# Patent

## Title
Modulation of eIF4E-BP2 expression

## Abstract

Compounds, compositions and methods are provided for modulating the expression of eIF4E-BP2. The compositions comprise oligonucleotides, targeted to nucleic acid encoding eIF4E-BP2. Methods of using these compounds for modulation of eIF4E-BP2 expression and for diagnosis and treatment of diseases and conditions associated with expression of eIF4E-BP2 are provided.

## Background
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of eIF4E-BP2. In particular, this invention relates to antisense compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding eIF4E-BP2. Such compounds are shown herein to modulate the expression of eIF4E-BP2.
BACKGROUND OF THE INVENTION
Eukaryotic gene expression must be regulated such that cells can rapidly respond to a wide range of different conditions. The process of mRNA translation is one step at which gene expression is highly regulated. In response to hormones, growth factors, cytokines and nutrients, animal cells generally activate translation in preparation for the proliferative response. The rate of protein synthesis typically decreases under stressful conditions, such as oxidative or osmotic stress, DNA damage or nutrient withdrawal. Activation or suppression of mRNA translation occurs within minutes and control over this process is thought to be exerted at the initiation phase of protein synthesis (Rosenwald et al., Oncogene, 1999, 18, 2507-2517; Strudwick and Borden, Differentiation, 2002, 70, 10-22).
Translation initiation necessitates the coordinated activities of several eukaryotic initiation factors (eIFs), proteins which are classically defined by their cytoplasmic location and ability to regulate the initiation phase of protein synthesis. One of these factors, eukaryotic initiation factor 4E (eIF4E), is present in limiting amounts relative to other initiation factors and is one component of the eIF4F initiation complex, which is also comprised of the scaffold protein eIF4G and the RNA helicase eIF4A. In the cytoplasm, eIF4E catalyzes the rate-limiting step of cap-dependent protein synthesis by specifically binding to the 5′ terminal 7-methyl GpppX cap structure present on nearly all mature cellular mRNAs, which serves to deliver the mRNAs to the eIF4F complex. Once bound, the eIF4F complex scans from the 5′ to the 3′ end of the cap, permitting the RNA helicase activity of eIF4A to resolve any secondary structure present in the 5′ untranslated region (UTR), thus revealing the translation initiation codon and facilitating ribosome loading onto the mRNA (Graff and Zimmer, Clin. Exp. Metastasis, 2003, 20, 265-273; Strudwick and Borden, Differentiation, 2002, 70, 10-22).
eIF4E availability for incorporation into the eIF4E complex is regulated through phosphorylation as well as through the binding of inhibitory proteins. eIF4E is a phosphoprotein that is phosphorylated on serine 209 by the mitogen-activated protein kinase-interacting kinase Mnk1, as well as by protein kinase C (Flynn and Proud, J. Biol. Chem., 1995, 270, 21684-21688; Wang et al., J. Biol. Chem., 1998, 273, 9373-9377; Waskiewicz et al., Embo J., 1997, 16, 1909-1920). The inhibitory eIF4E-binding proteins 1 and 2 (eIF4E-BP1 and eIF4E-BP2) act as effective inhibitors of cap-dependent translation by competing with eIF4G for binding to the dorsal surface of eIF4E (Pause et al., Nature, 1994, 371, 762-767; Ptushkina et al., Embo J., 1999, 18, 4068-4075). When complexed with bp1, eIF4E is not a substrate for phosphorylation by protein kinase C or Mnk1, indicating that dissociation of bp 1 from eIF4E is a prerequisite for eIF4E phosphorylation (Wang et al., J. Biol. Chem., 1998, 273, 9373-9377; Whalen et al., J Biol Chem, 1996, 271, 11831-11837). Phosphorylation of eIF4E increases its affinity for mRNA caps, thus elevating translation rates (Waskiewicz et al., Mol. Cell. Biol., 1999, 19, 1871-1880).
eIF4E-BP2 (also known as PHAS-II; 4EBP2; 4E-binding protein 2; EIF4EBP2) was cloned through use of the eIF4E protein in probing a cDNA expression library (Hu et al., Proc Natl Acad Sci U S A, 1994, 91, 3730-3734; Pause et al., Nature, 1994, 371, 762-767). eIF4E-BP2 is ubiquitously expressed in human tissues, including heart, brain, placenta, lung, liver, kidney and spleen, as well as adipose tissue and skeletal muscle, the major insulin-responsive tissues (Hu et al., Proc Natl Acad Sci U S A, 1994, 91, 3730-3734; Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The human gene maps to chromosome 10q21-q22 (Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The mouse bp1 gene consists of three exons, spans approximately 20 kb and maps to mouse chromosome 10 (Tsukiyama-Kohara et al., Genomics, 1996, 38, 353-363). The expression of eIF4E-BP2 does not appear to be altered in mice bearing a systemic disruption of bp1 (Blackshear et al., J Biol Chem, 1997, 272, 31510-31514).
Rather than preventing the binding of eIF4E to mRNA caps, eIF4E-BP2 prohibits the binding of eIF4E to eIF4G, thereby preventing formation of a complex that is necessary for efficient binding and proper positioning of the 40S ribosomal subunit on the target mRNA. When eIF4E-BP2 is bound to eIF4E, eIF4E does not serve as a substrate for phosphorylation by protein kinase C, suggesting that dissociation of eIF4E-BP2 from eIF4E is a prerequisite for phosphorylation of eIF4E (Whalen et al., J Biol Chem, 1996, 271, 11831-11837). The region to which eIF4E binds is a common motif shared by eIF4G and eIF4E-BP2, and point mutations in this region of eIF4E-BP2 abolish binding to eIF4E (Mader et al., Mol Cell Biol 1995, 15, 4990-4997). Two conserved motifs are present in the eIF4E-BP2: the RAIP motif, which is found in the NH2-terminal region of EIF4E-BP2 and the TOS motif, which is formed by the last five amino acids of eIF4E-BP2 (Schalm and Blenis, Curr Biol, 2002, 12, 632-639; Tee and Proud, Mol Cell Biol, 2002, 22, 1674-1683).
Like eIF4E-BP1, insulin stimulates the phosphorylation of eIF4E-BP2 in cultured cells, which promotes the release of eIF4E-BP2 from eIF4E and allows for cap-dependent translation to proceed Ferguson et al., J Biol Chem, 2003, 278, 47459-47465). Mitogen-activated protein kinase, the major insulin-stimulated kinase in rat adipocytes, can phosphorylate recombinant eIF4E-BP2 in vitro. However, treatment of 3T3-L1 rat adipocytes with rapamycin attenuates the effects of insulin on the phosphorylation of eIF4E-BP2, indicating that elements of the mTOR signaling pathway mediate the actions of insulin on eIF4E-BP2 (Lin and Lawrence, J Biol Chem, 1996, 271, 30199-30204). Additionally, serine-65 of eIF4E-BP2 represents an ideal consensus site for phosphorylation by cyclicAMP-dependent protein kinase. In rat 3T3-L1 adipocytes, where insulin or epidermal growth factor markedly increased the phosphorylation of eIF4E-BP2, compounds that increase cyclic AMP decrease the amount of radiolabeled phosphate incorporated into eIF4E-BP2, and attenuate the effects of insulin on increasing the phosphorylation of eIF4E-BP2. Incubation of eIF4E-BP2 with the catalytic subunit of cyclic AMP-dependent protein kinase results in the rapid phosphorylation of eIF4E-BP2. Together, these data suggest that increasing cyclic AMP may selectively increase eIF4E-BP2 phosphorylation (Lin and Lawrence, J Biol Chem, 1996, 271, 30199-30204).
Induction of cellular differentiation and reduction of cellular proliferation are concomitant with a reduction in translation rates, as is observed in conduction with differential regulation of eIF4E-BPs during human myeloid cell differentiation. When induced to differentiate into monocytes/macrophages, cells from the HL-60 promyelocytic leukemia cell or U-937 monoblastic cell lines exhibit a decrease in the phosphorylation of bp1. In contrast, when HL-60 cells are stimulated to differentiate into granulocytic cells, the amount of bp1 is decreased, whereas phosphorylation of bp1 is not affected. Conversely, eIF4E-BP2 levels are markedly increased. These findings suggest that translation machinery is differentially regulated during human myeloid cell differentiation (Grolleau et al., J Immunol, 1999, 162, 3491-3497).
The disregulation of signaling networks that promote cell proliferation is often observed in association with cancer (Lawrence and Abraham, Trends Biochem Sci, 1997, 22, 345-349). Expression of excess eIF4E-BP2 in cells transformed by eIF4E or v-src results in significant reversion of the transformed phenotype, demonstrating that eIF4E-BP2 can function as an inhibitor of cell growth (Rousseau et al., Oncogene, 1996, 13, 2415-2420).
The U.S. Pat. No. 6,410,715 describes a purified human nucleic acid sequence encoding a cellular component that binds to eIF4E comprising a coding sequence for the protein eIF4E-BP2, and discloses a method for screening a non-hormone agent potentially useful to treat a hormone disorder (Sonenberg et al., 2000).
Currently, there are no known therapeutic agents that target eIF4E-BP2. Consequently, there remains a long felt need for agents capable of effectively inhibiting eIF4E-BP2. Antisense technology is an effective means of reducing the expression of specific gene products and therefore is uniquely useful in a number of therapeutic, diagnostic and research applications for the modulation of eIF4E-BP2 expression.
The present invention provides compositions and methods for inhibiting eIF4E-BP2 expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding eIF4E-BP2, and which modulate the expression of eIF4E-BP2. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of eIF4E-BP2 and methods of modulating the expression of eIF4E-BP2 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 eIF4E-BP2 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
Provided herein are antisense compounds 12 to 80 nucleobases in length targeted to a nucleic acid molecule encoding eIF4E-BP2, wherein said compounds are complementary to said nucleic acid molecule encoding eIF4E-BP2 and inhibit the expression of eIF4E-BP2 mRNA. In one embodiment, the antisense compounds are 12 to 50 nucleobases in length or 15 to 30 nucleobases in length. In some embodiments, the antisense compound is an oligonucleotide, a DNA oligonucleotide, or an RNA oligonucleotide. In some embodiments, the antisense compound is a double-stranded oligonucleotide. In a preferred embodiment, the antisense compound is a chimeric oligonucleotide. In a preferred embodiment, at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
The antisense compounds of the invention may have at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% complementarity with said nucleic acid molecule encoding eIF4E-BP2. Also provided herein are antisense compounds having at least one modified internucleoside linkage, sugar moiety, or nucleobase. In one embodiment, the antisense compounds of the invention have at least one 2′-O-methoxyethyl sugar moiety or at least one phosphorothioate internucleoside linkage or both.
Provided herein are antisense nucleic acid molecules specifically hybridizable within a region of a nucleic acid molecule encoding eIF4E-BP2 selected from a 5′-untranslated region (5′UTR), a start region, a coding region, a stop region, or a 3′-untranslated region (3′UTR). Also provided are antisense compounds targeted to nucleotides 146-165 in the 5′ UTR, nucleotides 372-391, 420-520 or 544-593 in the coding region, nucleotides 589-608 in the stop codon region, nucleotides 623-766, 803-940, 1105-1599, 1868-1887, 1900-1919, 1962-1981, 2218-2242, 2377-2401, 2449-2490, 2536-2555 or 2578-2597 in the 3′ UTR, all of SEQ ID NO: 4; nucleotides 8892-8911 and 11559-11937 in intron 1, and nucleotides 17941-17960 in the intron 1:exon 2 junction, all of SEQ ID NO: 25; nucleotides 2088-2107 in the 3′ UTR of SEQ ID NO: 26; and nucleotides 697-716 in the 3′UTR of SEQ ID NO: 27, wherein the compounds inhibit the expression of human eI4E-BP2 mRNA. Further provided are antisense compounds targeted to nucleotides 9-105 in the 5′UTR; nucleotides 132480 in the coding region; nucleotides 473-492 in the stop codon region; and nucleotides 500-1175, 1222-1638, 1662-1780 in the 3′ UTR, all of SEQ ID NO: 11; nucleotides 365-384 in the 3′ UTR of SEQ ID NO: 107; and nucleotides 36-55 in the 5′ UTR of SEQ ID NO: 108; wherein the compounds inhibit the expression of mouse eIF4E-BP2 mRNA. Also provided herein are antisense compounds targeted to nucleotides 7-26 in the 5′UTR, nucleotides 7-151, 164-247, 270-313, or 303-388 in the coding region; nucleotides 390409 in the stop codon region and nucleotides 402-490 in the 3′ UTR, all of SEQ ID NO: 18; wherein the compounds inhibit the expression of rat eIF4E-BP2 mRNA.
In one embodiment, the antisense compound comprises at least an 8-nucleobase portion of SEQ ID NO 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 or 258. In another embodiment, the antisense compound has a sequence selected from the group consisting of SEQ ID NOs 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 and 258. In another embodiment, the antisense compound has a sequence selected from SEQ ID NO: 29, 31, 32, 33, 60, 61, 65, or 75.
Also provided herein are methods of treating an animal having a disease or condition associated with eIF4E-BP2 comprising administering to said animal a therapeutically or prophylactically effective amount of an antisense compound of the invention so that expression of eIF4E-BP2 is inhibited. In some embodiments, the animal is a human or a rodent. In some embodiments, the disease or condition is a metabolic disease or condition. In other embodiments, the disease or condition is diabetes, metabolic syndrome X, obesity, hyperlipidemia, prediabetes, or elevated blood triglycerides. In one embodiment, the disease or condition is Type 2 diabetes. Also provided herein are methods of decreasing blood glucose levels in an animal in need of such treatment, comprising administering to said animal an antisense compound of the invention. In some embodiments, the blood glucose levels are plasma glucose levels or serum glucose levels. In some embodiments, the animal is a diabetic animal.
Further provided are methods of improving insulin sensitivity in an animal in need of such treatment, comprising administering to said animal a compound of the invention. In some embodiments the animal is diabetic, obese, or hyperinsulinemic. Also provided are methods of decreasing blood lipid levels in an animal comprising administering to said animal an antisense compound of the invention. In some embodiments, the blood lipid levels are blood triglyceride levels. In some embodiments, the blood lipid levels are serum or plasma lipid levels.
Another aspect of the present invention is method of treating obesity, hyperglycemia, metabolic syndrome, diabetes, hyperlipidemia or insulin resistance in a subject comprising administering to said subject an antisense compound 12-80 nucleobases in length targeted to a nucleic acid molecule encoding eIF4E-BP2, wherein said compound inhibits the expression of eIF4E-BP2. In one embodiment the compound has at least 80% complementarity to a nucleic acid molecule encoding eIF4E-BP2 and comprising at least an 8-nucleobase portion of SEQ ID NO 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 or 258. In some embodiments, the antisense compound is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% complementary to a nucleic acid molecule encoding eIF4E-BP2.
Also provided are methods of decreasing body fat, decreasing hepatic triglyceride levels, improving glucose tolerance, and reducing lipogenesis in an animal comprising administering to said animal an antisense compound 12-80 nucleobases in length targeted to a nucleic acid molecule encoding eIF4E-BP2, wherein said compound inhibits the expression of eIF4E-BP2. In one embodiment the compound has at least with at least 80% complementarity to a nucleic acid molecule encoding eIF4E-BP2 and comprising at least an 8-nucleobase portion of SEQ ID NO 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 or 258. In some embodiments, the antisense compound is at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% complementary to a nucleic acid molecule encoding eIF4E-BP2. In some embodiments, the animal has steatosis. In some embodiments, the steatosis is steatohepatitis or NASH. Another aspect of the present invention is a method of reducing expression of genes involved in lipogenesis in the fat tissue of an animal comprising administering to said animal an antisense compound of the invention. In some embodiments, the genes involved in lipogenesis are DGAT2 and FAS. Also provided are methods of modulating hepatic expression of genes involved in glucose metabolism in an animal comprising administering to said animal an antisense compound of the invention. In one embodiment, the genes involved in glucose metabolism are glucose-6-phosphatase and glycogen synthase. In some embodiments, said animal has a metabolic disease or condition. In some embodiments, the metabolic disease or condition is obesity, hyperlipidemia, hyperglycemia, diabetes, metabolic syndrome, or insulin resistance. In a particular embodiment, the metabolic disease or condition is Type 2 diabetes.
In some embodiments, the antisense compound has the nucleobase sequence of SEQ ID NO 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 or 258. In some embodiments, the antisense compound is characterized by a 10-deoxynucleotide gap flanked on its 5′ and 3′ ends by 5 2′-MOE nucleotides. In some embodiments, at least one cytosine is a 5-methylcytosine or at least one internucleoside linkage is a phosphorothioate linkage. In one embodiment, all of the cytosines in the antisense compound are 5-methylcytosines. In another embodiment, all of the internucleoside linkages are phosphorothioate linkages.
A. Overview of the Invention
“Antisense mechanisms” encompass hybridization of an antisense compound, antisense oligonucleotide, or antisense oligonucleotide compound with a target nucleic acid, such that the outcome of hybridization is achievement of a desired effect. The desired effect can include, for example, target degradation, target occupancy with concomitant stalling of cellular machinery involving, for example, transcription or splicing, or other phenotypic effects.
“Antisense inhibition” refers to inhibition of target expression as a result of hybridization of an antisense oligonucleotide with the target nucleic acid. In some embodiments, antisense inhibition is evidenced by reduction in target nucleic acid levels, levels of protein encoded by the target nucleic acid, or degree of phenotypic change related to expression of target nucleic acid.
The present invention employs antisense compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding eIF4E-BP2. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding eIF4E-BP2. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding eIF4E-BP2” have been used for convenience to encompass DNA encoding eIF4E-BP2, 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”. 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.
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 eIF4E-BP2. 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.
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 which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
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, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
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 and 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.
“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 which 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.
It is understood in the art that the sequence of an antisense compound need not 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 (e.g., a loop structure or hairpin structure). It is preferred that 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, more preferably that they comprise at least 90% sequence complementarity and even more preferably 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 1-8 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).
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 preferred embodiments, homology, sequence identity or complementarity, is between about 70% and about 80%. In more preferred embodiments, homology, sequence identity or complementarity, is between about 80% and about 90%. In some preferred embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.
B. Compounds of the Invention
According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which 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, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. 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.
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.
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.
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 which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).
The antisense compounds of the present invention also include modified compounds in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenosine, modified compounds may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the antisense compound. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of eIF4E-BP2 mRNA.
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.
While oligonucleotides are a preferred form of the antisense compounds of this invention, the present invention comprehends other families of antisense compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.
The antisense 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.
In one preferred embodiment, the antisense compounds of the invention are 13 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 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.
In another preferred embodiment, the antisense 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.
Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 12 to about 30 nucleobases. In another embodiment, the compounds of the invention are oligonucleotides from about 20 to about 30 nucleobases in length. In another embodiment, the compounds of the invention are oligonucleotides from about 13 to about 30 nucleobases in length.
Antisense compounds 12-80 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.
Exemplary preferred 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 preferred 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). It is also understood that preferred antisense compounds may be represented by oligonucleotide sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of an illustrative preferred antisense compound, and may extend in either or both directions until the oligonucleotide contains about 8 to about 80 nucleobases.
Antisense compounds 12 to 80 nucleobases in length comprising a stretch of at least twelve (12) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well. It is understood that preferred antisense compounds may be represented by oligonucleotide sequences that comprise at least 12 consecutive nucleobases from an internal portion of the sequence of an illustrative preferred antisense compound, and may extend in either or both directions until the oligonucleotide contains about 12 to about 80 nucleobases.
One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.
C. Targets of the Invention
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 eIF4E-BP2.
The terms “Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all refer to any nucleic acid capable of being targeted by compounds and methods disclosed herein. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) or a nucleic acid molecule from an infectious organism. As disclosed herein, a target nucleic acid molecule encodes eIF4E-BP2 or a fragment thereof. As used herein, the terms “eIF4E-BP2 target nucleic acid” and “nucleic acid encoding eIF4E-BP2” encompass nucleic acid, including, for example, DNA (including, for example, cDNA), RNA (including, for example pre-mRNA, mRNA, and miRNA) transcribed from DNA encoding eIF4E-BP2, and also cDNA derived from such RNA. In one embodiment, a nucleic acid encoding human eIF4E-BP2 is GENBANK® Accession No. NM—004096.3, incorporated herein as SEQ ID NO: 4. In another embodiment, a nucleic acid encoding mouse eIF4E-BP2 is GENBANK® Accession No. NM—010124.1, incorporated herein as SEQ ID NO: 11. In another embodiment, a nucleic acid encoding rat eIF4E-BP2 is GENBANK® Accession No. NM—215414.1, incorporated herein as SEQ ID NO: 18. “eIF4E-BP2 mRNA” refers to an mRNA encoding a eIF4E-BP2 protein.
“Targeting” refers to the process of design and selection of a compound that will specifically hybridize to the target nucleic acid molecule and induce a desired effect. A compound designed or selected by this process is referred to as “targeted to” the target nucleic acid molecule. Targeting includes determination of at least one “target segment,” to which the compound hybridizes, such that a desired effect occurs. In some embodiments, the desired effect is reduction of target nucleic acid levels, reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid. “mRNA levels” refers to the relative amount of a particular mRNA in a particular sample, while “protein levels” refers to the relative amount of protein in a particular sample.
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.
“Targeting” refers to the process of design and selection of a compound that will hybridize to the target nucleic acid molecule and inducing a desired effect. A compound designed or selected by this process is referred to as “targeted to” the target nucleic acid molecule. Targeting includes determination of at least one “target segment,” to which the compound hybridizes, such that a desired effect occurs. In some embodiments, the desired effect is reduction of target nucleic acid levels, reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid. “mRNA levels” refers to the relative amount of a particular mRNA in a particular sample, while “protein levels” refers to the relative amount of protein in a particular sample.
As used herein “Target region” encompasses a fragment of a target nucleic acid to which a particular compound is targeted, containing one or more “target segments.” “Target segment” refers to the sequence of nucleotides of a target nucleic acid to which a particular compound is targeted. “5′ target site” refers to the 5′-most nucleotide of the particular target segment to which a compound is targeted. “3′ target site” refers to the 3′-most nucleotide of the particular target segment, to which a compound is targeted. In one embodiment, a target region is a structurally defined region of the nucleic acid. For example, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, a coding region, a translation initiation region, translation termination region, any combination thereof, or other defined nucleic acid region. In another embodiment, a target region is a region of the target nucleic acid encompassing a plurality of target segments. For example, a target region may be the sequence spanning the region from the 5′ target site of a first target segment within the region, to the 3′ target site of the final target segment within the region. Alternatively, a target region may be a region identified by other means. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. Thus, multiple antisense compounds may hybridize to overlapping sequences on a particular target region. Target segments may be contiguous. Target segments within a target region may be separated by nucleotides. In one embodiment, target segments within a target region are separated by no more than about 10 nucleotides on the target nucleic acid. In another embodiment, target segments within a target region are separated by no more than about 5 nucleotides on the target nucleic acid. Suitable target segments may be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, or an exon, or any combination thereof. Target segments containing a start codon or a stop codon are also suitable for targeting.
The determination of target segments or target regions for selecting antisense compounds includes a comparison of the target nucleic acid sequence to other nucleic acid sequences, to prevent the selection of antisense compound sequences that might hybridize in a non-specific manner to “non-target” sequences or “off target sequences.” Herein, “non-target” or “off-target” sequences encompass sequences other than those encoding eIF4E-BP2. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids.
As used herein, “active target region” refers to any target region on a target nucleic acid to which one or more “active antisense compounds” binds. An “active target segment” is the specific sequence of nucleic acids to which an active antisense compound binds. “Active antisense compounds” are those that hybridize to the target nucleic acid, causing a desired effect. In one embodiment, a desired effect is effectively reducing or inhibiting target expression. Effective reduction or inhibition can be evidenced by reduction in mRNA or protein levels or by phenotypic changes. In other embodiments, other desired effects may be used to determine an “active target region.”
Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding eIF4E-BP2, regardless of the sequence(s) of such codons. It is also known in the art that 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).
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 which may be targeted effectively with the antisense compounds of the present invention.
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.
Other target regions include 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), and 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. It is also preferred to target the 5′ cap region.
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. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.
It is also known in the art that 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.
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.
It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that 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 preferred target nucleic acids.
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 which are accessible for hybridization.
While the specific sequences of certain preferred 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 preferred target segments may be identified by one having ordinary skill.
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.
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). It is also understood that preferred antisense target segments may be represented by DNA or RNA sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of an illustrative preferred target segment, and may extend in either or both directions until the oligonucleotide contains about 8 to about 80 nucleobases. One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.
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.
The oligomeric antisense compounds may also be targeted to regions of the target nucleobase sequence (e.g., such as those disclosed in Example 13) comprising nucleobases 1-80, 81-160, 161-240, 241-320, 321-400, 401-480, 481-560, 561-640, 641-720, 721-800, 801-880, 881-960, 961-1040, 1041-1120, 1121-1200, 1201-1280, 1281-1360, 1361-1440, 1441-1520, 1521-1600, 1601-1680, 1681-1760, 1761-1840, 1841-1920, 1921-2000, 2001-2080, 2081-2160, 2161-2240, 2241-2320, 2321-2400, 2401-2480, 2481-2560, 2561-2640, 2641-2720, 2721-2782, or any combination thereof.
In one embodiment of the invention, the antisense compounds are targeted to a nucleic acid molecule encoding human eIF4E-BP2, for example nucleotides 146-165 in the 5′ UTR, nucleotides 372-391, 420-520 or 544-593 in the coding region, nucleotides 589-608 in the stop codon region, nucleotides 623-766, 803-940, 1105-1599, 1868-1887, 1900-1919, 1962-1981, 2218-2242, 2377-2401, 2449-2490, 2536-2555 or 2578-2597 in the 3′UTR, all of SEQ ID NO: 4; nucleotides 8892-8911 and 11559-11937 in intron 1, and nucleotides 17941-17960 in the intron 1:exon 2 junction, all of SEQ ID NO: 25; nucleotides 2088-2107 in the 3′ UTR of SEQ ID NO: 26; and nucleotides 697-716 in the 3′UTR of SEQ ID NO: 27, wherein said compound inhibits the expression of human eIF4E-BP2 mRNA.
In another embodiment of the invention, the antisense compounds are targeted to a nucleic acid molecule encoding mouse eIF4E-BP2, for example nucleotides 9-105 in the 5′UTR; nucleotides 132-480 in the coding region; nucleotides 473-492 in the stop codon region; and nucleotides 500-1175, 1222-1638, 1662-1780 in the 3′ UTR, all of SEQ ID NO: 11; nucleotides 365-384 in the 3′ UTR of SEQ ID NO: 107; and nucleotides 36-55 in the 5′ UTR of SEQ ID NO: 108; wherein said compound inhibits the expression of mouse eIF4E-BP2 mRNA.
In a further embodiment of the invention, antisense compounds are targeted to a nucleic acid molecule encoding rat eIF4E-BP2, for example nucleotides 7-26 in the 5′UTR, nucleotides 7-151, 164-247, 270-313, or 303-388 in the coding region; nucleotides 390-409 in the stop codon region and nucleotides 402-490 in the 3′ UTR, all of SEQ ID NO: 18; wherein said compound inhibits the expression of rat eIF4E-BP2 mRNA.
D. Screening and Target Validation
In a further embodiment, the “preferred target segments” identified herein may be employed in a screen for additional compounds that modulate the expression of eIF4E-BP2. “Modulators” are those compounds that decrease or increase the expression of a nucleic acid molecule encoding eIF4E-BP2 and which comprise at least an 8-nucleobase portion which 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 eIF4E-BP2 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 eIF4E-BP2. 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 eIF4E-BP2, the modulator may then be employed in further investigative studies of the function of eIF4E-BP2, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.
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.
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).
The antisense 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 eIF4E-BP2 and a disease state, phenotype, or condition. These methods include detecting or modulating eIF4E-BP2 comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of eIF4E-BP2 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
The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and 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.
For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used 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.
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 which affect expression patterns.
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).
The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding eIF4E-BP2. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective eIF4E-BP2 inhibitors will also be 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 eIF4E-BP2 and in the amplification of said nucleic acid molecules for detection or for use in further studies of eIF4E-BP2. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding eIF4E-BP2 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 eIF4E-BP2 in a sample may also be prepared.
The specificity and sensitivity of antisense is 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, including ribozymes, 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.
For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of eIF4E-BP2 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 eIF4E-BP2 inhibitor. The eIF4E-BP2 inhibitors of the present invention effectively inhibit the activity of the eIF4E-BP2 protein or inhibit the expression of the eIF4E-BP2 protein. In one embodiment, the activity or expression of eIF4E-BP2 in an animal is inhibited by about 10%. Preferably, the activity or expression of eIF4E-BP2 in an animal is inhibited by about 30%. More preferably, the activity or expression of eIF4E-BP2 in an animal is inhibited by 50% or more. Thus, the oligomeric antisense compounds modulate expression of eIF4E-BP2 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%.
For example, the reduction of the expression of eIF4E-BP2 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 eIF4E-BP2 protein and/or the eIF4E-BP2 protein itself.
The antisense 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.
The compounds of the present inventions are inhibitors of eIF4E-BP2 expression. Thus, the compounds of the present invention are believed to be useful for treating metabolic diseases and conditions, particularly diabetes, obesity, hyperlipidemia or metabolic syndrome X. The compounds of the invention are also believed to be useful for preventing or delaying the onset of metabolic diseases and conditions, particularly diabetes, obesity, hyperlipidemia or metabolic syndrome X. Metabolic syndrome, metabolic syndrome X or simply Syndrome X refers to a cluster of risk factors that include obesity, dyslipidemia, particularly high blood triglycerides, glucose intolerance, high blood sugar and high blood pressure. Scott, C. L., Am J Cardiol. 2003 Jul. 3;92(1A):35i−42i. The compounds of the invention have surprisingly been found to be effective for lowering blood glucose, including plasma glucose, and for lowering blood lipids, including serum lipids, particularly serum cholesterol and serum triglycerides. The compounds of the invention are therefore particularly useful for the treatment, prevention and delay of onset of type 2 diabetes, high blood glucose and hyperlipidemia.
F. Modifications
As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base sometimes referred to as a “nucleobase” or simply a “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)
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.
Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriaminoalkylphosphotriesters, 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 a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
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.
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 CH2 component parts.
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
In other preferred antisense compounds, e.g., 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.
Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —O—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] 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
Modified antisense compounds may also contain one or more substituted sugar moieties. Preferred are antisense compounds, preferably antisense oligonucleotides, comprising 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 C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, 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—CH2CH2OCH3, 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(CH2)2ON(CH3)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—CH2—O—CH2—N(CH3)2, also described in examples hereinbelow.
Other preferred modifications include 2′-methoxy (2′-O—CH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2), 2′-allyl (2′-CH2—CH═CH2), 2′-O-allyl (2′-O—CH2—CH═CH2) 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. Antisense compounds 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.
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 (—CH2—)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 WO 98/39352 and WO 99/14226.
Natural and Modified Nucleobases
Antisense compounds may also include nucleobase (often referred to in the art as heterocyclic base or 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≡C—CH3) 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-deaza-adenine, 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° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
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
Another modification of the antisense compounds of the invention involves chemically linking to the antisense compound one or more moieties or conjugates which 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, phenanthridine, 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 PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure 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-5-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. Antisense compounds 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-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, 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.
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
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.
The present invention also includes antisense compounds which 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. Chimeric antisense oligonucleotides are thus a form of antisense 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.
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
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.
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.
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. For oligonucleotides, presently preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. Sodium salts are presently believed to be more preferred.
The present invention also includes pharmaceutical compositions and formulations which 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.
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.
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 which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
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.
Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which 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.
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 which 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.
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in 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.
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.
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.
One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.
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. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
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.
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. application Ser. No. 09/108,673 (filed Jul. 1, 1998), U.S. application Ser. No. 09/315,298 (filed May 20, 1999) and U.S. application Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.
Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which 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.
Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which 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-chloroethylnitrosurea, 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-hydroxyperoxycyclophosphoramide, 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.
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. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.
H. Dosing
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 EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug 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 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.
NASH
The term “nonalcoholic fatty liver disease” (NAFLD) encompasses a disease spectrum ranging from simple triglyceride accumulation in hepatocytes (hepatic steatosis) to hepatic steatosis with inflammation (steatohepatitis), fibrosis, and cirrhosis. Hepatic steatosis is assessed by specifically measuring lipid content in the liver. Such assessment may be done through 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. Nonalcoholic steatohepatitis (NASH) occurs from progression of NAFLD beyond deposition of triglycerides in the liver. A second-hit capable of inducing necrosis, inflammation, and fibrosis is required for development of NASH. Candidates for the second-hit can be grouped into broad categories: factors causing an increase in oxidative stress and factors promoting expression of proinflammatory cytokines. It has been suggested that increased liver triglycerides lead to increased oxidative stress in hepatocytes of animals and humans, indicating a potential cause-and-effect relationship between hepatic triglyceride accumulation, oxidative stress, and the progression of hepatic steatosis to NASH (Browning and Horton, J. Clin. Invest., 2004, 114, 147-152). Hypertriglyceridemia and hyperfattyacidemia can cause triglyceride accumulation in peripheral tissues (Shimamura et al., Biochem. Biophys. Res. Commun., 2004, 322, 1080-1085).
As shown herein (see Example 16), reduction of 4E-BP2 expression improves liver steatosis. High-fat diet feeding in the C57BL/6 mouse strain, caused severe liver steatosis by showing an over 5-fold increase in liver TG content when comparing it to the low fat diet or lean-saline group. The control ASO treatment did not affect the content. However, 4E-BP2 ASO treatment dramatically reduced liver TG content as compared to both the saline-treated high-fat fed group or control ASO-treated high fat fed group (P<0.01; FIG. 3A).
Metabolic Syndrome
“Metabolic syndrome” is defined as a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. It has been closely linked to the generalized metabolic disorder known as insulin resistance. The National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATPIII) established criteria for diagnosis of metabolic syndrome when three or more of five risk determinants are present. The five risk determinants are abdominal obesity defined as waist circumference of greater than 102 cm for men or greater than 88 cm for women, triglyceride levels greater than or equal to 150 mg/dL, HDL cholesterol levels of less than 40 mg/dL for men and less than 50 mg/dL for women, blood pressure greater than or equal to 130/85 mm Hg and fasting glucose levels greater than or equal to 110 mg/dL. These determinants can be readily measured in clinical practice (JAMA, 2001, 285: 2486-2497).
The World Health Organization definition of metabolic syndrome is diabetes, impaired fasting glucose, impaired glucose tolerance, or insulin resistance (assessed by clamp studies) and at least two of the following criteria: waist-to-hip ratio greater than 0.90 in men or greater than 0.85 in women, serum triglycerides greater than or equal to 1.7 mmol/l or HDL cholesterol less than 0.9 mmol in men and less then 1.0 mmol in women, blood pressure greater than or equal to 140/90 mmHg, urinary albumin excretion rate greater than 20 μg/min or albumin-to-creatinine ratio greater than or equal to 30 mg/g (Diabetes Care, 2005, 28(9): 2289-2304).
A statement from the American Diabetes Association and the European Association for the Study of Diabetes comments on the construct of metabolic syndrome to denote risk factor clustering. In addition to suggestions for research of the underlying pathophysiology, the recommendations include individually and aggressively treating all cardiovascular disease risk factors (Diabetes Care, 2005, 28(9): 2289-2304). Therefore, another aspect of the present invention is a method of ameliorating any of the risk determinants or criteria associated with metabolic syndrome by administering an antisense compound of the invention.
As shown herein (see Example, 16), reduction of 4E-BP2 expression lowers blood glucose levels and improves insulin sensitivity. As compared to chow-fed mice, high-fat feeding not only caused hyperinsulinemia but also caused hyperglycemia. Treatment with control ASO did not cause any changes in these two parameters. However, 4E-BP2 ASO lowered both plasma insulin and glucose levels. These data indicate that 4E-BP2 ASO treatment improved insulin sensitivity. To further confirm this, a glucose tolerance test was carried out. As predicted, glucose excursion curve in 4E-BP2 ASO-treated mice was significant lower than that in either control group. Gene expression analysis found that the expression of hepatic G6Pase (glucose-6-phosphatase) was over 65% lower whereas the expression of hepatic GS (glycogen synthase) was over 30% higher in 4E-BP2 ASO-treated mice, implying a decreased hepatic glucose output in these mice.
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, and the like recited in the present application is incorporated herein by reference in its entirety.
EXAMPLES
Example 1
Design and Screening of Duplexed Antisense Compounds Targeting eIF4E-BP2
In accordance with the present invention, a series of duplexes, including dsRNA and mimetics thereof, comprising the compounds of the present invention and their complements can be designed to target eIF4E-BP2. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an antisense oligonucleotide targeted to eIF4E-BP2 as described herein. 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. The antisense and sense strands of the duplex comprise from about 17 to 25 nucleotides or from about 19 to 23 nucleotides. Alternatively, the antisense and sense strands comprise 20, 21, or 22 nucleotides.
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.
For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 261) and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:




Overhangs can range from 2 to 6 nucleobases and these nucleobases may or may not be complementary to the target nucleic acid. In another embodiment, the duplexes can have an overhang on only one terminus.
In another embodiment, a duplex comprising an antisense strand having the same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 261) may be prepared with blunt ends-(no single stranded overhang) as shown:




The RNA duplex can be unimolecular or bimolecular; i.e., the two strands can be part of a single molecule or may be separate molecules.

RNA strands of the duplex can be synthesized by methods routine to the skilled artisan 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 500mM. Once diluted, 30 μL of each strand is combined with 15 μL of a 5× 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 μL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 μM.
Once prepared, the duplexed compounds are evaluated for their ability to modulate target mRNA levels. When cells reach 80% confluency, they are treated with duplexed compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM® 1 reduced-serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM® 1 containing 5 μg/mL LIPOFECTAMINE 2000TM (Invitrogen Life Technologies, Carlsbad, Calif.) and the duplex antisense compound at the desired final concentration. After about 4 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 quantitative real-time PCR as described herein.
Example 2
Oligonucleotide Isolation
After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH4OAc 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 was 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 3
Oligonucleotide Synthesis-96 Well Plate Format
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.
Oligonucleotides were cleaved from support and deprotected with concentrated NH4OH at elevated temperature (55-60° 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 4
Oligonucleotide Analysis—96-Well Plate Format
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/ACETM MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACETM 5000, 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 5
Cell Culture and Oligonucleotide Treatment
The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. 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:
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 micrograms per 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.
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:
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 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.
NHDF Cells:
Human neonatal dermal fibroblast (NHDF) 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:
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.
b.END Cells:
The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cells were routinely cultured in DMEM, high glucose (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 3000 cells/well for use in RT-PCR analysis.
For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.
A10 Cells:
The rat aortic smooth muscle cell line A10 was obtained from the American Type Culture Collection (Manassas, Va.). A10 cells were routinely cultured in DMEM, high glucose (American Type Culture Collection, Manassas, Va.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 80% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 2500 cells/well for use in RT-PCR analysis.
For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.
EMT-6 Cells:
The mouse mammary epithelial carcinoma cell line EMT-6 was obtained from American Type Culture Collection (Manassus, Va.). They were grown in serial monolayer culture in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum, (Invitrogen Life Technologies, Carlsbad, Calif.), 100 ug/ml penicillin and 100 ug/ml streptomycin (Invitrogen Life Technologies, Carlsbad, Calif.) in a humidified atmosphere of 90% air-10% CO2 at 37° C. Cells were routinely passaged by trypsinization and dilution when they reached 85-90% confluencey. Cells were seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of 1000 cells/well for use in antisense oligonucleotide transfection.
Treatment with Antisense Compounds:
When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEMTM-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEMTM-1 containing 3.75 μg/mL LIPOFECTINTM (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° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.
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 6
Analysis of Oligonucleotide Inhibition of eIF4E-BP2 Expression
Antisense modulation of eIF4E-BP2 expression can be assayed in a variety of ways known in the art. For example, eIF4E-BP2 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 PRISMTM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
Protein levels of eIF4E-BP2 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 eIF4E-BP2 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 7
Design of Phenotypic Assays for the Use of eIF4E-BP2 Inhibitors
Phenotypic Assays
Once eIF4E-BP2 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 eIF4E-BP2 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.).
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 eIF4E-BP2 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.
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.
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 eIF4E-BP2 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.
Example 8
RNA Isolation
Poly(A)+ mRNA Isolation
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 μL cold PBS. 60 μL 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 μL 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 μL 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 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a -90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.
Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.
Total RNA Isolation
Total RNA was isolated using an RNEASY 96TM 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 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL 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 96TM well plate attached to a QIAVACTM manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96TM plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96TM plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY96TM 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 QIAVACTM manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVACTM manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.
The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (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 9
Real-time Quantitative PCR Analysis of eIF4E-BP2 mRNA Levels
Quantitation of eIF4E-BP2 mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISMTM 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 PRISMTM 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.
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.
PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl2, 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 mM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
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 RiboGreenT (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 RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreenTM are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).
In this assay, 170 μL of RiboGreenTM working reagent (RiboGreenTM reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.
Probes and primers to human eIF4E-BP2 were designed to hybridize to a human eIF4E-BP2 sequence, using published sequence information (GenBank accession number NM—004096.3, incorporated herein as SEQ ID NO: 4). For human eIF4E-BP2 the PCR primers were: forward primer: CCTCTAGTTTTGGGTGTGCATGT (SEQ ID NO: 5)

    reverse primer: CCCATAGCAAGGCAGAATGG (SEQ ID NO: 6) and the PCR probe was: FAM-TGGAGTTTGTAGTGGGTGGTTTGTAAAACTGG-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.


Probes and primers to mouse eIF4E-BP2 were designed to hybridize to a mouse eIF4E-BP2 sequence, using published sequence information (GenBank accession number NM—010124.1, incorporated herein as SEQ ID NO: 11). For mouse eIF4E-BP2 the PCR primers were:

    forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 12)
    reverse primer: CGGACAGACGGACGATGAG (SEQ ID NO: 13) and the PCR probe was: FAM-CCTCCCAGGTCTCTCGCCCT-TAMRA (SEQ ID NO: 14) where FAM is the fluorescent reporter dye and TAMRA is the quencher dye. For mouse GAPDH the PCR primers were:
    forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 15)
    reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 16) and the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 17) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.


Probes and primers to rat eIF4E-BP2 were designed to hybridize to a rat eIF4E-BP2 sequence, using published sequence information (GenBank accession number XM—215414.1, incorporated herein as SEQ ID NO: 18). For rat eIF4E-BP2 the PCR primers were:

    forward primer: AGTGAACAACTTGAACAACCTGAACA (SEQ ID NO: 19)
    reverse primer: ACTGCAGCAGGGTCAGATGTC (SEQ ID NO: 20) and the PCR probe was: FAM-TCACGACAGGAAGCACGCAGTTGG-TAMRA (SEQ ID NO: 21) where FAM is the fluorescent reporter dye and TAMRA is the quencher dye. For rat GAPDH the PCR primers were:
    forward primer: TGTTCTAGAGACAGCCGCATCTT (SEQ ID NO: 22)
    reverse primer: CACCGACCTTCACCATCTTGT (SEQ ID NO: 23) and the PCR probe was: 5′ JOE-TTGTGCAGTGCCAGCCTCGTCTCA-TAMRA 3′ (SEQ ID NO: 24) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.


Example 10
Northern Blot Analysis of eIF4E-BP2 mRNA Levels
Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOLTM (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 HYBONDTM-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 STRATALINERTM UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYBTM hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.
To detect human eIF4E-BP2, a human eIF4E-BP2 specific probe was prepared by PCR using the forward primer CCTCTAGTTTGGGTGTGCATGT (SEQ ID NO: 5) and the reverse primer CCCATAGCAAGGCAGAATGG (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.).
To detect mouse eIF4E-BP2, a mouse eIF4E-BP2 specific probe was prepared by PCR using the forward primer AGAGCAGCACAGGCTAAGACAGT (SEQ ID NO: 12) and the reverse primer CGGACAGACGGACGATGAG (SEQ ID NO: 13). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
To detect rat eIF4E-BP2, a rat eIF4E-BP2 specific probe was prepared by PCR using the forward primer AGTGAACAACTTGAACAACCTGAACA (SEQ ID NO: 19) and the reverse primer ACTGCAGCAGGGTCAGATGTC (SEQ ID NO: 20). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGERTM and IMAGEQUANTTM Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.
Example 11
Antisense Inhibition of Human eIF4E-BP2 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap
In accordance with the present invention, a series of antisense compounds was designed to target different regions of the human eIF4E-BP2 RNA, using published sequences (GenBank accession number NM—004096.3, incorporated herein as SEQ ID NO: 4, nucleotides 20714677 to 20740000 of the sequence with GenBank accession number NT—008583.16, incorporated herein as SEQ ID NO: 25, GenBank accession number AK057643.1, incorporated herein as SEQ ID NO: 26, GenBank accession number AK001936.1, incorporated herein as SEQ ID NO: 27, and GenBank accession number BF686401.1, incorporated herein as SEQ ID NO: 28). 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═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human eIF4E-BP2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which A549 cells were treated with 75 nM of the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”.








TABLE 1







Inhibition of human eIF4E-BP2 mRNA levels by chimeric phosphoro-



thioate oligonucleotides having 2′-MOE wings and a deoxy gap

















TARGET



SEQ
CONTROL





SEQ ID
TARGET

%
ID
SEQ ID


ISIS #
REGION
NO
SITE
SEQUENCE
INHIB
NO
NO





232773
Coding
 4
  420
gccatgggagaattgcgacg
68
 29
2



 


232776
Coding
 4
  493
tttggagtcttcaattaagg
31
 30
2


 


232777
Coding
 4
  498
tctactttggagtcttcaat
 2
 31
2


 


232828
3′UTR
 4
 1962
gtctgtagtcatcttaaaaa
52
 32
2


 


322947
Coding
 4
  501
acttctactttggagtcttc
60
 33
2


 


347546
Intron 1
25
 1836
tagaccgcaggagctgcgaa
 0
 34
2


 


347547
Intron 1
25
 8892
agtgattctcaaactgcaga
38
 35
2


 


347548
Intron 1
25
11559
tcttctgatccatggccacc
52
 36
2


 


347549
Intron 1
25
11918
tcagcactatctgttgaaaa
39
 37
2


 


347550
Intron 1:
25
16139
attcgagttcctggaaaaca
 0
 38
2



Exon 2



junction


 


347551
Exon 2:
25
16324
ttctcttaccaactgcatgt
 0
 39
2



Intron 2



junction


 


347552
Intron 2:
25
17941
gcatcatcccctagttagga
27
 40
2



exon 3



junction


 


347553
5′UTR
 4
  146
cctcaggcggacggaaaagc
39
 41
2


 


347554
Coding
 4
  332
cgggcgtggtgcaatagtca
63
 42
2


 


347555
Coding
 4
  372
attcgagttcctcccggtgt
55
 43
2


 


347556
Coding
 4
  392
gaaactttctgtcataaatg
 0
 44
2


 


347557
Coding
 4
  397
caacagaaactttctgtcat
15
 45
2


 


347558
Coding
 4
  474
gtgccagggctagtgactcc
43
 46
2


 


347559
Coding
 4
  526
attgttcaagttgttcaaat
 0
 47
2


 


347560
Coding
 4
  544
tgcatgtttcctgtcgtgat
59
 48
2


 


347561
Coding
 4
  549
ccaactgcatgtttcctgtc
54
 49
2


 


347562
Coding
 4
  558
gcatcatccccaactgcatg
54
 50
2


 


347563
Coding
 4
  574
gtccatctcgaactgagcat
46
 51
2


 


347564
Stop Codon
 4
  589
gcaggagagtcagatgtcca
47
 52
2


 


347565
3′UTR
 4
  623
aagtatcagtgttgctgctt
45
 53
2


 


347566
3′UTR
 4
  635
tcaggtgcacacaagtatca
43
 54
2


 


347567
3′UTR
 4
  734
atcatttggcacccagagga
54
 55
2


 


347568
3′UTR
 4
  747
agctcatcttcccatcattt
38
 56
2


 


347569
3′UTR
 4
  772
acagggagaagaaatggtca
13
 57
2


 


347570
3′UTR
 4
  803
taacctgtttaactgggaag
59
 58
2


 


347571
3′UTR
 4
  829
cagaaatacagcaagggcct
70
 59
2


 


347572
3′UTR
 4
  851
ctctaagggctgcttagctc
71
 60
2


 


347573
3′UTR
 4
  868
agagttgaactgttttcctc
78
 61
2


 


347574
3′UTR
 4
  921
caaaattacagggtatgagg
63
 62
2


 


347575
3′UTR
 4
 1085
aagaccccaagcccagactc
 9
 63
2


 


347576
3′UTR
 4
 1105
atttccccctgctggtttta
62
 64
2


 


347577
3′UTR
 4
 1130
aagggaaagcagctctcttt
70
 65
2


 


347578
3′UTR
 4
 1180
agagttgcacaagctgtgct
40
 66
2


 


347579
3′UTR
 4
 1217
agtggacctcaaaacagtgt
64
 67
2


 


347580
3′UTR
 4
 1303
tctgcacaaatgcactaagt
65
 68
2


 


347581
3′UTR
 4
 1350
aaaactggttaccaagggct
34
 69
2


 


347582
3′UTR
 4
 1357
gaagagcaaaactggttacc
27
 70
2


 


347583
3′UTR
 4
 1393
ccagcaacgagatgcaagca
65
 71
2


 


347584
3′UTR
 4
 1410
agtacaagaggactctgcca
56
 72
2


 


347585
3′UTR
 4
 1458
tggtatggacctgctctagg
51
 73
2


 


347586
3′UTR
 4
 1472
gtgcctctattacttggtat
48
 74
2


 


347587
3′UTR
 4
 1533
ttcttaggcattatctgaca
70
 75
2


 


347588
3′UTR
 4
 1541
agcggtcattcttaggcatt
59
 76
2


 


347589
3′UTR
 4
 1580
acgactgagaccgggtactc
67
 77
2


 


347590
3′UTR
 4
 1614
acaactaccacaatgctcac
 0
 78
2


 


347591
3′UTR
 4
 1664
attctgaaaatcaacttcaa
 0
 79
2


 


347592
3′UTR
 4
 1724
tcccagcagccaaacaaagc
 0
 80
2


 


347593
3′UTR
 4
 1868
atttgaaaaatggcctggta
47
 81
2


 


347594
3′UTR
 4
 1892
acacttcaggtatctttgat
 6
 82
2


 


347595
3′UTR
 4
 1900
agataccaacacttcaggta
49
 83
2


 


347596
3′UTR
 4
 1912
acagatattctcagatacca
 0
 84
2


 


347597
3′UTR
 4
 2018
atgtttaattaaaaagttgc
 0
 85
2


 


347598
3′UTR
 4
 2028
acactggaagatgtttaatt
17
 86
2


 


347599
3′UTR
 4
 2173
cagttttacaaaccacccac
 0
 87
2


 


347600
3′UTR
 4
 2218
aagaatgaggctttcttgaa
47
 88
2


 


347601
3′UTR
 4
 2223
cagaaaagaatgaggctttc
34
 89
2


 


347602
3′UTR
 4
 2246
tgaatgcaaaagcgaaaggg
 0
 90
2


 


347603
3′UTR
 4
 2301
tcccgggattattatgctgc
 0
 91
2


 


347604
3′UTR
 4
 2377
gaaattcccaggacaccagt
63
 92
2


 


347605
3′UTR
 4
 2382
aaccagaaattcccaggaca
47
 93
2


 


347606
3′UTR
 4
 2389
caaatccaaccagaaattcc
 0
 94
2


 


347607
3′UTR
 4
 2449
ccaaatggcctgttactctc
26
 95
2


 


347608
3′UTR
 4
 2471
aacaaacaggtttctttctt
40
 96
2


 


347609
3′UTR
 4
 2492
cttttcatagttcaaaagaa
19
 97
2


 


347610
3′UTR
 4
 2536
cagacatccttcctctcttt
33
 98
2


 


347611
3′UTR
 4
 2564
ttgtggcagaaaacagaaca
 0
 99
2


 


347612
3′UTR
 4
 2578
aactattcacatttttgtgg
62
100
2


 


347613
3′UTR
 4
 2632
tggagatccagcttattcct
49
101
2


 


347614
3′UTR
26
 1189
aagaatgaaaagcttcattc
 0
102
2


 


347615
3′UTR
26
 1336
tttaaatccattcctcaccg
 0
103
2


 


347616
3′UTR
26
 2088
ataactaatacaggtggaag
41
104
2


 


347617
3′UTR
27
  697
ggtcatctgaaatctctaaa
45
105
2


 


347618
3′UTR
28
  464
gcctcccacccttagaaagg
 2
106
2









As shown in Table 1, SEQ ID NOs 29, 30, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104 and 105 demonstrated at least 25% inhibition of human eIF4E-BP2 expression in this assay and are therefore preferred. 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 5. 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 disclosed herein. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 5 is the species in which each of the preferred target segments was found.
SEQ ID NOs 29, 30, 31 and 32 are cross species oligonucleotides which are also complementary to the mouse eIF4E-BP2 nucleic acid target. SEQ ID NOs 29 and 33 are cross species oligonucleotides which are also complementary to rat eIF4E-BP2.
Example 12
Antisense Inhibition of Mouse eIF4E-BP2 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap
In accordance with the present invention, a second series of antisense compounds was designed to target different regions of the mouse eIF4E-BP2 RNA, using published sequences (GenBank accession number NM—010124.1, incorporated herein as SEQ ID NO: 11, GenBank accession number B1696127.1, incorporated herein as SEQ ID NO: 107, and GenBank accession number BE332409.1, incorporated herein as SEQ ID NO: 108). The compounds are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the compound binds. All compounds in Table 2 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═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on mouse eIF4E-BP2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data, shown in Table 2, are averages from two experiments in which b.END cells were treated with 150 nM of the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”.








TABLE 2







Inhibition of mouse eIF4E-BP2 mRNA levels in b.END cells by



chimeric phosphorothioate oligonucleotides having 2′-MOE wings and


a deoxy gap

















TARGET



SEQ
CONTROL





SEQ ID
TARGET

%
ID
SEQ ID


ISIS #
REGION
NO
SITE
SEQUENCE
INHIB
NO
NO





232759
5′UTR
 11
   9
tctcaactcgcctgctctcg
92
109
2



 


232760
5′UTR
 11
  26
ggctcctcacgctcggctct
81
110
2


 


232761
5′UTR
 11
  86
tcgaggctttgtgcagcagc
64
111
2


 


232762
Coding
 11
 132
gctggtggctaccaccggcc
49
112
2


 


232763
Coding
 11
 137
gctgggctggtggctaccac
72
113
2


 


232764
Coding
 11
 179
gtcgctgatagccacggtgc
77
114
2


 


232765
Coding
 11
 201
agtcctgaggtagctgcgcg
79
115
2


 


232766
Coding
 11
 211
gtggtgcagtagtcctgagg
81
116
2


 


232767
Coding
 11
 264
cataaatgattcgtgttcct
73
117
2


 


232768
Coding
 11
 269
tcggtcataaatgattcgtg
86
118
2


 


232769
Coding
 11
 274
aactttcggtcataaatgat
52
119
2


 


232770
Coding
 11
 281
caacagaaactttcggtcat
72
120
2


 


232771
Coding
 11
 286
cggtccaacagaaactttcg
84
121
2


 


232772
Coding
 11
 299
gggagaattgcgacggtcca
80
122
2


 


232773
Coding
 11
 304
gccatgggagaattgcgacg
83
 29
2


 


232774
Coding
 11
 309
tctgcgccatgggagaattg
66
123
2


 


232775
Coding
 11
 354
caggactggtgactccaggg
87
124
2


 


232776
Coding
 11
 377
tttggagtcttcaattaagg
24
 30
2


 


232777
Coding
 11
 382
tctactttggagtcttcaat
69
 31
2


 


232778
Coding
 11
 388
ttcacttctactttggagtc
71
125
2


 


232779
Coding
 11
 449
aaactgagcctcatccccaa
89
126
2


 


232780
Coding
 11
 454
atctcaaactgagcctcatc
85
127
2


 


232781
Coding
 11
 461
gatgtccatctcaaactgag
73
128
2


 


232782
Stop Codon
 11
 473
tggcagtagtcagatgtcca
91
129
2


 


232783
3′UTR
 11
 500
ggctgctccacgaggcctcc
90
130
2


 


232784
3′UTR
 11
 521
tgggccagtcaggtgcacac
77
131
2


 


232785
3′UTR
 11
 540
ctgtacactgtgttcctact
87
132
2


 


232786
3′UTR
 11
 607
atgtgatcagacagtgcaca
67
133
2


 


232787
3′UTR
 11
 614
cgggaagatgtgatcagaca
59
134
2


 


232788
3′UTR
 11
 696
ttcttctgtggactgtcagc
44
135
2


 


232789
3′UTR
 11
 787
gtgctgcttggagactgccc
54
136
2


 


232790
3′UTR
 11
 798
tacaagcagaggtgctgctt
47
137
2


 


232791
3′UTR
 11
 827
ggcactaaacctccttcacc
87
138
2


 


232792
3′UTR
 11
 835
acacaatgggcactaaacct
68
139
2


 


232793
3′UTR
 11
 845
gagcccaggaacacaatggg
61
140
2


 


232794
3′UTR
 11
 900
aatgtcccccacatccagcg
88
141
2


 


232795
3′UTR
 11
 909
ctgaggacaaatgtccccca
81
142
2


 


232796
3′UTR
 11
 927
caggactgtgctccagagct
78
143
2


 


232797
3′UTR
 11
 934
ggaggtacaggactgtgctc
69
144
2


 


232798
3′UTR
 11
 975
gaggctgctgtcacatgtcc
68
145
2


 


232799
3′UTR
 11
 998
aagccttcctcccagagaaa
81
146
2


 


232800
3′UTR
 11
1020
tatcacacccaagacaagac
70
147
2


 


232801
3′UTR
 11
1030
gatgatgagctatcacaccc
83
148
2


 


232802
3′UTR
 11
1093
cccttcaggagggcttaaaa
70
149
2


 


232803
3′UTR
 11
1127
cagacaggcaaagaccagct
85
150
2


 


232804
3′UTR
 11
1156
tgcctacgggatgcaggtag
71
151
2


 


232805
3′UTR
 11
1204
cttctgctctaaaagcagac
 1
152
2


 


232806
3′UTR
 11
1222
caggccaaggtgttggcact
57
153
2


 


232807
3′UTR
 11
1250
gctgagagcaggctggactc
66
154
2


 


232808
3′UTR
 11
1263
tctcaggcagaccgctgaga
54
155
2


 


232809
3′UTR
 11
1276
gcccctgatgtattctcagg
72
156
2


 


232810
3′UTR
 11
1282
tcagaggcccctgatgtatt
51
157
2


 


232811
3′UTR
 11
1289
gtcctcttcagaggcccctg
89
158
2


 


232812
3′UTR
 11
1303
tgcacggcggctcagtcctc
69
159
2


 


232813
3′UTR
 11
1308
ctggctgcacggcggctcag
71
160
2


 


232814
3′UTR
 11
1327
aaaaccatgacccccgaggc
92
161
2


 


232815
3′UTR
 11
1340
tacacctggttttaaaacca
67
162
2


 


232816
3′UTR
 11
1355
acacccaacgtaaggtacac
86
163
2


 


232817
3′UTR
 11
1361
tgcaggacacccaacgtaag
85
164
2


 


232818
3′UTR
 11
1381
aaactcaaggtatagtaacc
73
165
2


 


232819
3′UTR
 11
1392
aagtcgactttaaactcaag
66
166
2


 


232820
3′UTR
 11
1399
taagaggaagtcgactttaa
75
167
2


 


232821
3′UTR
 11
1455
ctgtgctgctctctcagcag
21
168
2


 


232822
3′UTR
 11
1467
cactgtcttagcctgtgctg
90
169
2


 


232823
3′UTR
 11
1584
tggaaaatggcccggtggaa
82
170
2


 


232824
3′UTR
 11
1619
tactaacatgggaggcatct
84
171
2


 


232825
3′UTR
 11
1646
tgataaggagagactgatat
28
172
2


 


232826
3′UTR
 11
1662
taaaaggtctctcctctgat
33
173
2


 


232827
3′UTR
 11
1668
taaaaataaaaggtctctcc
24
174
2


 


232828
3′UTR
 11
1682
gtctgtagtcatcttaaaaa
93
 32
2


 


232829
3′UTR
 11
1699
aacttatctaaaaataggtc
30
175
2


 


232830
3′UTR
 11
1708
tgtactgaaaacttatctaa
74
176
2


 


232831
3′UTR
 11
1749
atactggaagatgttttgtt
70
177
2


 


232832
3′UTR
 11
1761
ataaccttcccaatactgga
82
178
2


 


232833
3′UTR
107
 365
acagctcatggcaaggcaga
79
179
2


 


232834
3′UTR
107
 437
aactgctcttctatgtgtgg
 4
180
2


 


232835
3′UTR
107
 454
tcgctgatagtctcttgaac
 0
181
2


 


232836
5′UTR
108
  36
ggctcttcacgctcggctct
73
182
2









As shown in Table 2, SEQ ID NOs 29, 31, 32, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 169, 170, 171, 176, 177, 178, 179 and 182 demonstrated at least 44% inhibition of mouse eIF4E-BP2 expression in this experiment and are therefore preferred. 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 4. 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 disclosed herein. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 5 is the species in which each of the preferred target segments was found.
In a further embodiment, antisense oligonucleotides targeting mouse eIF4E-BP2 were tested in EMT-6 cells. The compounds were analyzed for their effect on mouse eIF4E-BP2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data, shown in Table 3, are averages from two experiments in which EMT-6 cells were treated with 150 nM of the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”.








TABLE 3







Inhibition of mouse eIF4E-BP2 mRNA levels in EMT-6 cells by chi-



meric phosphorothioate oligonucleotides having 2′-MOE wings and


a deoxy gap

















TARGET



SEQ
CONTROL





SEQ ID
TARGET

%
ID
SEQ ID


ISIS #
REGION
NO
SITE
SEQUENCE
INHIB
NO
NO





232759
5′UTR
 11
   9
tctcaactcgcctgctctcg
95
109
2



 


232760
5′UTR
 11
  26
ggctcctcacgctcggctct
93
110
2


 


232761
5′UTR
 11
  86
tcgaggctttgtgcagcagc
96
111
2


 


232762
Coding
 11
 132
gctggtggctaccaccggcc
88
112
2


 


232763
Coding
 11
 137
gctgggctggtggctaccac
94
113
2


 


232764
Coding
 11
 179
gtcgctgatagccacggtgc
95
114
2


 


232765
Coding
 11
 201
agtcctgaggtagctgcgcg
97
115
2


 


232766
Coding
 11
 211
gtggtgcagtagtcctgagg
93
116
2


 


232767
Coding
 11
 264
cataaatgattcgtgttcct
92
117
2


 


232768
Coding
 11
 269
tcggtcataaatgattcgtg
98
118
2


 


232769
Coding
 11
 274
aactttcggtcataaatgat
80
119
2


 


232770
Coding
 11
 281
caacagaaactttcggtcat
84
120
2


 


232771
Coding
 11
 286
cggtccaacagaaactttcg
97
121
2


 


232772
Coding
 11
 299
gggagaattgcgacggtcca
95
122
2


 


232773
Coding
 11
 304
gccatgggagaattgcgacg
96
 29
2


 


232774
Coding
 11
 309
tctgcgccatgggagaattg
93
123
2


 


232775
Coding
 11
 354
caggactggtgactccaggg
98
124
2


 


232776
Coding
 11
 377
tttggagtcttcaattaagg
73
 30
2


 


232777
Coding
 11
 382
tctactttggagtcttcaat
85
 31
2


 


232778
Coding
 11
 388
ttcacttctactttggagtc
93
125
2


 


232779
Coding
 11
 449
aaactgagcctcatccccaa
93
126
2


 


232780
Coding
 11
 454
atctcaaactgagcctcatc
92
127
2


 


232781
Coding
 11
 461
gatgtccatctcaaactgag
89
128
2


 


232782
Stop Codon
 11
 473
tggcagtagtcagatgtcca
95
129
2


 


232783
3′UTR
 11
 500
ggctgctccacgaggcctcc
98
130
2


 


232784
3′UTR
 11
 521
tgggccagtcaggtgcacac
95
131
2


 


232785
3′UTR
 11
 540
ctgtacactgtgttcctact
98
132
2


 


232786
3′UTR
 11
 607
atgtgatcagacagtgcaca
89
133
2


 


232787
3′UTR
 11
 614
cgggaagatgtgatcagaca
75
134
2


 


232788
3′UTR
 11
 696
ttcttctgtggactgtcagc
59
135
2


 


232789
3′UTR
 11
 787
gtgctgcttggagactgccc
77
136
2


 


232790
3′UTR
 11
 798
tacaagcagaggtgctgctt
87
137
2


 


232791
3′UTR
 11
 827
ggcactaaacctccttcacc
91
138
2


 


232792
3′UTR
 11
 835
acacaatgggcactaaacct
87
139
2


 


232793
3′UTR
 11
 845
gagcccaggaacacaatggg
89
140
2


 


232794
3′UTR
 11
 900
aatgtcccccacatccagcg
95
141
2


 


232795
3′UTR
 11
 909
ctgaggacaaatgtccccca
92
142
2


 


232796
3′UTR
 11
 927
caggactgtgctccagagct
95
143
2


 


232797
3′UTR
 11
 934
ggaggtacaggactgtgctc
91
144
2


 


232798
3′UTR
 11
 975
gaggctgctgtcacatgtcc
95
145
2


 


232799
3′UTR
 11
 998
aagccttcctcccagagaaa
83
146
2


 


232800
3′UTR
 11
1020
tatcacacccaagacaagac
80
147
2


 


232801
3′UTR
 11
1030
gatgatgagctatcacaccc
91
148
2


 


232802
3′UTR
 11
1093
cccttcaggagggcttaaaa
85
149
2


 


232803
3′UTR
 11
1127
cagacaggcaaagaccagct
94
150
2


 


232804
3′UTR
 11
1156
tgcctacgggatgcaggtag
95
151
2


 


232805
3′UTR
 11
1204
cttctgctctaaaagcagac
36
152
2


 


232806
3′UTR
 11
1222
caggccaaggtgttggcact
83
153
2


 


232807
3′UTR
 11
1250
gctgagagcaggctggactc
82
154
2


 


232808
3′UTR
 11
1263
tctcaggcagaccgctgaga
74
155
2


 


232809
3′UTR
 11
1276
gcccctgatgtattctcagg
93
156
2


 


232810
3′UTR
 11
1282
tcagaggcccctgatgtatt
86
157
2


 


232811
3′UTR
 11
1289
gtcctCttcagaggcccctg
95
158
2


 


232812
3′UTR
 11
1303
tgcacggcggctcagtcctc
86
159
2


 


232813
3′UTR
 11
1308
ctggctgcacggcggctcag
91
160
2


 


232814
3′UTR
 11
1327
aaaaccatgacccccgaggc
96
161
2


 


232815
3′UTR
 11
1340
tacacctggttttaaaacca
93
162
2


 


232816
3′UTR
 11
1355
acacccaacgtaaggtacac
95
163
2


 


232817
3′UTR
 11
1361
tgcaggacacccaacgtaag
97
164
2


 


232818
3′UTR
 11
1381
aaactcaaggtatagtaacc
89
165
2


 


232819
3′UTR
 11
1392
aagtcgactttaaactcaag
96
166
2


 


232820
3′UTR
 11
1399
taagaggaagtcgactttaa
96
167
2


 


232821
3′UTR
 11
1455
ctgtgctgctctctcagcag
79
168
2


 


232822
3′UTR
 11
1467
cactgtcttagcctgtgctg
96
169
2


 


232823
3′UTR
 11
1584
tggaaaatggcccggtggaa
96
170
2


 


232824
3′UTR
 11
1619
tactaacatgggaggcatct
95
171
2


 


232825
3′UTR
 11
1646
tgataaggagagactgatat
60
172
2


 


232826
3′UTR
 11
1662
taaaaggtctctcctctgat
67
173
2


 


232827
3′UTR
 11
1668
taaaaataaaaggtctctcc
23
174
2


 


232828
3′UTR
 11
1682
gtctgtagtcatcttaaaaa
98
 32
2


 


232829
3′UTR
 11
1699
aacttatctaaaaataggtc
69
175
2


 


232830
3′UTR
 11
1708
tgtactgaaaacttatctaa
97
176
2


 


232831
3′UTR
 11
1749
atactggaagatgttttgtt
89
177
2


 


232832
3′UTR
 11
1761
ataaccttcccaatactgga
95
178
2


 


232833
3′UTR
107
 365
acagctcatggcaaggcaga
96
179
2


 


232834
3′UTR
107
 437
aactgctcttctatgtgtgg
40
180
2


 


232835
3′UTR
107
 454
tcgctgatagtctcttgaac
23
181
2


 


232836
5′UTR
108
  36
ggctcttcacgctcggctct
88
182
2









As shown in Table 3, SEQ ID NOs 29, 30, 31, 32, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179 and 182 demonstrated at least 67% inhibition of mouse eIF4E-BP2 expression in this assay and are therefore preferred.
Example 13
Antisense Inhibition of Rat eIF4E-BP2 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-M10E Wings and a Deoxy Gap
In accordance with the present invention, a third series of antisense compounds was designed to target different regions of the rat eIF4E-BP2 RNA, using published sequences (GenBank accession number XM—215414.1, incorporated herein as SEQ ID NO: 18). The compounds are shown in Table 4. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the compound binds. All compounds in Table 4 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═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on rat eIF4E-BP2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data, shown in Table 4, are averages from two experiments in which A10 cells were treated with 50 nM of the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”.








TABLE 4







Inhibition of rat eIF4E-BP2 mRNA levels by chimeric phosphorothio-



ate oligonucleotides having 2′-MOE wings and a deoxy gap

















TARGET



SEQ
CONTROL





SEQ ID
TARGET

%
ID
SEQ ID


ISIS #
REGION
NO
SITE
SEQUENCE
INHIB
NO
NO





232773
Coding
11
304
gccatgggagaattgcgacg
90
 29
2



 


322907
5′UTR
18
  7
ggctcgtggctttgtgcagc
48
183
2


 


322908
Coding
18
 48
tggtgtccaccaccggccga
49
184
2


 


322909
Coding
18
 57
tggctgggctggtgtccacc
65
185
2


 


322910
Coding
18
 59
tctggctgggctggtgtcca
50
186
2


 


322911
Coding
18
 71
gaatggcgcggctctggctg
65
187
2


 


322912
Coding
18
 93
ctaatagccacggtgcgtgt
65
188
2


 


322913
Coding
18
 97
gtcgctaatagccacggtgc
83
189
2


 


322914
Coding
18
102
gctgcgtcgctaatagccac
80
190
2


 


322915
Coding
18
114
tgaggtagctgcgctgcgtc
62
191
2


 


322916
Coding
18
116
cctgaggtagctgcgctgcg
68
192
2


 


322917
Coding
18
120
tagtcctgaggtagctgcgc
77
193
2


 


322918
Coding
18
122
agtagtcctgaggtagctgc
75
194
2


 


322919
Coding
18
125
tgcagtagtcctgaggtagc
80
195
2


 


322920
Coding
18
127
ggtgcagtagtcctgaggta
85
196
2


 


322921
Coding
18
130
cgtggtgcagtagtcctgag
78
197
2


 


322922
Coding
18
132
ggcgtggtgcagtagtcctg
74
198
2


 


322923
Coding
18
159
ggtgttgtggagaacagcgt
35
199
2


 


322924
Coding
18
164
ctcccggtgttgtggagaac
48
200
2


 


322925
Coding
18
168
gttcctcccggtgttgtgga
78
201
2


 


322926
Coding
18
193
aaactttcggtcataaatga
53
202
2


 


322927
Coding
18
195
agaaactttcggtcataaat
41
203
2


 


322928
Coding
18
197
acagaaactttcggtcataa
65
204
2


 


322929
Coding
18
198
aacagaaactttcggtcata
79
205
2


 


322930
Coding
18
201
tccaacagaaactttcggtc
83
206
2


 


322931
Coding
18
203
ggtccaacagaaactttcgg
83
207
2


 


322932
Coding
18
208
gcgacggtccaacagaaact
80
208
2


 


322933
Coding
18
210
ttgcgacggtccaacagaaa
76
209
2


 


322934
Coding
18
213
gaattgcgacggtccaacag
78
210
2


 


322935
Coding
18
215
gagaattgcgacggtccaac
75
211
2


 


322936
Coding
18
218
tgggagaattgcgacggtcc
36
212
2


 


322937
Coding
18
223
cgccatgggagaattgcgac
73
213
2


 


322938
Coding
18
225
tgcgccatgggagaattgcg
52
214
2


 


322939
Coding
18
228
gtctgcgccatgggagaatt
67
215
2


 


322940
Coding
18
250
attgggcagatggcaaggtg
33
216
2


 


322941
Coding
18
265
ggtgactccagggatattgg
35
217
2


 


322942
Coding
18
270
ggactggtgactccagggat
74
218
2


 


322943
Coding
18
275
cgccaggactggtgactcca
83
219
2


 


322944
Coding
18
292
ggagtcttccattaaggcgc
64
220
2


 


322945
Coding
18
294
ttggagtcttccattaaggc
66
221
2


 


322946
Coding
18
298
tactttggagtcttccatta
27
222
2


 


322947
Coding
18
303
acttctactttggagtcttc
68
 33
2


 


322948
Coding
18
304
cacttctactttggagtctt
64
223
2


 


322949
Coding
18
308
tgttcacttctactttggag
87
224
2


 


322950
Coding
18
313
caagttgttcacttctactt
80
225
2


 


322951
Coding
18
316
gttcaagttgttcacttcta
82
226
2


 


322952
Coding
18
323
tcaggttgttcaagttgttc
83
227
2


 


322953
Coding
18
326
tgttcaggttgttcaagttg
84
228
2


 


322954
Coding
18
329
gattgttcaggttgttcaag
68
229
2


 


322955
Coding
18
332
cgtgattgttcaggttgttc
95
230
2


 


322956
Coding
18
335
tgtcgtgattgttcaggttg
95
231
2


 


322957
Coding
18
339
ttcctgtcgtgattgttcag
88
232
2


 


322958
Coding
18
341
gcttcctgtcgtgattgttc
95
233
2


 


322959
Coding
18
343
gtgcttcctgtcgtgattgt
92
234
2


 


322960
Coding
18
348
actgcgtgcttcctgtcgtg
97
235
2


 


322961
Coding
18
350
ccccaactgcgtgcttcctg
91
236
2


 


322962
Coding
18
353
ccccaactgcgtgcttcctg
85
237
2


 


322963
Coding
18
355
atccccaactgcgtgcttcc
48
238
2


 


322964
Coding
18
358
ctcatccccaactgcgtgct
83
239
2


 


322965
Coding
18
360
gcctcatccccaactgcgtg
90
240
2


 


322966
Coding
18
362
gagcctcatccccaactgcg
94
241
2


 


322967
Coding
18
364
ctgagcctcatccccaactg
89
242
2


 


322968
Coding
18
369
tcaaactgagcctcatcccc
50
243
2


 


322969
Stop Codon
18
390
cagcagggtcagatgtccat
81
244
2


 


322970
3′UTR
18
402
ccttcgacactgcagcaggg
88
245
2


 


322971
3′UTR
18
406
gccgccttcgacactgcagc
83
246
2


 


322972
3′UTR
18
428
gtgcacacgggccgtgtcag
76
247
2


 


322973
3′UTR
18
436
ccagtcaggtgcacacgggc
84
248
2


 


322974
3′UTR
18
439
ggtccagtcaggtgcacacg
86
249
2


 


322975
3′UTR
18
443
tactggtccagtcaggtgca
80
250
2


 


322976
3′UTR
18
446
tcctactggtccagtcaggt
72
251
2


 


322977
3′UTR
18
450
gtgttcctactggtccagtc
84
252
2


 


322978
3′UTR
18
454
cacggtgttcctactggtcc
83
253
2


 


322979
3′UTR
18
458
tgtacacggtgttcctactg
76
254
2


 


322980
3′UTR
18
462
tctctgtacacggtgttcct
89
255
2


 


322981
3′UTR
18
464
cttctctgtacacggtgttc
90
256
2


 


322982
3′UTR
18
469
tggagcttctctgtacacgg
90
257
2


 


322983
3′UTR
18
471
actggagcttctctgtacac
85
258
2









As shown in Table 4, SEQ ID NOs 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 and 258 demonstrated at least 48% inhibition of rat eI4E-BP2 expression in this experiment and are therefore preferred. 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 5. 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 tables above. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the compound binds.
“Preferred target segments,” as described in Table 5 of U.S. Patent Application No. 60/538,752, filed Jan. 22, 2004, 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 eIF4E-BP2.
According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.
Example 14
Western Blot Analysis of eIF4E-BP2 Protein Levels
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 ul/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 eIF4E-BP2 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORMAGERTM (Molecular Dynamics, Sunnyvale Calif.).
Example 15
Reduction of Blood Glucose Levels in ob/ob Mice by Antisense Inhibition of eIF4E-BP2
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 diabetes and treatments designed to treat these conditions. In accordance with the present invention, compounds targeted to eIF4E-BP2 are tested in the ob/ob model of obesity and diabetes.
Seven-week old male C57B1/6J-Lepr 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 at a dose of 25 mg/kg two times per week for 4 weeks. Saline-injected animals, leptin wildtype littermates (i.e. lean littermates) and ob/ob mice fed a standard rodent diet 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.
To assess the physiological effects resulting from antisense inhibition of target mRNA, the ob/ob mice that receive antisense oligonucleotide treatment are typically further evaluated at the end of the treatment period for serum lipids, serum free fatty acids, serum cholesterol, liver triglycerides, fat tissue triglycerides and liver enzyme levels. Hepatic steatosis, or accumulation of lipids in the liver, is assessed by measuring the liver triglyceride content. As such, hepatic steatosis is assessed in animal models 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.
The effects of target inhibition on glucose and insulin metabolism are evaluated in the ob/ob mice treated with antisense oligonucleotides. Plasma glucose is measured at the start of the antisense oligonucleotide treatment and following two and four weeks of treatment. Both fed and fasted plasma glucose levels were measured. At start of study, the treatment groups of mice are chosen to have an average fed plasma glucose level of about 350 mg/dL. Plasma insulin is also measured at the beginning 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.
In mice treated with ISIS 232828 (SEQ ID NO: 32), an antisense inhibitor of eIF4E-BP2, fed plasma glucose levels were approximately 355 mg/dL at week 0, 295 mg/dL at week 2 and 210 mg/dL at week 4. In contrast, mice treated with saline alone had fed plasma glucose levels of approximately 365 mg/dL at week 0, 425 mg/dL at week 2 and 410 mg/dL at week 4. Mice treated with a positive control oligonucleotide, ISIS 116847 (CTGCTAGCCTCTGGATTTGA; SEQ ID NO: 447), targeted to PTEN, had fed plasma glucose levels of approximately 360 mg/dL at week 0, 215 mg/dL at week 2 and 180 mg/dL at week 4.
Fasted plasma glucose was measured at week 3 of antisense treatment. Plasma glucose was approximately 330 mg/dL in saline treated mice, 245 mg/dL in mice treated with ISIS 232828 (inhibitor of eIF4E-BP2) and 195 mg/dL in mice treated with the positive control oligonucleotide, ISIS 116847.
At the end of the four week study, average liver weights were approximately 3.6 grams for saline treated mice, 3.2 grams for ISIS 232828-treated mice and 4.1 grams for positive control (ISIS 116847) treated mice. White adipose tissue weights were approximately 3.9 grams for saline treated mice, 3.8 grams for ISIS 232828-treated mice and 3.7 grams for positive control (ISIS 116847) treated mice.
At the end of the study, liver transaminases were found to be lower in mice treated with antisense to eIF4E-BP2 (ISIS 232828) than in mice treated with saline or the positive control oligonucleotide (ISIS 116847). AST levels were approximately 330 IU/L for saline treated mice, 110 IU/L for ISIS 232828-treated mice and 430 IU/L for ISIS 116847-treated mice. ALT levels were approximately 435 UI/L for saline treated mice, 140 IU/L for ISIS 232828-treated mice and 710 IU/L for ISIS 116847-treated mice.
Serum lipids were also measured at the end of the study. Cholesterol levels were approximately 230 mg/dL for saline treated mice, 210 mg/dL for ISIS 232828-treated mice and 260 mg/dL for ISIS 116847-treated mice. Triglycerides were approximately 135 mg/dL for saline treated mice, 80 mg/dL for ISIS 232828-treated mice and 110 mg/dL for ISIS 116847-treated mice.
eIF4E-BP2 mRNA levels in liver were measured at the end of study using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) as taught in previous examples above. eIF4E-BP2 mRNA levels were reduced by approximately 90% in mice treated with ISIS 232828, compared to saline treatment. Target reduction in mice treated with ISIS 116847 was approximately 30%.
Example 16
Effect of Antisense Inhibition of eIF4E-BP2 in High-Fat Diet-Induced Obese Mice
The C57BL/6 mouse strain is reported to be susceptible to hyperlipidemia-induced atherosclerotic plaque formation. Consequently, when these mice are fed a high-fat diet, they develop diet-induced obesity. Accordingly these mice are a useful model for the investigation of obesity and treatments designed to treat this conditions. In a further embodiment of the present invention, the oligomeric compounds of the invention were tested in a model of diet-induced obesity.
Male C57BL/6 mice (4-weeks old) received a 60% fat diet for 3 months, after which mice were subcutaneously injected with ISIS 232828 (SEQ ID NO: 32) at a dose of 25 mg/kg two times per week for 6 weeks. Saline-injected and control compound-injected animals also fed a high-fat diet as described served as controls. The control compound was ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, incorporated herein as SEQ ID NO: 260). ISIS 141923 is a chimeric oligonucleotide comprising a central gap region consisting of ten 2′-deoxynucleotides, which is flanked its 5′ and 3′ ends by five 2′-MOE nucleotides. The internucleoside linkages are phosphorothioate throughout the oligonucleotide and all cytidine residues are 5-methylcytidines. As another control, animals fed normal chow were injected with saline. Each treatment group was comprised of 7 animals, and data from this study presented herein is generally the average from 6 or 7 animals per treatment group.
After the treatment period, mice were sacrificed and eIF4E-BP2 target levels are evaluated in liver and white adipose tissue (WAT). RNA isolation and target mRNA expression level quantitation are performed as described by other examples herein. Average results for each treatment group are shown in Table 5 as percent inhibition of target expression as compared to saline-treated high-fat fed controls.







TABLE 5







eIF4E-BP2 Target Reduction in Liver and WAT










% Inhibition












Treatment group
Liver
WAT















ISIS 141923
0
8



ISIS 232828
74
84



Saline, normal chow
0
0










As shown in Table 5, treatment with ISIS 232828 reduces eIF4E-BP2 levels in liver and adipose tissue.
The effects of target inhibition on glucose and insulin metabolism are also evaluated in the diet-induced obese mice treated with the oligomeric compounds of the invention. Plasma glucose was measured via methods routine in the art (for example, with a YSI glucose analyzer, YSI Scientific, Yellow Springs, Ohio) prior to the start of treatment (Week 0), after about 3.5 weeks of treatment (Week 3.5), and at the end of the treatment period (Week 6). Results are shown in Table 6 as the average plasma glucose level for each treatment group.







TABLE 6







Effects of antisense oligonucleotides targeting


eIF4E-BP2 on plasma glucose levels









Plasma Glucose (mg/dL)












Treatment group
Week 0
Week 3.5
Week 6







Saline, high fat fed
198
221
256



ISIS 141923
197
208
254



ISIS 232828
208
208
213



Saline, chow
160
180
198










As shown in Table 6, animals treated with ISIS 232828 did not display the increase in plasma glucose levels observed in saline treated high-fat fed control animals.
Insulin levels were measured using methods known in the art (for example, with an Alpco insulin-specific ELISA kit, Windham, N.H.) at the time points indicated. Results are shown in Table 7 as the average insulin level for each treatment group.







TABLE 7







Effects of antisense oligonucleotides targeting


eIF4E-BP2 on plasma insulin levels









Plasma Insulin (ng/mL)












Treatment group
Week 0
Week 3.5
Week 6







Saline, high fat fed
1.4
1.6
2.5



ISIS 141923
1.4
1.5
2.3



ISIS 232828
1.4
1.5
1.2



Saline, chow
0.3
1.2
1.1










As shown in Table 7, treatment with ISIS 232828 lowered insulin levels by Week 6.
Glucose and insulin tolerance tests were also administered. Mice received intraperitoneal injections of either glucose (1 mg/kg) or insulin (0.5 U/kg), and blood glucose levels were measured before the insulin or glucose challenge and at 30 minute intervals for up to 2 hours. The insulin tolerance test was performed after about 4.5 weeks of treatment. The glucose tolerance test was administered after about 5.5 weeks of treatment. Average results at each time point are shown in Tables 8 (glucose tolerance test) and 9 (insulin tolerance test) for each treatment group.







TABLE 8







Glucose tolerance test in high-fat fed mice









Blood glucose (mg/dL)



Time (min.)












Treatment group
0
30
60
90
120





Saline, high fat fed
112
346
264
219
194


ISIS 141923
112
317
243
210
185


ISIS 232828
101
270
204
186
164


Saline, chow
100
249
184
151
133
















TABLE 9







Insulin tolerance test in high-fat fed mice









Blood glucose (mg/dL)



Time (min.)












Treatment group
0
30
60
90
120















Saline, high fat fed
218
122
123
129
154


ISIS 141923
208
127
108
116
145


ISIS 232828
194
121
100
96
114









A plot of the blood glucose levels shown in Table 8 over time reveals that treatment with ISIS 232848 improved glucose tolerance in the high-fat fed mice.
Body fat content was assessed using MRI at the beginning of the study (Week 0) and after treatment (Week 6). The averages for each treatment group at Week 0 were about 33 to 34% body fat. At Week 6, high-fat fed animals treated with saline alone or ISIS 141923 had about 35% body fat and animals treated with ISIS 232848 had about 29% body fat. Thus, treatment with ISIS 232848 decreases body fat content. Lean mass and body weight were not significantly altered.
Liver triglyceride levels were measured at the end of the study using a commercially available kit (for example, the Triglyceride GPO Assay from Roche Diagnostics, Indianapolis, Ind.). On average, liver triglyceride content was about 66 mg/g for saline treated high-fat fed control animals, about 56 mg/g for high-fat fed animals treated with ISIS 141923, about 26 mg/g for high-fat fed animals treated with ISIS 232828, and about 12 mg/g for saline treated animals fed normal chow. Thus, treatment with ISIS 232828 reduces liver triglyceride content.
Quantitative gene expression analysis using real-time RT-PCR was performed on liver, brown adipose tissue (BAT), and WAT from the animals in this study. Expression of low-molecular weight phosphatase (LMW-PTPase), glucose-6-phosphatase, PEPCK, glycogen phosphorylase, HMGCoA reductase, stearoyl CoA desaturase 1 (SCD1), pyruvate dehydrogenase alpha, glycogen synthase, fatty acid synthase (FAS), and diacylglycerol acyltransferase 2 (DGAT2) was examined in liver tissue. Expression of perilipin, FAS, diacylglycerol acyltransferase 1 (DGAT1), DGAT2, lipoprotein lipase, lipotransin, 11-beta hydroxysteroid dehydrogenase 1, adronal receptor beta3, GLUT4, ACRP30 (also known as adiponectin), glycerol kinase, and adipocyte fatty acid-binding protein (FABP) aP2 was examined in WAT. LMW-PTPase, DGAT1, DGAT2, FAS, peroxisome proliferative activated receptor-gamma coactivator 1, UCP1, and UCP2 expression were examined in BAT.
Through these analyses, it was shown that treatment with ISIS 232828 secondarily reduced expression of DGAT2 and FAS in fat, suggesting a reduction in lipogenesis. The treatment also caused a reduction of hepatic glucose-6-phosphatase expression and an increase in hepatic glycogen synthase expression.

## Claims

1. A method of decreasing hepatic triglyceride levels in an animal comprising administering to said animal an antisense compound 12-80 nucleobases in length targeted to a nucleic acid molecule encoding eIF4E-BP2, wherein said compound inhibits the expression of eIF4E-BP2.

2. The method of claim 1, wherein said compound has an oligonucleotide sequence comprising at least an 8-nucleobase portion of SEQ ID NO 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 or 258.

3. The method of claim 1 wherein said animal has steatosis.

4. The method of claim 3 wherein the steatosis is steatohepatitis.

5. The method of claim 3 wherein the steatosis is NASH.

6. A method of decreasing body fat in an animal comprising administering to said animal an antisense compound 12-80 nucleobases in length targeted to a nucleic acid molecule encoding eIF4E-BP2, wherein said compound inhibits the expression of eIF4E-BP2.

7. The method of claim 6, wherein said compound has an oligonucleotide sequence comprising at least an 8-nucleobase portion of SEQ ID NO 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 or 258.

8. A method of improving glucose tolerance in an animal comprising administering to said animal an antisense compound 12-80 nucleobases in length targeted to a nucleic acid molecule encoding eIF4E-BP2, wherein said compound inhibits the expression of eIF4E-BP2.

9. The method of claim 8, wherein said compound has an oligonucleotide sequence comprising at least an 8-nucleobase portion of SEQ ID NO 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 or 258.

10. A method of reducing lipogenesis in an animal comprising administering to said animal an antisense compound 12-80 nucleobases in length targeted to a nucleic acid molecule encoding eIF4E-BP2, wherein said compound inhibits the expression of eIF4E-BP2.

11. The method of claim 10, wherein said compound has an oligonucleotide sequence comprising at least an 8-nucleobase portion of SEQ ID NO 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 or 258.

12. A method of reducing expression of genes involved in lipogenesis in the fat tissue of an animal comprising administering to said animal an antisense compound 12-80 nucleobases in length targeted to a nucleic acid molecule encoding eIF4E-BP2, wherein said compound inhibits the expression of eIF4E-BP2.

13. The method of claim 12, wherein said compound has an oligonucleotide sequence comprising at least an 8-nucleobase portion of SEQ ID NO 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 or 258.

14. The method of claim 12 wherein the genes involved in lipogenesis are DGAT2 and FAS.

15. A method of modulating hepatic expression of genes involved in glucose metabolism in an animal comprising administering to said animal an antisense compound 12-80 nucleobases in length targeted to a nucleic acid molecule encoding eIF4E-BP2, wherein said compound inhibits the expression of eIF4E-BP2.

16. The method of claim 15, wherein said compound has an oligonucleotide sequence comprising at least an 8-nucleobase portion of SEQ ID NO 29, 30, 31, 32, 33, 35, 36, 37, 40, 41, 42, 43, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 81, 83, 88, 89, 92, 93, 95, 96, 98, 100, 101, 104, 105, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 175, 176, 177, 178, 179, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200, 201, 202, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 218, 219, 220, 221, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 or 258.

17. The method of claim 15 wherein the genes involved in glucose metabolism are glucose-6-phosphatase and glycogen synthase.

18. The method of any one of claims 1-17 wherein said animal has a metabolic disease or condition.

19. The method of claim 18 wherein said metabolic disease or condition is obesity, hyperlipidemia, hyperglycemia, diabetes, metabolic syndrome, or insulin resistance.

20. The method of claim 18 wherein the metabolic disease or condition is Type 2 diabetes.