneously injected with oligonucleotides of this invention or control oligonucleotide at a dose of 5, 10 or 25 mg/kg two times per week for 4 weeks. Saline-injected animals serve as a control. After the treatment period, mice are sacrificed and apolipoprotein(a) levels are evaluated in liver, brown adipose tissue (BAT) and white adipose tissue (WAT). RNA isolation and apolipoprotein(a) mRNA expression level quantitation are performed as described by other examples herein.

[0212] To assess the physiological effects resulting from antisense inhibition of apolipoprotein(a) mRNA, the lean mice that receive antisense oligonucleotide treatment are further evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol (CHOL), liver triglycerides, and fat tissue triglycerides. Serum components are measured on routine clinical diagnostic instruments. Tissue triglycerides are extracted using an acetone extraction technique known in the art, and subsequently measured by ELISA. The presence of the Lp(a) particle in the serum is measured using a commercially available ELISA kit (ALerCHEK Inc., Portland, Me.). Hepatic steatosis, i.e., clearing of lipids from the liver, is assessed by measuring the liver triglyceride content. Hepatic steatosis is also assessed by routine histological analysis of frozen liver tissue sections stained with oil red O stain, which is commonly used to visualize lipid deposits, and counterstained with hematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.

[0213] The effects of apolipoprotein(a) inhibition on glucose and insulin metabolism are also evaluated in the lean mice treated with antisense oligonucleotides of this invention. Plasma glucose is measured at the start of the antisense oligonucleotide treatment and after 2 weeks and 4 weeks of treatment. Plasma insulin is similarly at the beginning to of the treatment, and following 2 weeks and 4 weeks of treatment. Glucose and insulin tolerance tests are also administered in fed and fasted mice. Mice receive intraperitoneal injections of either glucose or insulin, and the blood glucose levels are measured before the insulin or glucose challenge and 15, 30, 60, 90 and 120 minutes following the injection.

[0214] To assess the metabolic rate of lean mice treated with antisense oligonucleotides of this invention, the respiratory quotient and oxygen consumption of the mice can also be measured.

[0215] The lean mice that received antisense oligonucleotide treatment are further evaluated at the end of the treatment period for the effects of apolipoprotein(a) inhibition on the expression of genes that participate in lipid metabolism, cholesoterol biosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesis and glucose metabolism. These genes include, but are not limited to, HMG-CoA reductase, acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2, carnitine palmitoyltransferase I and glycogen phosphorylase, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1, lipoprotein lipase and hormone sensitive lipase. mRNA levels in liver and white and brown adipose tissue are quantitated by real-time PCR as described in other examples herein, employing primer-probe sets that were generated using published sequences of each gene of interest.

Sequence CWU 1

1

73120DNAArtificial SequenceAntisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20220DNAArtificial SequenceAntisense Oligonucleotide 2gtgcgcgcga gcccgaaatc 20320DNAArtificial SequenceAntisense Oligonucleotide 3atgcattctg cccccaagga 20413938DNAHomo sapiensCDS(46)..(13692) 4ctgggattgg gacacacttt ctggacactg ctggccagtc ccaaa atg gaa cat aag 57 Met Glu His Lys 1 gaa gtg gtt ctt cta ctt ctt tta ttt ctg aaa tca gca gca cct gag 105Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser Ala Ala Pro Glu 5 10 15 20 caa agc cat gtg gtc cag gat tgc tac cat ggt gat gga cag agt tat 153Gln Ser His Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr 25 30 35 cga ggc acg tac tcc acc act gtc aca gga agg acc tgc caa gct tgg 201Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp 40 45 50 tca tct atg aca cca cat caa cat aat agg acc aca gaa aac tac cca 249Ser Ser Met Thr Pro His Gln His Asn Arg Thr Thr Glu Asn Tyr Pro 55 60 65 aat gct ggc ttg atc atg aac tac tgc agg aat cca gat gct gtg gca 297Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 70 75 80 gct cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac tgc 345Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys 85 90 95 100 aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg 393Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro 105 110 115 act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa caa gca ccg 441Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro 120 125 130 act gag caa agg cct ggg gtg cag gag tgc tac cat ggt aat gga cag 489Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln 135 140 145 agt tat cga ggc aca tac tcc acc act gtc aca gga aga acc tgc caa 537Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln 150 155 160 gct tgg tca tct atg aca cca cac tcg cat agt cgg acc cca gaa tac 585Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr 165 170 175 180 tac cca aat gct ggc ttg atc atg aac tac tgc agg aat cca gat gct 633Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala 185 190 195 gtg gca gct cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag 681Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu 200 205 210 tac tgc aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg 729Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala 215 220 225 cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa caa 777Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln 230 235 240 gca ccg act gag caa agg cct ggg gtg cag gag tgc tac cat ggt aat 825Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn 245 250 255 260 gga cag agt tat cga ggc aca tac tcc acc act gtc aca gga aga acc 873Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr 265 270 275 tgc caa gct tgg tca tct atg aca cca cac tcg cat agt cgg acc cca 921Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro 280 285 290 gaa tac tac cca aat gct ggc ttg atc atg aac tac tgc agg aat cca 969Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro 295 300 305 gat gct gtg gca gct cct tat tgt tat acg agg gat ccc ggt gtc agg 1017Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg 310 315 320 tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaa ggg act gcc 1065Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala 325 330 335 340 gtc gcg cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tcc 1113Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser 345 350 355 gaa caa gca ccg act gag caa agg cct ggg gtg cag gag tgc tac cat 1161Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His 360 365 370 ggt aat gga cag agt tat cga ggc aca tac tcc acc act gtc aca gga 1209Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly 375 380 385 aga acc tgc caa gct tgg tca tct atg aca cca cac tcg cat agt cgg 1257Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg 390 395 400 acc cca gaa tac tac cca aat gct ggc ttg atc atg aac tac tgc agg 1305Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg 405 410 415 420 aat cca gat gct gtg gca gct cct tat tgt tat acg agg gat ccc ggt 1353Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly 425 430 435 gtc agg tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaa ggg 1401Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly 440 445 450 act gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta gag gct 1449Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala 455 460 465 cct tcc gaa caa gca ccg act gag caa agg cct ggg gtg cag gag tgc 1497Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys 470 475 480 tac cat ggt aat gga cag agt tat cga ggc aca tac tcc acc act gtc 1545Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val 485 490 495 500 aca gga aga acc tgc caa gct tgg tca tct atg aca cca cac tcg cat 1593Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His 505 510 515 agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg aac tac 1641Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr 520 525 530 tgc agg aat cca gat gct gtg gca gct cct tat tgt tat acg agg gat 1689Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp 535 540 545 ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc tca gac gca 1737Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala 550 555 560 gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta 1785Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu 565 570 575 580 gag gct cct tcc gaa caa gca ccg act gag caa agg cct ggg gtg cag 1833Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln 585 590 595 gag tgc tac cat ggt aat gga cag agt tat cga ggc aca tac tcc acc 1881Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr 600 605 610 act gtc aca gga aga acc tgc caa gct tgg tca tct atg aca cca cac 1929Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His 615 620 625 tcg cat agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg 1977Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met 630 635 640 aac tac tgc agg aat cca gat gct gtg gca gct cct tat tgt tat acg 2025Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr 645 650 655 660 agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc tca 2073Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser 665 670 675 gac gca gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg gtt cca 2121Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro 680 685 690 agc cta gag gct cct tcc gaa caa gca ccg act gag caa agg cct ggg 2169Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly 695 700 705 gtg cag gag tgc tac cat ggt aat gga cag agt tat cga ggc aca tac 2217Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr 710 715 720 tcc acc act gtc aca gga aga acc tgc caa gct tgg tca tct atg aca 2265Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr 725 730 735 740 cca cac tcg cat agt cgg acc cca gaa tac tac cca aat gct ggc ttg 2313Pro His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu 745 750 755 atc atg aac tac tgc agg aat cca gat gct gtg gca gct cct tat tgt 2361Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys 760 765 770 tat acg agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa 2409Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln 775 780 785 tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg 2457Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro 790 795 800 gtt cca agc cta gag gct cct tcc gaa caa gca ccg act gag caa agg 2505Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg 805 810 815 820 cct ggg gtg cag gag tgc tac cat ggt aat gga cag agt tat cga ggc 2553Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly 825 830 835 aca tac tcc acc act gtc aca gga aga acc tgc caa gct tgg tca tct 2601Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser 840 845 850 atg aca cca cac tcg cat agt cgg acc cca gaa tac tac cca aat gct 2649Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala 855 860 865 ggc ttg atc atg aac tac tgc agg aat cca gat gct gtg gca gct cct 2697Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro 870 875 880 tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg 2745Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu 885 890 895 900 acg caa tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg act gtt 2793Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Val 905 910 915 acc ccg gtt cca agc cta gag gct cct tcc gaa caa gca ccg act gag 2841Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu 920 925 930 caa agg cct ggg gtg cag gag tgc tac cat ggt aat gga cag agt tat 2889Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr 935 940 945 cga ggc aca tac tcc acc act gtc aca gga aga acc tgc caa gct tgg 2937Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp 950 955 960 tca tct atg aca cca cac tcg cat agt cgg acc cca gaa tac tac cca 2985Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro 965 970 975 980 aat gct ggc ttg atc atg aac tac tgc agg aat cca gat gct gtg gca 3033Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 985 990 995 gct cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac 3078Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr 1000 1005 1010 tgc aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg 3123Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala 1015 1020 1025 cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa 3168Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu 1030 1035 1040 caa gca ccg act gag caa agg cct ggg gtg cag gag tgc tac cat 3213Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His 1045 1050 1055 ggt aat gga cag agt tat cga ggc aca tac tcc acc act gtc aca 3258Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr 1060 1065 1070 gga aga acc tgc caa gct tgg tca tct atg aca cca cac tcg cat 3303Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His 1075 1080 1085 agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg aac 3348Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn 1090 1095 1100 tac tgc agg aat cca gat gct gtg gca gct cct tat tgt tat acg 3393Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr 1105 1110 1115 agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc 3438Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys 1120 1125 1130 tca gac gca gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg 3483Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro 1135 1140 1145 gtt cca agc cta gag gct cct tcc gaa caa gca ccg act gag caa 3528Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln 1150 1155 1160 agg cct ggg gtg cag gag tgc tac cat ggt aat gga cag agt tat 3573Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr 1165 1170 1175 cga ggc aca tac tcc acc act gtc aca gga aga acc tgc caa gct 3618Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala 1180 1185 1190 tgg tca tct atg aca cca cac tcg cat agt cgg acc cca gaa tac 3663Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr 1195 1200 1205 tac cca aat gct ggc ttg atc atg aac tac tgc agg aat cca gat 3708Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp 1210 1215 1220 gct gtg gca gct cct tat tgt tat acg agg gat ccc ggt gtc agg 3753Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg 1225 1230 1235 tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaa ggg act 3798Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr 1240 1245 1250 gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta gag gct 3843Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala 1255 1260 1265 cct tcc gaa caa gca ccg act gag caa agg cct ggg gtg cag gag 3888Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu 1270 1275 1280 tgc tac cat ggt aat gga cag agt tat cga ggc aca tac tcc acc 3933Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr 1285 1290 1295 act gtc aca gga aga acc tgc caa gct tgg tca tct atg aca cca 3978Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro 1300 1305 1310 cac tcg cat agt cgg acc cca gaa tac tac cca aat gct ggc ttg 4023His Ser His Ser

Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu 1315 1320 1325 atc atg aac tac tgc agg aat cca gat gct gtg gca gct cct tat 4068Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr 1330 1335 1340 tgt tat acg agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg 4113Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu 1345 1350 1355 acg caa tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg act 4158Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr 1360 1365 1370 gtt acc ccg gtt cca agc cta gag gct cct tcc gaa caa gca ccg 4203Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro 1375 1380 1385 act gag caa agg cct ggg gtg cag gag tgc tac cat ggt aat gga 4248Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly 1390 1395 1400 cag agt tat cga ggc aca tac tcc acc act gtc aca gga aga acc 4293Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr 1405 1410 1415 tgc caa gct tgg tca tct atg aca cca cac tcg cat agt cgg acc 4338Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr 1420 1425 1430 cca gaa tac tac cca aat gct ggc ttg atc atg aac tac tgc agg 4383Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg 1435 1440 1445 aat cca gat gct gtg gca gct cct tat tgt tat acg agg gat ccc 4428Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro 1450 1455 1460 ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc tca gac gca 4473Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala 1465 1470 1475 gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc 4518Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser 1480 1485 1490 cta gag gct cct tcc gaa caa gca ccg act gag caa agg cct ggg 4563Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly 1495 1500 1505 gtg cag gag tgc tac cat ggt aat gga cag agt tat cga ggc aca 4608Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr 1510 1515 1520 tac tcc acc act gtc aca gga aga acc tgc caa gct tgg tca tct 4653Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser 1525 1530 1535 atg aca cca cac tcg cat agt cgg acc cca gaa tac tac cca aat 4698Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn 1540 1545 1550 gct ggc ttg atc atg aac tac tgc agg aat cca gat gct gtg gca 4743Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 1555 1560 1565 gct cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac 4788Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr 1570 1575 1580 tgc aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg 4833Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala 1585 1590 1595 cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa 4878Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu 1600 1605 1610 caa gca ccg act gag caa agg cct ggg gtg cag gag tgc tac cat 4923Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His 1615 1620 1625 ggt aat gga cag agt tat cga ggc aca tac tcc acc act gtc aca 4968Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr 1630 1635 1640 gga aga acc tgc caa gct tgg tca tct atg aca cca cac tcg cat 5013Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His 1645 1650 1655 agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg aac 5058Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn 1660 1665 1670 tac tgc agg aat cca gat gct gtg gca gct cct tat tgt tat acg 5103Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr 1675 1680 1685 agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc 5148Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys 1690 1695 1700 tca gac gca gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg 5193Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro 1705 1710 1715 gtt cca agc cta gag gct cct tcc gaa caa gca ccg act gag caa 5238Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln 1720 1725 1730 agg cct ggg gtg cag gag tgc tac cat ggt aat gga cag agt tat 5283Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr 1735 1740 1745 cga ggc aca tac tcc acc act gtc aca gga aga acc tgc caa gct 5328Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala 1750 1755 1760 tgg tca tct atg aca cca cac tcg cat agt cgg acc cca gaa tac 5373Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr 1765 1770 1775 tac cca aat gct ggc ttg atc atg aac tac tgc agg aat cca gat 5418Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp 1780 1785 1790 gct gtg gca gct cct tat tgt tat acg agg gat ccc ggt gtc agg 5463Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg 1795 1800 1805 tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaa ggg act 5508Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr 1810 1815 1820 gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta gag gct 5553Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala 1825 1830 1835 cct tcc gaa caa gca ccg act gag caa agg cct ggg gtg cag gag 5598Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu 1840 1845 1850 tgc tac cat ggt aat gga cag agt tat cga ggc aca tac tcc acc 5643Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr 1855 1860 1865 act gtc aca gga aga acc tgc caa gct tgg tca tct atg aca cca 5688Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro 1870 1875 1880 cac tcg cat agt cgg acc cca gaa tac tac cca aat gct ggc ttg 5733His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu 1885 1890 1895 atc atg aac tac tgc agg aat cca gat gct gtg gca gct cct tat 5778Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr 1900 1905 1910 tgt tat acg agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg 5823Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu 1915 1920 1925 acg caa tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg act 5868Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr 1930 1935 1940 gtt acc ccg gtt cca agc cta gag gct cct tcc gaa caa gca ccg 5913Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro 1945 1950 1955 act gag caa agg cct ggg gtg cag gag tgc tac cat ggt aat gga 5958Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly 1960 1965 1970 cag agt tat cga ggc aca tac tcc acc act gtc aca gga aga acc 6003Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr 1975 1980 1985 tgc caa gct tgg tca tct atg aca cca cac tcg cat agt cgg acc 6048Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr 1990 1995 2000 cca gaa tac tac cca aat gct ggc ttg atc atg aac tac tgc agg 6093Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg 2005 2010 2015 aat cca gat gct gtg gca gct cct tat tgt tat acg agg gat ccc 6138Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro 2020 2025 2030 ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc tca gac gca 6183Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala 2035 2040 2045 gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc 6228Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser 2050 2055 2060 cta gag gct cct tcc gaa caa gca ccg act gag caa agg cct ggg 6273Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly 2065 2070 2075 gtg cag gag tgc tac cat ggt aat gga cag agt tat cga ggc aca 6318Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr 2080 2085 2090 tac tcc acc act gtc aca gga aga acc tgc caa gct tgg tca tct 6363Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser 2095 2100 2105 atg aca cca cac tcg cat agt cgg acc cca gaa tac tac cca aat 6408Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn 2110 2115 2120 gct ggc ttg atc atg aac tac tgc agg aat cca gat gct gtg gca 6453Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 2125 2130 2135 gct cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac 6498Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr 2140 2145 2150 tgc aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg 6543Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala 2155 2160 2165 cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa 6588Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu 2170 2175 2180 caa gca ccg act gag caa agg cct ggg gtg cag gag tgc tac cat 6633Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His 2185 2190 2195 ggt aat gga cag agt tat cga ggc aca tac tcc acc act gtc aca 6678Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr 2200 2205 2210 gga aga acc tgc caa gct tgg tca tct atg aca cca cac tcg cat 6723Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His 2215 2220 2225 agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg aac 6768Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn 2230 2235 2240 tac tgc agg aat cca gat gct gtg gca gct cct tat tgt tat acg 6813Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr 2245 2250 2255 agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc 6858Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys 2260 2265 2270 tca gac gca gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg 6903Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro 2275 2280 2285 gtt cca agc cta gag gct cct tcc gaa caa gca ccg act gag caa 6948Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln 2290 2295 2300 agg cct ggg gtg cag gag tgc tac cat ggt aat gga cag agt tat 6993Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr 2305 2310 2315 cga ggc aca tac tcc acc act gtc aca gga aga acc tgc caa gct 7038Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala 2320 2325 2330 tgg tca tct atg aca cca cac tcg cat agt cgg acc cca gaa tac 7083Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr 2335 2340 2345 tac cca aat gct ggc ttg atc atg aac tac tgc agg aat cca gat 7128Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp 2350 2355 2360 gct gtg gca gct cct tat tgt tat acg agg gat ccc ggt gtc agg 7173Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg 2365 2370 2375 tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaa ggg act 7218Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr 2380 2385 2390 gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta gag gct 7263Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala 2395 2400 2405 cct tcc gaa caa gca ccg act gag caa agg cct ggg gtg cag gag 7308Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu 2410 2415 2420 tgc tac cat ggt aat gga cag agt tat cga ggc aca tac tcc acc 7353Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr 2425 2430 2435 act gtc aca gga aga acc tgc caa gct tgg tca tct atg aca cca 7398Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro 2440 2445 2450 cac tcg cat agt cgg acc cca gaa tac tac cca aat gct ggc ttg 7443His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu 2455 2460 2465 atc atg aac tac tgc agg aat cca gat gct gtg gca gct cct tat 7488Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr 2470 2475 2480 tgt tat acg agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg 7533Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu 2485 2490 2495 acg caa tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg act 7578Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr 2500 2505 2510 gtt acc ccg gtt cca agc cta gag gct cct tcc gaa caa gca ccg 7623Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro 2515 2520 2525 act gag caa agg cct ggg gtg cag gag tgc tac cat ggt aat gga 7668Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly 2530 2535 2540 cag agt tat cga ggc aca tac tcc acc act gtc aca gga aga acc 7713Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr 2545 2550 2555 tgc caa gct tgg tca tct atg aca cca cac tcg cat agt cgg acc 7758Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr 2560 2565 2570 cca gaa tac tac cca aat gct ggc ttg atc atg aac tac tgc agg 7803Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg 2575 2580 2585 aat cca gat gct gtg gca gct cct tat tgt tat acg agg gat ccc 7848Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro 2590 2595 2600 ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc tca gac gca 7893Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala

2605 2610 2615 gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc 7938Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser 2620 2625 2630 cta gag gct cct tcc gaa caa gca ccg act gag cag agg cct ggg 7983Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly 2635 2640 2645 gtg cag gag tgc tac cac ggt aat gga cag agt tat cga ggc aca 8028Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr 2650 2655 2660 tac tcc acc act gtc act gga aga acc tgc caa gct tgg tca tct 8073Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser 2665 2670 2675 atg aca cca cac tcg cat agt cgg acc cca gaa tac tac cca aat 8118Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn 2680 2685 2690 gct ggc ttg atc atg aac tac tgc agg aat cca gat gct gtg gca 8163Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 2695 2700 2705 gct cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac 8208Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr 2710 2715 2720 tgc aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg 8253Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala 2725 2730 2735 cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa 8298Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu 2740 2745 2750 caa gca ccg act gag caa agg cct ggg gtg cag gag tgc tac cat 8343Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His 2755 2760 2765 ggt aat gga cag agt tat cga ggc aca tac tcc acc act gtc aca 8388Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr 2770 2775 2780 gga aga acc tgc caa gct tgg tca tct atg aca cca cac tcg cat 8433Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His 2785 2790 2795 agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg aac 8478Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn 2800 2805 2810 tac tgc agg aat cca gat gct gtg gca gct cct tat tgt tat acg 8523Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr 2815 2820 2825 agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc 8568Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys 2830 2835 2840 tca gac gca gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg 8613Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro 2845 2850 2855 gtt cca agc cta gag gct cct tcc gaa caa gca ccg act gag caa 8658Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln 2860 2865 2870 agg cct ggg gtg cag gag tgc tac cat ggt aat gga cag agt tat 8703Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr 2875 2880 2885 cga ggc aca tac tcc acc act gtc aca gga aga acc tgc caa gct 8748Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala 2890 2895 2900 tgg tca tct atg aca cca cac tcg cat agt cgg acc cca gaa tac 8793Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr 2905 2910 2915 tac cca aat gct ggc ttg atc atg aac tac tgc agg aat cca gat 8838Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp 2920 2925 2930 gct gtg gca gct cct tat tgt tat acg agg gat ccc ggt gtc agg 8883Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg 2935 2940 2945 tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaa ggg act 8928Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr 2950 2955 2960 gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta gag gct 8973Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala 2965 2970 2975 cct tcc gaa caa gca ccg act gag cag agg cct ggg gtg cag gag 9018Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu 2980 2985 2990 tgc tac cac ggt aat gga cag agt tat cga ggc aca tac tcc acc 9063Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr 2995 3000 3005 act gtc act gga aga acc tgc caa gct tgg tca tct atg aca cca 9108Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro 3010 3015 3020 cac tcg cat agt cgg acc cca gaa tac tac cca aat gct ggc ttg 9153His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu 3025 3030 3035 atc atg aac tac tgc agg aat cca gat gct gtg gca gct cct tat 9198Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr 3040 3045 3050 tgt tat acg agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg 9243Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu 3055 3060 3065 acg caa tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg act 9288Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr 3070 3075 3080 gtt acc ccg gtt cca agc cta gag gct cct tcc gaa caa gca ccg 9333Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro 3085 3090 3095 act gag cag agg cct ggg gtg cag gag tgc tac cac ggt aat gga 9378Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly 3100 3105 3110 cag agt tat cga ggc aca tac tcc acc act gtc act gga aga acc 9423Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr 3115 3120 3125 tgc caa gct tgg tca tct atg aca cca cac tcg cat agt cgg acc 9468Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr 3130 3135 3140 cca gaa tac tac cca aat gct ggc ttg atc atg aac tac tgc agg 9513Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg 3145 3150 3155 aat cca gat gct gtg gca gct cct tat tgt tat acg agg gat ccc 9558Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro 3160 3165 3170 ggt gtc agg tgg gag tac tgc aac ctg acg caa tgc tca gac gca 9603Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala 3175 3180 3185 gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc 9648Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser 3190 3195 3200 cta gag gct cct tcc gaa caa gca ccg act gag cag agg cct ggg 9693Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly 3205 3210 3215 gtg cag gag tgc tac cac ggt aat gga cag agt tat cga ggc aca 9738Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr 3220 3225 3230 tac tcc acc act gtc act gga aga acc tgc caa gct tgg tca tct 9783Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser 3235 3240 3245 atg aca cca cac tcg cat agt cgg acc cca gaa tac tac cca aat 9828Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn 3250 3255 3260 gct ggc ttg atc atg aac tac tgc agg aat cca gat gct gtg gca 9873Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 3265 3270 3275 gct cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac 9918Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr 3280 3285 3290 tgc aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg 9963Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala 3295 3300 3305 cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa 10008Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu 3310 3315 3320 caa gca ccg act gag cag agg cct ggg gtg cag gag tgc tac cac 10053Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His 3325 3330 3335 ggt aat gga cag agt tat cga ggc aca tac tcc acc act gtc act 10098Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr 3340 3345 3350 gga aga acc tgc caa gct tgg tca tct atg aca cca cac tcg cat 10143Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His 3355 3360 3365 agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg aac 10188Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn 3370 3375 3380 tac tgc agg aat cca gat cct gtg gca gcc cct tat tgt tat acg 10233Tyr Cys Arg Asn Pro Asp Pro Val Ala Ala Pro Tyr Cys Tyr Thr 3385 3390 3395 agg gat ccc agt gtc agg tgg gag tac tgc aac ctg aca caa tgc 10278Arg Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys 3400 3405 3410 tca gac gca gaa ggg act gcc gtc gcg cct cca act att acc ccg 10323Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Ile Thr Pro 3415 3420 3425 att cca agc cta gag gct cct tct gaa caa gca cca act gag caa 10368Ile Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln 3430 3435 3440 agg cct ggg gtg cag gag tgc tac cac gga aat gga cag agt tat 10413Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr 3445 3450 3455 caa ggc aca tac ttc att act gtc aca gga aga acc tgc caa gct 10458Gln Gly Thr Tyr Phe Ile Thr Val Thr Gly Arg Thr Cys Gln Ala 3460 3465 3470 tgg tca tct atg aca cca cac tcg cat agt cgg acc cca gca tac 10503Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Ala Tyr 3475 3480 3485 tac cca aat gct ggc ttg atc aag aac tac tgc cga aat cca gat 10548Tyr Pro Asn Ala Gly Leu Ile Lys Asn Tyr Cys Arg Asn Pro Asp 3490 3495 3500 cct gtg gca gcc cct tgg tgt tat aca aca gat ccc agt gtc agg 10593Pro Val Ala Ala Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg 3505 3510 3515 tgg gag tac tgc aac ctg aca cga tgc tca gat gca gaa tgg act 10638Trp Glu Tyr Cys Asn Leu Thr Arg Cys Ser Asp Ala Glu Trp Thr 3520 3525 3530 gcc ttc gtc cct ccg aat gtt att ctg gct cca agc cta gag gct 10683Ala Phe Val Pro Pro Asn Val Ile Leu Ala Pro Ser Leu Glu Ala 3535 3540 3545 ttt ttt gaa caa gca ctg act gag gaa acc ccc ggg gta cag gac 10728Phe Phe Glu Gln Ala Leu Thr Glu Glu Thr Pro Gly Val Gln Asp 3550 3555 3560 tgc tac tac cat tat gga cag agt tac cga ggc aca tac tcc acc 10773Cys Tyr Tyr His Tyr Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr 3565 3570 3575 act gtc aca gga aga act tgc caa gct tgg tca tct atg aca cca 10818Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro 3580 3585 3590 cac cag cat agt cgg acc cca gaa aac tac cca aat gct ggc ctg 10863His Gln His Ser Arg Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu 3595 3600 3605 acc agg aac tac tgc agg aat cca gat gct gag att cgc cct tgg 10908Thr Arg Asn Tyr Cys Arg Asn Pro Asp Ala Glu Ile Arg Pro Trp 3610 3615 3620 tgt tac acc atg gat ccc agt gtc agg tgg gag tac tgc aac ctg 10953Cys Tyr Thr Met Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu 3625 3630 3635 aca caa tgc ctg gtg aca gaa tca agt gtc ctt gca act ctc acg 10998Thr Gln Cys Leu Val Thr Glu Ser Ser Val Leu Ala Thr Leu Thr 3640 3645 3650 gtg gtc cca gat cca agc aca gag gct tct tct gaa gaa gca cca 11043Val Val Pro Asp Pro Ser Thr Glu Ala Ser Ser Glu Glu Ala Pro 3655 3660 3665 acg gag caa agc ccc ggg gtc cag gat tgc tac cat ggt gat gga 11088Thr Glu Gln Ser Pro Gly Val Gln Asp Cys Tyr His Gly Asp Gly 3670 3675 3680 cag agt tat cga ggc tca ttc tct acc act gtc aca gga agg aca 11133Gln Ser Tyr Arg Gly Ser Phe Ser Thr Thr Val Thr Gly Arg Thr 3685 3690 3695 tgt cag tct tgg tcc tct atg aca cca cac tgg cat cag agg aca 11178Cys Gln Ser Trp Ser Ser Met Thr Pro His Trp His Gln Arg Thr 3700 3705 3710 aca gaa tat tat cca aat ggt ggc ctg acc agg aac tac tgc agg 11223Thr Glu Tyr Tyr Pro Asn Gly Gly Leu Thr Arg Asn Tyr Cys Arg 3715 3720 3725 aat cca gat gct gag att agt cct tgg tgt tat acc atg gat ccc 11268Asn Pro Asp Ala Glu Ile Ser Pro Trp Cys Tyr Thr Met Asp Pro 3730 3735 3740 aat gtc aga tgg gag tac tgc aac ctg aca caa tgt cca gtg aca 11313Asn Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Pro Val Thr 3745 3750 3755 gaa tca agt gtc ctt gcg acg tcc acg gct gtt tct gaa caa gca 11358Glu Ser Ser Val Leu Ala Thr Ser Thr Ala Val Ser Glu Gln Ala 3760 3765 3770 cca acg gag caa agc ccc aca gtc cag gac tgc tac cat ggt gat 11403Pro Thr Glu Gln Ser Pro Thr Val Gln Asp Cys Tyr His Gly Asp 3775 3780 3785 gga cag agt tat cga ggc tca ttc tcc acc act gtt aca gga agg 11448Gly Gln Ser Tyr Arg Gly Ser Phe Ser Thr Thr Val Thr Gly Arg 3790 3795 3800 aca tgt cag tct tgg tcc tct atg aca cca cac tgg cat cag aga 11493Thr Cys Gln Ser Trp Ser Ser Met Thr Pro His Trp His Gln Arg 3805 3810 3815 acc aca gaa tac tac cca aat ggt ggc ctg acc agg aac tac tgc 11538Thr Thr Glu Tyr Tyr Pro Asn Gly Gly Leu Thr Arg Asn Tyr Cys 3820 3825 3830 agg aat cca gat gct gag att cgc cct tgg tgt tat acc atg gat 11583Arg Asn Pro Asp Ala Glu Ile Arg Pro Trp Cys Tyr Thr Met Asp 3835 3840 3845 ccc agt gtc aga tgg gag tac tgc aac ctg acg caa tgt cca gtg 11628Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Pro Val 3850 3855 3860 atg gaa tca act ctc ctc aca act ccc acg gtg gtc cca gtt cca 11673Met Glu Ser Thr Leu Leu Thr Thr Pro Thr Val Val Pro Val Pro 3865 3870 3875 agc aca gag ctt cct tct gaa gaa gca cca act gaa aac agc act 11718Ser Thr Glu Leu Pro Ser Glu Glu Ala Pro Thr Glu Asn Ser Thr 3880 3885 3890

ggg gtc cag gac tgc tac cga ggt gat gga cag agt tat cga ggc 11763Gly Val Gln Asp Cys Tyr Arg Gly Asp Gly Gln Ser Tyr Arg Gly 3895 3900 3905 aca ctc tcc acc act atc aca gga aga aca tgt cag tct tgg tcg 11808Thr Leu Ser Thr Thr Ile Thr Gly Arg Thr Cys Gln Ser Trp Ser 3910 3915 3920 tct atg aca cca cat tgg cat cgg agg atc cca tta tac tat cca 11853Ser Met Thr Pro His Trp His Arg Arg Ile Pro Leu Tyr Tyr Pro 3925 3930 3935 aat gct ggc ctg acc agg aac tac tgc agg aat cca gat gct gag 11898Asn Ala Gly Leu Thr Arg Asn Tyr Cys Arg Asn Pro Asp Ala Glu 3940 3945 3950 att cgc cct tgg tgt tac acc atg gat ccc agt gtc agg tgg gag 11943Ile Arg Pro Trp Cys Tyr Thr Met Asp Pro Ser Val Arg Trp Glu 3955 3960 3965 tac tgc aac ctg aca cga tgt cca gtg aca gaa tcg agt gtc ctc 11988Tyr Cys Asn Leu Thr Arg Cys Pro Val Thr Glu Ser Ser Val Leu 3970 3975 3980 aca act ccc aca gtg gcc ccg gtt cca agc aca gag gct cct tct 12033Thr Thr Pro Thr Val Ala Pro Val Pro Ser Thr Glu Ala Pro Ser 3985 3990 3995 gaa caa gca cca cct gag aaa agc cct gtg gtc cag gat tgc tac 12078Glu Gln Ala Pro Pro Glu Lys Ser Pro Val Val Gln Asp Cys Tyr 4000 4005 4010 cat ggt gat gga cgg agt tat cga ggc ata tcc tcc acc act gtc 12123His Gly Asp Gly Arg Ser Tyr Arg Gly Ile Ser Ser Thr Thr Val 4015 4020 4025 aca gga agg acc tgt caa tct tgg tca tct atg ata cca cac tgg 12168Thr Gly Arg Thr Cys Gln Ser Trp Ser Ser Met Ile Pro His Trp 4030 4035 4040 cat cag agg acc cca gaa aac tac cca aat gct ggc ctg acc gag 12213His Gln Arg Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Glu 4045 4050 4055 aac tac tgc agg aat cca gat tct ggg aaa caa ccc tgg tgt tac 12258Asn Tyr Cys Arg Asn Pro Asp Ser Gly Lys Gln Pro Trp Cys Tyr 4060 4065 4070 aca acc gat ccg tgt gtg agg tgg gag tac tgc aat ctg aca caa 12303Thr Thr Asp Pro Cys Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln 4075 4080 4085 tgc tca gaa aca gaa tca ggt gtc cta gag act ccc act gtt gtt 12348Cys Ser Glu Thr Glu Ser Gly Val Leu Glu Thr Pro Thr Val Val 4090 4095 4100 cca gtt cca agc atg gag gct cat tct gaa gca gca cca act gag 12393Pro Val Pro Ser Met Glu Ala His Ser Glu Ala Ala Pro Thr Glu 4105 4110 4115 caa acc cct gtg gtc cgg cag tgc tac cat ggt aat ggc cag agt 12438Gln Thr Pro Val Val Arg Gln Cys Tyr His Gly Asn Gly Gln Ser 4120 4125 4130 tat cga ggc aca ttc tcc acc act gtc aca gga agg aca tgt caa 12483Tyr Arg Gly Thr Phe Ser Thr Thr Val Thr Gly Arg Thr Cys Gln 4135 4140 4145 tct tgg tca tcc atg aca cca cac cgg cat cag agg acc cca gaa 12528Ser Trp Ser Ser Met Thr Pro His Arg His Gln Arg Thr Pro Glu 4150 4155 4160 aac tac cca aat gat ggc ctg aca atg aac tac tgc agg aat cca 12573Asn Tyr Pro Asn Asp Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro 4165 4170 4175 gat gcc gat aca ggc cct tgg tgt ttt acc atg gac ccc agc atc 12618Asp Ala Asp Thr Gly Pro Trp Cys Phe Thr Met Asp Pro Ser Ile 4180 4185 4190 agg tgg gag tac tgc aac ctg acg cga tgc tca gac aca gaa ggg 12663Arg Trp Glu Tyr Cys Asn Leu Thr Arg Cys Ser Asp Thr Glu Gly 4195 4200 4205 act gtg gtc gct cct ccg act gtc atc cag gtt cca agc cta ggg 12708Thr Val Val Ala Pro Pro Thr Val Ile Gln Val Pro Ser Leu Gly 4210 4215 4220 cct cct tct gaa caa gac tgt atg ttt ggg aat ggg aaa gga tac 12753Pro Pro Ser Glu Gln Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr 4225 4230 4235 cgg ggc aag aag gca acc act gtt act ggg acg cca tgc cag gaa 12798Arg Gly Lys Lys Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Glu 4240 4245 4250 tgg gct gcc cag gag ccc cat aga cac agc acg ttc att cca ggg 12843Trp Ala Ala Gln Glu Pro His Arg His Ser Thr Phe Ile Pro Gly 4255 4260 4265 aca aat aaa tgg gca ggt ctg gaa aaa aat tac tgc cgt aac cct 12888Thr Asn Lys Trp Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro 4270 4275 4280 gat ggt gac atc aat ggt ccc tgg tgc tac aca atg aat cca aga 12933Asp Gly Asp Ile Asn Gly Pro Trp Cys Tyr Thr Met Asn Pro Arg 4285 4290 4295 aaa ctt ttt gac tac tgt gat atc cct ctc tgt gca tcc tct tca 12978Lys Leu Phe Asp Tyr Cys Asp Ile Pro Leu Cys Ala Ser Ser Ser 4300 4305 4310 ttt gat tgt ggg aag cct caa gtg gag ccg aag aaa tgt cct gga 13023Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly 4315 4320 4325 agc att gta ggg ggg tgt gtg gcc cac cca cat tcc tgg ccc tgg 13068Ser Ile Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp 4330 4335 4340 caa gtc agt ctc aga aca agg ttt gga aag cac ttc tgt gga ggc 13113Gln Val Ser Leu Arg Thr Arg Phe Gly Lys His Phe Cys Gly Gly 4345 4350 4355 acc tta ata tcc cca gag tgg gtg ctg act gct gct cac tgc ttg 13158Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu 4360 4365 4370 aag aag tcc tca agg cct tca tcc tac aag gtc atc ctg ggt gca 13203Lys Lys Ser Ser Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala 4375 4380 4385 cac caa gaa gtg aac ctc gaa tct cat gtt cag gaa ata gaa gtg 13248His Gln Glu Val Asn Leu Glu Ser His Val Gln Glu Ile Glu Val 4390 4395 4400 tct agg ctg ttc ttg gag ccc aca caa gca gat att gcc ttg cta 13293Ser Arg Leu Phe Leu Glu Pro Thr Gln Ala Asp Ile Ala Leu Leu 4405 4410 4415 aag cta agc agg cct gcc gtc atc act gac aaa gta atg cca gct 13338Lys Leu Ser Arg Pro Ala Val Ile Thr Asp Lys Val Met Pro Ala 4420 4425 4430 tgt ctg cca tcc cca gac tac atg gtc acc gcc agg act gaa tgt 13383Cys Leu Pro Ser Pro Asp Tyr Met Val Thr Ala Arg Thr Glu Cys 4435 4440 4445 tac atc act ggc tgg gga gaa acc caa ggt acc ttt ggg act ggc 13428Tyr Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Thr Gly 4450 4455 4460 ctt ctc aag gaa gcc cag ctc ctt gtt att gag aat gaa gtg tgc 13473Leu Leu Lys Glu Ala Gln Leu Leu Val Ile Glu Asn Glu Val Cys 4465 4470 4475 aat cac tat aag tat att tgt gct gag cat ttg gcc aga ggc act 13518Asn His Tyr Lys Tyr Ile Cys Ala Glu His Leu Ala Arg Gly Thr 4480 4485 4490 gac agt tgc cag ggt gac agt gga ggg cct ctg gtt tgc ttc gag 13563Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu 4495 4500 4505 aag gac aaa tac att tta caa gga gtc act tct tgg ggt ctt ggc 13608Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly 4510 4515 4520 tgt gca cgc ccc aat aag cct ggt gtc tat gct cgt gtt tca agg 13653Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Ala Arg Val Ser Arg 4525 4530 4535 ttt gtt act tgg att gag gga atg atg aga aat aat taa ttggacggga 13702Phe Val Thr Trp Ile Glu Gly Met Met Arg Asn Asn 4540 4545 gacagagtga agcatcaacc tacttagaag ctgaaacgtg ggtaaggatt tagcatgctg 13762gaaataatag acagcaatca aacgaagaca ctgttcccag ctaccagcta tgccaaacct 13822tggcattttt ggtatttttg tgtataagct tttaaggtct gactgacaaa ttctgtatta 13882aggtgtcata gctatgacat ttgttaaaaa taaactctgc acttattttg atttga 13938525DNAArtificial SequencePCR Primer 5cagctcctta ttgttatacg aggga 25618DNAArtificial SequencePCR Primer 6tgcgtctgag cattgcgt 18724DNAArtificial SequencePCR Probe 7cccggtgtca ggtgggagta ctgc 24819DNAArtificial SequencePCR Primer 8gaaggtgaag gtcggagtc 19920DNAArtificial SequencePCR Primer 9gaagatggtg atgggatttc 201020DNAArtificial SequencePCR Probe 10caagcttccc gttctcagcc 201120DNAArtificial SequenceAntisense Oligonucleotide 11ggcaggtcct tcctgtgaca 201220DNAArtificial SequenceAntisense Oligonucleotide 12tctgcgtctg agcattgcgt 201320DNAArtificial SequenceAntisense Oligonucleotide 13aagcttggca ggttcttcct 201420DNAArtificial SequenceAntisense Oligonucleotide 14tcggaggcgc gacggcagtc 201520DNAArtificial SequenceAntisense Oligonucleotide 15cggaggcgcg acggcagtcc 201620DNAArtificial SequenceAntisense Oligonucleotide 16ggcaggttct tcctgtgaca 201720DNAArtificial SequenceAntisense Oligonucleotide 17ataacaataa ggagctgcca 201820DNAArtificial SequenceAntisense Oligonucleotide 18gaccaagctt ggcaggttct 201920DNAArtificial SequenceAntisense Oligonucleotide 19taacaataag gagctgccac 202020DNAArtificial SequenceAntisense Oligonucleotide 20tgaccaagct tggcaggttc 202120DNAArtificial SequenceAntisense Oligonucleotide 21ttctgcgtct gagcattgcg 202220DNAArtificial SequenceAntisense Oligonucleotide 22aacaataagg agctgccaca 202320DNAArtificial SequenceAntisense Oligonucleotide 23acctgacacc gggatccctc 202420DNAArtificial SequenceAntisense Oligonucleotide 24ctgagcattg cgtcaggttg 202520DNAArtificial SequenceAntisense Oligonucleotide 25agtagttcat gatcaagcca 202620DNAArtificial SequenceAntisense Oligonucleotide 26gacggcagtc ccttctgcgt 202720DNAArtificial SequenceAntisense Oligonucleotide 27ggcaggttct tccagtgaca 202820DNAArtificial SequenceAntisense Oligonucleotide 28tgaccaagct tggcaagttc 202920DNAArtificial SequenceAntisense Oligonucleotide 29tataacacca aggactaatc 203020DNAArtificial SequenceAntisense Oligonucleotide 30ccatctgaca ttgggatcca 203120DNAArtificial SequenceAntisense Oligonucleotide 31tgtggtgtca tagaggacca 203220DNAArtificial SequenceAntisense Oligonucleotide 32atgggatcct ccgatgccaa 203320DNAArtificial SequenceAntisense Oligonucleotide 33acaccaaggg cgaatctcag 203420DNAArtificial SequenceAntisense Oligonucleotide 34ttctgtcact ggacatcgtg 203520DNAArtificial SequenceAntisense Oligonucleotide 35cacacggatc ggttgtgtaa 203620DNAArtificial SequenceAntisense Oligonucleotide 36acatgtcctt cctgtgacag 203720DNAArtificial SequenceAntisense Oligonucleotide 37cagaaggagg ccctaggctt 203820DNAArtificial SequenceAntisense Oligonucleotide 38ctggcggtga ccatgtagtc 203920DNAArtificial SequenceAntisense Oligonucleotide 39tctaagtagg ttgatgcttc 204020DNAArtificial SequenceAntisense Oligonucleotide 40tccttaccca cgtttcagct 204120DNAArtificial SequenceAntisense Oligonucleotide 41ggaacagtgt cttcgtttga 204220DNAArtificial SequenceAntisense Oligonucleotide 42gtttggcata gctggtagct 204320DNAArtificial SequenceAntisense Oligonucleotide 43accttaaaag cttatacaca 204420DNAArtificial SequenceAntisense Oligonucleotide 44atacagaatt tgtcagtcag 204520DNAArtificial SequenceAntisense Oligonucleotide 45gtcatagcta tgacacctta 204620DNAHomo sapiens 46tgtcacagga aggacctgcc 204720DNAHomo sapiens 47acgcaatgct cagacgcaga 204820DNAHomo sapiens 48gactgccgtc gcgcctccga 204920DNAHomo sapiens 49tgtcacagga agaacctgcc 205020DNAHomo sapiens 50tggcagctcc ttattgttat 205120DNAHomo sapiens 51agaacctgcc aagcttggtc 205220DNAHomo sapiens 52gtggcagctc cttattgtta 205320DNAHomo sapiens 53cgcaatgctc agacgcagaa 205420DNAHomo sapiens 54gagggatccc ggtgtcaggt 205520DNAHomo sapiens 55tggcttgatc atgaactact 205620DNAHomo sapiens 56tggatcccaa tgtcagatgg 205720DNAHomo sapiens 57tggtcctcta tgacaccaca 205820DNAHomo sapiens 58ttggcatcgg aggatcccat 205920DNAHomo sapiens 59ctgagattcg cccttggtgt 206020DNAHomo sapiens 60cacgatgtcc agtgacagaa 206120DNAHomo sapiens 61ttacacaacc gatccgtgtg 206220DNAHomo sapiens 62ctgtcacagg aaggacatgt 206320DNAHomo sapiens 63gactacatgg tcaccgccag 206420DNAHomo sapiens 64gaagcatcaa cctacttaga 206520DNAHomo sapiens 65agctgaaacg tgggtaagga 206620DNAHomo sapiens 66tcaaacgaag acactgttcc 206720DNAHomo sapiens 67agctaccagc tatgccaaac 206820DNAHomo sapiens 68tgtgtataag cttttaaggt 206920DNAHomo sapiens 69taaggtgtca tagctatgac 207020DNAArtificial SequenceAntisense Oligonucleotide 70ctcttactgt gctgtggaca 207120DNAArtificial SequencePCR Primer 71ggcaaattca acggcacagt 207220DNAArtificial SequencePCR Primer 72gggtctcgct cctggaagat

207327DNAArtificial SequencePCR Probe 73aaggccgaga atgggaagct tgtcatc 27

Claims

1. A method of inhibiting apolipoprotein (a) (apo(a)) expression in an animal comprising administering to the animal a compound comprising a modified oligonucleotide 15-30 linked nucleosides in length targeted to apo(a), wherein expression of apo(a) is reduced by at least 30% in the animal.

2. The method of claim 1, wherein reducing apo(a) expression in the animal (a) reduces Lp(a) levels; (b) reduces cholesterol levels; and/or (c) treats a cardiovascular disease.

3. The method of claim 1, wherein the modified oligonucleotide has a nucleobase sequence at least 90%, at least 95% or 100% complementary to SEQ ID NO: 4, as measured over the entirety of said modified oligonucleotide.

4. The method of claim 1, wherein the modified oligonucleotide has a nucleobase sequence comprising at least 8 contiguous nucleobases selected from any of SEQ ID NOs: 12-14, 16-19, 21, 25, 30-36, 38, 41-43 or 45.

5. The method of claim 1, wherein at least one internucleoside linkage of said modified oligonucleotide comprises a modified internucleoside linkage, at least one nucleoside of said modified oligonucleotide comprises a modified sugar and/or at least one nucleoside of said modified oligonucleotide comprises a modified nucleobase.

6. The method of claim 5, wherein at least one internucleoside linkage comprises a phosphorothioate internucleoside linkage.

7. The method of claim 5, wherein at least one modified sugar comprises a bicyclic sugar.

8. The method of claim 5, wherein at least one modified sugar comprises a 2'-O-methoxyethyl.

9. The method of claim 5, wherein the modified nucleobase is a 5-methylcytosine.

10. The method of claim 1, wherein the modified oligonucleotide consists of 20 linked nucleosides.

11. The method of claim 1, wherein the modified oligonucleotide comprises: a. a gap segment consisting of linked deoxynucleosides; b. a 5' wing segment consisting of linked nucleosides; and c. a 3' wing segment consisting of linked nucleosides; wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

12. The method of claim 1, wherein the modified oligonucleotide consists of 20 linked nucleosides and comprises: a. a gap segment consisting of ten linked deoxynucleosides; b. a 5' wing segment consisting of five linked nucleosides; and c. a 3' wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.

13. The method of claim 1, wherein the apo(a) levels are reduced by at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90%.

14. A compound comprising a modified oligonucleotide 15 to 30 linked nucleosides in length targeting apo(a) which comprises: a. a gap segment consisting of linked deoxynucleosides; b. a 5' wing segment consisting of linked nucleosides; and c. a 3' wing segment consisting of linked nucleosides; wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

15. The compound of claim 14, wherein the modified oligonucleotide targeting apo(a) has a nucleobase sequence comprising at least 8 contiguous nucleobases selected from any of SEQ ID NOs: 12-14, 16-19, 21, 25, 30-36, 38, 41-43 or 45.

16. The compound of claim 14, wherein the modified oligonucleotide has a nucleobase sequence at least 90%, at least 95% or 100% complementary to SEQ ID NO: 4, as measured over the entirety of said modified oligonucleotide.

17. The compound of claim 14, wherein the modified sugar comprises a bicyclic sugar.

18. The compound of claim 14, wherein the modified sugar comprises a 2'-O-methoxyethyl.

19. The compound of claim 14, wherein at least one internucleoside linkage is a phosphorothioate internucleoside linkage.

20. The compound of claim 14, further comprising a modified nucleobase.

21. The compound of claim 20, wherein the modified nucleobase is a 5-methylcytosine.