ENHANCED ANTISENSE OLIGONUCLEOTIDES
Described herein are gap-widened antisense oligonucleotides having improved therapeutic index as compared to 5-10-5 MOE gapmer antisense oligonucleotides of the same sequence. Also described are methods of reducing a target RNA in an animal using the gap-widened antisense oligonucleotides of the present invention. Further, are methods for selecting a gap-widened antisense oligonucleotides.
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This application is a continuation of U.S. application Ser. No. 11/231,243, filed Sep. 19, 2005, allowed Oct. 19, 2010, which claims priority under 35 USC 119(e) to U.S. Application No. 60/611,100, filed on Sep. 17, 2004 and to U.S. Application No. 60/663,442, filed on Mar. 18, 2005, each of which is herein incorporated by reference in its entirety. The instant application is also related to U.S. Application 60/718,685, filed Sep. 19, 2005, and U.S. Application 60/718,684, filed Sep. 19, 2005, each of which is herein incorporated by reference in its entirety.
SEQUENCE LISTINGThe present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0051USC1SEQ.txt, created on Jan. 17, 2011 which is 92 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTIONThe present invention provides chimeric antisense compounds having enhanced in vivo potency and thus an improved therapeutic index. The compounds described herein have widened deoxy gaps and enhanced in vivo potency which is unexpected based on their in vitro activity.
BACKGROUND OF THE INVENTIONAntisense oligonucleotides are accepted therapeutic modalities and many thousands of patients have been treated with antisense compounds. The original “first generation” antisense compounds employed in the first antisense clinical trials were oligodeoxynucleotides having 2′-deoxy ribonucleotides and phosphorothioate intemucleoside linkages. Subsequently, chimeric “second generation” antisense oligonucleotides exhibited a marked improvement in potency over first generation antisense oligonucleotides. Second generation antisense oligonucleotides are chimeric oligonucleotides typically having a 2′-deoxy “gap” region flanked by “wings” having nucleotides with 2′-modified ribonucleotides, referred to as “gapmers.” The most widely used of the “second generation” antisense motifs is often referred to as a “MOE gapmer” in which the 2′-modified ribonucleotide is a 2′-O-methoxyethyl (2′-MOE or simply MOE) modification, and each of the internucleotide linkages is a phosphorothioate. Predominantly, second generation oligonucleotides have a length of 20 nucleotides of which the 5 nucleotides at each terminus are 2′-MOE nucleotides and the center ten nucleotides are 2′-deoxyribonucleotides. These second generation oligonucleotides are referred to as “5-10-5 MOE gapmers” have a 5-10-5 wing-gap-wing motif. Chimeric antisense compounds with other arrangements of modifications have also been made. “Hemimers,” are chimeric compounds in which there is a single 2′-modified “wing” adjacent to (on either the 5′, or the 3′ side of) a 2′-deoxy gap have been described (Geary et al., 2001, J. Pharm. Exp. Therap., 296, 898-904).
SUMMARY OF THE INVENTIONThe present invention is directed to “gap-widened” antisense oligonucleotides having a gap region of greater than 11 2′deoxyribonucleotides flanked by two “wing” regions having from one to eight nucleotides which do not support RNase H activity. The gap-widened antisense oligonucleotide of the present invention have been shown to have an improved therapeutic index as compared to a corresponding antisense oligonucleotide having a 5-10-5 MOE gamer antisense oligonucletide with the same sequence. The gap-widened antisense oligonucleotides of the present invention exhibit increased in vivo potency or improved tissue exposure as compared with the corresponding 5-10-5 MOE gapmer antisense oligonucleotide with the same sequence. Most interestingly, there is a lack of correlation between the in vitro potency and the in vivo potency of the gap-widened antisense oligonucleotides described herein. The gap-widened antisense oligonucleotides of the present invention are 18 to 24 nucleotides in length. In particular, the gap-widened antisense oligonucleotides of the present invention have wing regions having 2′-O-(2-methoxyethyl)ribonucleotides.
In an additional embodiment of the present invention is a method of reducing expression of a target RNA in an animal in need of reducing expression of said target RNA, comprising administering to said animal a gap-widened antisense oligonucleotide 18 to 24 nucleotides in length comprising: a gap region having greater than 11 contiguous 2′-deoxyribonucleotides; and a first wing region and a second wing region flanking the gap region, wherein each of said first and second wing regions independently have 1 to 8 2′-O-(2-methoxyethyl)ribonucleotides, having an improved therapeutic index as compared to a corresponding 5-10-5 MOE gapmer antisense oligonucleotide having a gap region of 10 contiguous 2′-deoxyribonucleotides and a first wing region and a second wing region flanking the gap region of 5 2′-O-(2-methoxyethyl)ribonucleotides. The improvement in therapeutic index is characterized by equal or increased potency coupled with a reduction in tissue concentration, or increased potency coupled with equal tissue exposures as compared to a corresponding 5-10-5 MOE gapmer antisense oligonucleotide of the same sequence. In addition, the improvement in therapeutic index may be characterized by an increased liver to kidney concentration ratio as compared to a corresponding 5-10-5 MOE gapmer antisense oligonucleotide of the same sequence. In particular, the method of the present invention is useful in reducing the expression of RNA targets expressed in the kidney, liver, or adipose tissues. The method of the present invention is also useful in reducing the expression of target RNA associated with a metabolic or cardiovascular disease or condition. The method of the present invention is useful wherein the metabolic disease or condition is selected from diabetes, hepatic steatosis, fatty liver disease, non-alcoholic steatohepatitis, metabolic syndrome, obesity, or the like. In addition, the method of the present invention is useful wherein the cardiovascular disease or condition is selected from hypercholesterolemia, atherosclerosis, hyperlipidemia, familial hypercholesterolemia, or the like.
An additional method of the present invention is a method of selecting a gap-widened antisense oligonucleotide with an improved therapeutic index, the method comprising:
screening in vitro a plurality of antisense oligonucleotides targeting a human RNA and having a single wing-gap-wing motif;
identifying a parent antisense oligonucleotide from the plurality of antisense oligonucleotides having a potent in vitro activity;
synthesizing a plurality of gap-widened antisense oligonucleotides having the same sequence as the parent antisense oligonucleotide, wherein said gap-widened antisense oligonucleotide is 18 to 24 nucleotides in length comprising a gap region having greater than 11 contiguous 2′-deoxyribonucleotides; and a first wing region and a second wing region flanking the gap region, wherein each of said first and second wing regions independently has 1 to 8 2′-O-(2-methoxyethyl)ribonucleotides;
testing said plurality of gap-widened antisense oligonucleotides in a plurality of animals;
obtaining potency and tissue concentration data from said testing step; and
determining an optimized gap-widened oligonucleotide wing-gap-wing motif with an improved therapeutic index, improved potency, reduced tissue exposure, or reduced toxicity, or a combination thereof as compared to the parent antisense oligonucleotide.
In one embodiment, the method of selecting a gap-widened antisense oligonucleotide further comprises the step of designing a rodent sequence analogous or a non-human primate sequence to said parent antisense oligonucleotide. In one embodiment, the step of determining the optimized gap-widened antisense oligonucleotide wing-gap-wing motif with an improved therapeutic index includes identifying a gap-widened antisense oligonucleotide which has equal or increased potency as compared to the parent antisense oligonucleotide.
In the step of screening, each of said antisense oligonucleotides has the same wing-gap-wing motif selected from 2-16-2, 3-14-3, 4-12-4, or 5-10-5. In a further embodiment, the wing portions of the gap-widened antisense oligonucleotides are 2′-O-(2-methoxyethyl)ribonucleotides. In particular, the step of screening is performed in primary hepatocytes, HepG2, bEND, or HeLa cells. In the step of identifying, the potent in vitro activity is greater than 50% reduction in the target mRNA expression as compared to a saline control. In alternate embodiments, in the step of identifying, the potent in vitro activity is greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%
In the step of synthesizing, the gap-widened antisense oligonucleotides each have different wing-gap-wing motifs. In particular, the gap-widened antisense oligonucleotides have gaps of 12, 13, 14, 15, 16, 17, or 18 2′-deoxyribonucleotides in length. In the step of testing, the animals are selected from rodents such as mice and rats, and non-human primates, such as cynomolgous monkeys.
In the step of obtaining, the tissue concentration data are concentrations of full-length gap-widened antisense oligonucleotides particularly measured in the liver, kidney, or adipose tissue. In one embodiment, each optimized gap-widened antisense oligonucleotide is selected because of equal or improved potency data. In another embodiment, each optimized gap-widened antisense oligonucleotide is selected because of reduced tissue exposure. In another embodiment, each optimized gap-widened antisense oligonucleotide is selected because of reduced toxicity. In another embodiment, each optimized gap-widened antisense oligonucleotide is selected because of improved therapeutic index. In another embodiment, each optimized gap-widened antisense oligonucleotide is selected because of reduced tissue exposure, reduced toxicity, improved potency, or a combination thereof.
The gap-widened antisense oligonucleotides described herein may have various wing-gap-wing motifs selected from: 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12-5, 5-12-1, 2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2, 1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14-1, 2-14-3, 3-14-2, 1-13-5, 5-13-1, 2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3, 1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-11-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 5-14-1, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 4-12-4, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 4-12-4, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-19-1, 1-18-2, 2-18-1, 1-17-3, 3-17-1, 2-17-2, 1-16-4, 4-16-1, 2-16-3, 3-16-2, 1-15-5, 2-15-4, 4-15-2, 3-15-3, 1-14-6, 6-14-1, 2-14-5, 5-14-2, 3-14-4, 4-14-3, 1-13-7, 7-13-1, 2-13-6, 6-13-2, 3-13-5, 5-13-3, 4-13-4, 1-12-8, 8-12-1, 2-12-7, 7-12-2, 3-12-6, 6-12-3, 4-12-5, 5-12-4, 2-11-8, 8-11-2, 3-11-7, 7-11-3, 4-11-6, 6-11-4, 5-11-5, 1-20-1, 1-19-2, 2-19-1, 1-18-3, 3-18-1, 2-18-2, 1-17-4, 4-17-1, 2-17-3, 3-17-2, 1-16-5, 2-16-4, 4-16-2, 3-16-3, 1-15-6, 6-15-1, 2-15-5, 5-15-2, 3-15-4, 4-15-3, 1-14-7, 7-14-1, 2-14-6, 6-14-2, 3-14-5, 5-14-3, 4-14-4, 1-13-8, 8-13-1, 2-13-7, 7-13-2, 3-13-6, 6-13-3, 4-13-5, 5-13-4, 2-12-8, 8-12-2, 3-12-7, 7-12-3, 4-12-6, 6-12-4, 5-12-5, 3-11-8, 8-11-3, 4-11-7, 7-11-4, 5-11-6, 6-11-5, 1-21-1, 1-20-2, 2-20-1, 1-20-3, 3-19-1, 2-19-2, 1-18-4, 4-18-1, 2-18-3, 3-18-2, 1-17-5, 2-17-4, 4-17-2, 3-17-3, 1-16-6, 6-16-1, 2-16-5, 5-16-2, 3-16-4, 4-16-3, 1-15-7, 7-15-1, 2-15-6, 6-15-2, 3-15-5, 5-15-3, 4-15-4, 1-14-8, 8-14-1, 2-14-7, 7-14-2, 3-14-6, 6-14-3, 4-14-5, 5-14-4, 2-13-8, 8-13-2, 3-13-7, 7-13-3, 4-13-6, 6-13-4, 5-13-5, 1-12-10, 10-12-1, 2-12-9, 9-12-2, 3-12-8, 8-12-3, 4-12-7, 7-12-4, 5-12-6, 6-12-5, 4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1-15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7, or 7-11-6. In a particular embodiment, the gap-widened antisense oligonucleotides of the present invention have a 2-16-2, 3-14-3, or 4-12-4 wing-gap-wing motif.
Another aspect of the present invention is the use of a gap-widened antisense oligonucleotide 18-24 nucleotides in length comprising: a gap region having greater than 11 contiguous 2′-deoxyribonucleotides; and a first wing region and a second wing region flanking the gap region, wherein each of said first and second wing regions independently have 1 to 8 2′-O-(2-methoxyethyl)ribonucleotides, having an improved therapeutic index as compared to a corresponding 5-10-5 antisense oligonucleotide having a gap region of 10 contiguous 2′-deoxyribonucleotides and a first wing region and a second wing region flanking the gap region of 5 2′-O-(2-methoxyethyl)ribonucleotides in the manufacture of a medicament for the treatment of disorders and diseases related to target RNA levels. Another embodiment of the present invention is a pharmaceutical composition comprising a gap-widened antisense oligonucleotide 18-24 nucleotides in length comprising: a gap region having greater than 11 contiguous 2′-deoxyribonucleotides; and a first wing region and a second wing region flanking the gap region, wherein each of said first and second wing regions independently have 1 to 8 2′-O-(2-methoxyethyl)ribonucleotides, having an improved therapeutic index as compared to a corresponding 5-10-5 antisense oligonucleotide having a gap region of 10 contiguous 2′-deoxyribonucleotides and a first wing region and a second wing region flanking the gap region of 5 2′-O-(2-methoxyethyl)ribonucleotides and optionally a pharmaceutically acceptable carrier, diluent, enhancer or excipient. Another embodiment of the present invention is a gap-widened antisense oligonucleotide 18-24 nucleotides in length comprising: a gap region having greater than 11 contiguous 2′-deoxyribonucleotides; and a first wing region and a second wing region flanking the gap region, wherein each of said first and second wing regions independently have 1 to 8 2′-O-(2-methoxyethyl)ribonucleotides, having lower kidney accumulation as compared to a corresponding 5-10-5 antisense oligonucleotide having a gap region of 10 contiguous 2′-deoxyribonucleotides and a first wing region and a second wing region flanking the gap region of 5 2′-O-(2-methoxyethyl)ribonucleotides as measured by plasma protein binding capacity of said gap-widened antisense oligonucleotide. Also provided is a method of modulating gene expression in an animal comprising the step of contacting said animal with the pharmaceutical composition. Another embodiment is a method of modulating gene expression in an animal comprising the step of contacting said animal with a gap-widened antisense oligonucleotide of the invention wherein the accumulation of the gap-widened antisense oligonucleotide in the kidney is less compared to a corresponding 5-10-5 antisense oligonucleotide having a gap region of 10 contiguous 2′-deoxyribonucleotides and a first wing region and a second wing region flanking the gap region of 5 2′-O-(2-methoxyethyl)ribonucleotides. In one embodiment, the kidney accumulation is measured by plasma protein binding capacity of said gap-widened antisense oligonucleotide.
Another embodiment of the present invention is a method of reducing levels of a preselected RNA target in the liver of an animal comprising administering to said animal a chimeric antisense compound 11 to 80 nucleobases in length which is targeted to said preselected RNA target wherein said chimeric antisense compound comprises a first gap region consisting of at least 10 contiguous 2′-deoxynucleotides and a wing region which consists of from 1 to 4 contiguous nucleosides or nucleoside analogs which are not substrates for RNaseH. In particular embodiments, said first gap region consists of at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 contiguous 2′-deoxynucleotides. In one embodiment, the chimeric antisense compound comprises second wing region which consists of from 1 to 7 contiguous nucleosides or nucleoside analogs which are not substrates for RNase H, and wherein said gap region is located between said first wing region and said second wing region. In another embodiment, the chimeric antisense compound is a chimeric antisense oligonucleotide, and the nucleosides or nucleoside analog which is not a substrate for RNase H is a nucleotide having a 2′ modification of the sugar moiety. In one embodiment, the nucleotide having a 2′ modification of the sugar moiety is a 2′-O-methoxyethyl nucleotide. In some embodiments the compound is a 2-16-2 MOE gapmer, a 3-12-3 MOE gapmer, a 3-10-7 MOE gapmer or a 7-10-3 MOE gapmer. In one embodiment, the chimeric antisense oligonucleotide has at least one phosphorothioate backbone linkage.
Another embodiment of the present invention is a pharmaceutical composition for use in reducing levels of a preselected RNA target in the liver of an animal comprising a chimeric antisense compound targeted to said preselected RNA target, wherein said chimeric antisense compound comprises a first gap region consisting of at least 10 contiguous 2′-deoxynucleotides and a wing region which consists of from 1 to 4 contiguous nucleosides or nucleoside analogs which are not substrates for RNase H.
Certain gap sizes are optimal for in vivo efficacy of antisense compounds. Surprisingly, improved potency (3-10× improvement) in mouse or rat liver has been demonstrated for gap-widened antisense oligonucleotides compared to standard 5-10-5 MOE gapmer (for example, 2-16-2, 2-14-2, 3-12-3 gapmers) antisense oligonucleotides. This has been shown for several distinct antisense targets and this improved potency is not observed in cultured cells transfected with the same gap-widened antisense oligonucleotides. Thus the “gap-widened” motifs appear to convey some benefit to in vivo potency, particularly in the liver. It is demonstrated herein that chimeric antisense compounds having a gap of greater than eleven contiguous deoxynucleotides flanked by wing regions consisting of from 1 to 4 nucleotides which are not substrates for RNase H are particularly effective at reducing target RNA levels in vivo, particularly in the liver.
Therapeutic IndexTherapeutic index is a measure which relates the dose of a drug required to produce a specified effect to that which produces an undesired effect. In one embodiment, improved therapeutic index of a gap-widened antisense oligonucleotide is characterized by equal or increased potency and a reduction in tissue concentration. In another embodiment, improved therapeutic index of a gap-widened antisense oligonucleotide is characterized by increased potency and equal tissue concentrations as compared to a corresponding 5-10-5 antisense oligonucleotide. In another embodiment, improved therapeutic index of a gap-widened antisense oligonucleotide is characterized by increased potency and decreased toxicity as compared to a corresponding 5-10-5 antisense oligonucleotide. In another embodiment, improved therapeutic index of a gap-widened antisense oligonucleotide is characterized by comparable potency and decreased toxicity as compared to a corresponding 5-10-5 antisense oligonucleotide. In some embodiments, the toxicity is renal toxicity. In some embodiments, the toxicity is hepatic toxicity.
IndicationsAn embodiment of the present invention is a method of treating a disease or condition wherein a target RNA is associated with said disease or condition by administering a compound of the invention. Another embodiment of the present invention is a method of preventing or delaying the onset of a disease or condition wherein a target RNA is associated with said disease or condition by administering a compound of the invention. Diseases or conditions include metabolic and cardiovascular diseases or conditions. In some embodiments, the disease or condition is metabolic syndrome, diabetes, obesity, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, Type 2 diabetes, diet-induced obesity, hyperglycemia, or insulin resistance. In one embodiment, the disease or condition is hepatic steatosis. In some embodiments, the steatosis is steatohepatitis or NASH. In some embodiments, the disease or condition is familial hypercholesterolemia, nonfamilial hypercholesterolemia, mixed dyslipidemia, dysbetalipoproteinemia, atherosclerosis, coronary artery disease, myocardial infarction, hypertension, carotid artery diseases, stroke, cerebrovascular disease, carotid artery disease, stroke, cerebrovascular disease, peripheral vascular disease, thrombosis, or arterial aneurism.
NAFLD and Metabolic SyndromeThe 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. Nonalcoholic steatohepatitis (NASH) occurs from progression of NAFLD beyond deposition of triglycerides. 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).
“Metabolic syndrome” is defined as a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. It is 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 metaolic syndrome when three or more of five risk determinants are present. The five risk determinants are abdominal obesity defined as waist circumference of greater than102 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).
HbA1cHbA1c is a stable minor hemoglobin variant formed in vivo via posttranslational modification by glucose, and it contains predominantly glycated NH2-terminal β-chains. There is a strong correlation between levels of HbA1c and the average blood glucose levels over the previous 3 months. Thus HbA1c is often viewed as the “gold standard” for measuring sustained blood glucose control (Bunn, H. F. et al., 1978, Science. 200, 21-7). HbA1c can be measured by ion-exchange HPLC or immunoassay; home blood collection and mailing kits for HbA1c measurement are now widely available. Serum fructosamine is another measure of stable glucose control and can be measured by a colorimetric method (Cobas Integra, Roche Diagnostics).
Cardiovascular Risk ProfileConditions associated with risk of developing a cardiovascular disease include, but are not limited to, history of myocardial infarction, unstable angina, stable angina, coronary artery procedures (angioplasty or bypass surgery), evidence of clinically significant myocardial ischemia, noncoronary forms of atherosclerotic disease (peripheral arterial disease, abdominal aortic aneurysm, carotid artery disease), diabetes, cigarette smoking, hypertension, low HDL cholesterol, family history of premature CHD, obesity, physical inactivity, elevated triglyceride, or metabolic syndrome (Jama, 2001, 285, 2486-2497; Grundy et al., Circulation, 2004, 110, 227-239).
EXAMPLES Example 1 Oligonucleotide Sequences and Targets
Modulation of target RNA expression can be assayed in a variety of ways known in the art. GCCR mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA by methods known in the art. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.
Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
Levels of proteins encoded by a target RNA can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to a protein encoded by a target RNA 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 antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.
Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.
The effect of oligomeric compounds of the present invention on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. The effect of oligomeric compounds of the present invention on target nucleic acid expression can be routinely determined using, for example, PCR or Northern blot analysis. Cell lines are derived from both normal tissues and cell types and from cells associated with various disorders (e.g. hyperproliferative disorders). Cell lines derived from multiple tissues and species can be obtained from American Type Culture Collection (ATCC, Manassas, Va.), the Japanese Cancer Research Resources Bank (Tokyo, Japan), or the Centre for Applied Microbiology and Research (Wiltshire, United Kingdom).
Primary cells, or those cells which are isolated from an animal and not subjected to continuous culture, can be prepared according to methods known in the art or obtained from various commercial suppliers. Additionally, primary cells include those obtained from donor human subjects in a clinical setting (i.e. blood donors, surgical patients).
Cell TypesThe effects of oligomeric compounds on target nucleic acid expression were tested in the following cell types:
b.END Cells:The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany). b.END cells were routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 3000 cells/well for use in oligomeric compound transfection experiments.
HepG2 Cells:The human hepatoblastoma cell line HepG2 was obtained from the American Type Culture Collection (Manassas, Va.). HepG2 cells were routinely cultured in Eagle's MEM supplemented with 10% fetal bovine serum, 1 mM non-essential amino acids, and 1 mM sodium pyruvate (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Multiwell culture plates are prepared for cell culture by coating with a 1:100 dilution of type 1 rat tail collagen (BD Biosciences, Bedford, Mass.) in phosphate-buffered saline. The collagen-containing plates were incubated at 37° C. for approximately 1 hour, after which the collagen was removed and the wells were washed twice with phosphate-buffered saline. Cells were seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 8,000 cells/well for use in oligomeric compound transfection experiments.
Primary Rat Hepatocytes:Primary rat hepatocytes are prepared from Sprague-Dawley rats purchased from Charles River Labs (Wilmington, Mass.) and are routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.), 100 units per mL penicillin, and 100 μg/mL streptomycin (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 4,000-6,000 cells/well treatment with the oligomeric compounds of the invention.
Treatment with Oligomeric Compounds
When cells reached appropriate confluency, they were treated with oligonucleotide using a transfection method as described. Other suitable transfection reagents known in the art include, but are not limited to, LIPOFECTAMINE™, CYTOFECTIN™, OLIGOFECTAMINE™, and FUGENE™. Other suitable transfection methods known in the art include, but are not limited to, electroporation.
LIPOFECTIN™When cells reach 65-75% confluency, they are treated with oligonucleotide. Oligonucleotide is mixed with UPOFECTIN™ Invitrogen Life Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired concentration of oligonucleotide and a LIPOFECTIN™ concentration of 2.5 or 3 μg/mL per 100 nM oligonucleotide. This transfection mixture is incubated at room temperature for approximately 0.5 hours. For cells grown in 96-well plates, wells are washed once with 100 μL OPTI-MEM™-1 and then treated with 130 μL of the transfection mixture. Cells grown in 24-well plates or other standard tissue culture plates are treated similarly, using appropriate volumes of medium and oligonucleotide. Cells are treated and data are obtained in duplicate or triplicate. After approximately 4-7 hours of treatment at 37° C., the medium containing the transfection mixture is replaced with fresh culture medium. Cells are harvested 16-24 hours after oligonucleotide treatment.
Example 3 Real-Time Quantitative PCR Analysis of GCCR mRNA LevelsQuantitation of GCCR mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions.
Gene target quantities obtained by RT, real-time PCR were normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). Total RNA was quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was pipetted into a 96-well plate containing 30 μL purified cellular RNA. The plate was read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.
GAPDH expression was quantified by RT, real-time PCR, either simultaneously with the quantification of the target or separately. For measurement simultaneous with measurement of target levels, primer-probe sets specific to the target gene being measured were evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction prior to quantitative PCR analysis. Multiplexing refers to the detection of multiple DNA species, in this case the target and endogenous GAPDH control, in a single tube, which requires that the primer-probe set for GAPDH does not interfere with amplification of the target.
Probes and primers for use in real-time PCR were designed to hybridize to target-specific sequences. Methods of primer and probe design are known in the art. Design of primers and probes for use in real-time PCR can be carried out using commercially available software, for example Primer Express®, PE Applied Biosystems, Foster City, Calif. The target-specific PCR probes have FAM covalently linked to the 5′ end and TAMRA or MGB covalently linked to the 3′ end, where FAM is the fluorescent dye and TAMRA or MGB is the quencher dye.
After isolation, the RNA is subjected to sequential reverse transcriptase (RT) reaction and real-time PCR, both of which are performed in the same well. RT and PCR reagents were obtained from Invitrogen Life Technologies (Carlsbad, Calif.). RT, real-time PCR was carried out in the same 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 nM 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).
Example 4 Increased Potency of ISIS 344266 (2-16-2) In Vivo Compared to 5-10-5 Compound is not due to Enhanced Oligonucleotide AccumulationMice were dosed with ISIS 116847 (SEQ ID NO: 1) or ISIS 344266 (SEQ ID NO: 1) at 6, 3, 1.5 or 0.75 micromol/kg (approx 40, 20, 10 or 5 mg per kg), twice a week for three weeks and sacrificed 48 hours after the last dose was given. The left panel of
The ID50 (dose resulting in 50% reduction of PTEN RNA) calculated from these results was 1.9 micromol/kg for 116847 and 0.63 micromol/kg for 344266. The IC50 for ISIS 116847 was also over three-fold that of ISIS 344266. These results indicate that the gap-widened antisense oligonucleotide is three-fold more potent than the 5-10-5 compound of equivalent sequence.
ISIS 344266 (2-16-2) supports similar persistence of action compared to ISIS 116847 (5-10-5). Mice were treated as described above with ISIS 344266 (1.5 or 6 micromol/kg) or ISIS 116847 (6 micromol/kg), or with saline. PTEN RNA levels were measured in mouse liver at days 1, 7, 14 and 28. As shown in
The advantage conveyed by the gap-widened antisense oligonucleotides of the present invention for target reduction in vivo is surprising because it is not observed in vitro. An in vitro comparison of the same PTEN oligonucleotides, ISIS 116847 (5-10-5) and ISIS 344266 (2-16-2) was performed in cultured mouse bEND cells. Cells were transfected with oligonucleotide at doses of 0.1 nM, 0.3 nM, 0.9 nM, 2.7 nM, 8.1 nM and 24.3 nM in the presence of 3 microgram/ml LIPOFECTIN. Reduction of target expression was assayed by quantitative RT real-time PCR as described herein.
The enhanced potency of the gap-widened (2-16-2) PTEN antisense oligonucleotide in liver is not due to increased concentrations in liver compared to the 5-10-5 gapmer. Oligonucleotide concentration in kidney and liver tissue from mice treated as described above with ISIS 116847 or ISIS 344266 were determined. Methods to determine oligonucleotide concentration in tissues are known in the art (Geary et al., Anal Biochem, 1999, 274, 241-248). Oligonucleotide concentrations (micrograms/gram) in mouse liver and kidney were determined. As shown in
Serum transaminases (AST/ALT) were higher for mice treated with 2-16-2 compound (ISIS 344266) than for those treated with ISIS 116847. However, because ISIS 344266 is more potent (active at lower doses), the therapeutic window for the two compounds is roughly comparable.
Example 5 Effect of Gap Size on In Vitro and In Vivo PotencyA series of MOE gapmers (2-14-2 through 6-6-6) were designed to target mouse TRADD (consensus sequence built from mouse ESTs: aa013629,aa914725,aa013699, aa122508, aa881900, aa423244, aa930854, w13708, aa201054, ai122320, aa611848, aa546092, and aa939422, incoporated herein as SEQ ID NO: 104). As shown in Table 2, a series of 18 mer chimeric antisense oligonucleotides were synthesized, all having the same sequence (GCTCATACTCGTAGGCCA, incorporated herein as SEQ ID NO: 3). Plain text indicates a deoxynucleotide, and nucleobases designated with bold, underlined text are 2′-O-(2-methoxyethyl)nucleotides. Internucleoside linkages are phosphorothioate throughout, and all cytosines are 5-methylcytosines. Indicated in Table 2 is the “motif' of each compound indicative of chemically distinct regions comprising the oligonucleotide.
The compounds were tested in vitro in mouse bEND cells at concentrations of 0.1 nM, 0.5 nM, 2.5 nM, 12.5 nM and 62.5 nM for their ability to reduce target mRNA levels using real-time PCR as described herein. As shown in
When these compounds were tested in vivo, a different rank order potency was observed. Mice were treated with TRADD gapmer oligos (described above) ranging from 2-14-2 chimeras to 6-6-6 chimeras, each at doses of 1.56 micromole/kg, 3.12 micromol/kg and 6.24 micromol/kg. The negative control was ISIS 29837 (SEQ ID NO: 4) and animals treated with saline alone served as the control group to which data were normalized. As shown in
The effect of these gapmer compounds on mouse body weight, liver weight and spleen weights was compared and no meaningful differences were seen. Mice gained weight at roughly the same rate (indicating general good health) and liver and spleen weights were comparable to saline in all the treatment groups.
Example 6 Antisense Inhibition of Human GCCR Expression by 5-10-5 Gapmers or 2-16-2 Gapmers In VitroA series of oligomeric compounds was designed to target different regions of human GCCR, using published sequences (GENBANK® accession no: NM—000176.1, incoporated herein as SEQ ID NO: 105). The compounds are shown in Table 3. All compounds in Table 3 are chimeric oligonucleotides (”gapmers“) 20 nucleotides in length, composed of a central “gap” region consisting of 10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) by five-nucleotide “wings”. The wings are composed of 2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. Shown in Table 3 is the sequence of the oligonucleotide, and the target site which is the first (5′ most) position on the target sequence to which the compound binds. The compounds were analyzed for their effect on gene target mRNA levels by quantitative real-time PCR as described in other examples herein, using a primer-probe set designed to hybridize to human GCCR.
Data are averages from three experiments in which HepG2 cells were treated with 50 nM of the disclosed oligomeric compounds using LIPOFECTIN™. A reduction in expression is expressed as percent inhibition in Table 3. If present, “N.D.” indicates “not determined”. The target regions to which these oligomeric compounds are inhibitory are herein referred to as “validated target segments.”
Gap-widened oligonucleotides having the same sequences as the compounds described in Table 4 were also tested. All compounds in Table 4 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of 16 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) by two-nucleotide “wings”. The wings are composed of 2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. Shown in Table 4 is the sequence of the oligonucleotide, and the target site which is the first (5′ most) position on the target sequence to which the compound binds. The 2-16-2 motif compounds were analyzed for their effect on gene target mRNA levels by quantitative real-time PCR as described herein.
Data are averages from three experiments in which HepG2 cells were treated with 50 nM of the disclosed oligomeric compounds using LIPOFECTIN™. A reduction in expression is expressed as percent inhibition in Table 4. If present, “N.D.” indicates “not determined”. The target regions to which these oligomeric compounds are inhibitory are herein referred to as “validated target segments.”
The 2-16-2 oligonucleotides shown in Table 4 and the 5-10-5 oligonucleotides shown in Table 3 which reduced GCCR expression by at least 30% are preferred. The target segments 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.
Example 7 Cross-Species Oligonucleotides Targeting GCCRSome oligonucleotides described in the previous example are complementary across species and are therefore expected to reduce expression of glucocorticoid receptor across species. Shown in Table 5 is the sequence of such cross-species oligonucleotides, and the ISIS numbers of the 5-10-5 motif version and the 2-16-2 motif version of the oligonucleotide. Also indicated for each sequence is the target site which is the first (5′ most) position on the human target sequence (NM—000176.1, incorporated herein as SEQ ID NO: 105) to which the compound binds. The complementarity for human, cynomolgus monkey, rat, and mouse GCCR mRNA is indicated (“yes” means perfect complementarity and “1 mm” means one mismatch from perfect complementarity).
In a further embodiment of the present invention, eleven oligonucleotides were selected for additional dose-response studies. Primary rat hepatocytes were treated with 5, 10, 25, 50, 100 or 200 nM of ISIS 180274, ISIS 180275, ISIS 180276, ISIS 180281, ISIS 180304, ISIS 361137, ISIS 361141, ISIS 361151, ISIS 361156, ISIS 345198, ISIS 361137 or the negative control oligonucleotide ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, incorporated herein as SEQ ID NO: 2), and mRNA levels were measured as described in other examples herein. ISIS 141923 is a 5-10-5 gapmer comprising a ten deoxynucleotide gap flanked by 2′-MOE wings and a phosphorothioate backbone. All cytosines are 5-methylcytosines. Untreated cells served as the control to which the data were normalized.
Results of these studies are shown in Table 6. Target mRNA levels were measured by real-time PCR as described herein. Data are averages from three experiments and are expressed as percent inhibition relative to untreated control.
In a further embodiment of the present invention, the same oligonucleotides were tested in the human HepG2 cell line for their ability to reduce GCCR mRNA expression at the indicated doses. Untreated cells served as the control to which the data were normalized.
Results of these studies are shown in Table 7. Target mRNA levels were measured by real-time PCR as described herein. Data are averages from three experiments and are expressed as percent inhibition relative to untreated control.
As shown in Table 6 and Table 7, antisense oligonucleotides targeting GCCR are effective at reducing both human and rat target mRNA levels in a dose-dependent manner in vitro.
Example 9 Antisense Inhibition of Rat GCCR mRNA Levels—In Vivo Dose-Response Studies with 5-10-5 GapmersFive of the 5-10-5 gapmer motif oligonucleotides (ISIS 180281, ISIS 361137, ISIS 345198, ISIS 180304, and ISIS 361141) were evaluated at various doses in rats for their ability to reduce GCCR mRNA levels in liver. Eight week-old Sprague Dawley rats were divided into treatment groups which received doses of 50, 25 or 12.5 mg/kg of one the indicated oligonucleotides via injection. Each treatment group was comprised of four animals, and was dosed twice weekly for 3 weeks. Animals injected with saline alone served as a control group. The animals were evaluated weekly for standard blood parameters (ALT/AST, cholesterol, triglycerides, and glucose). Animals were sacrificed at the end of the study and liver tissue was collected and analyzed for target reduction using real-time PCR analysis methods described herein. Results are shown in Tables 8a and 8b (separate experiments) as the percentage reduction in GCCR mRNA measured after treatment with the indicated doses of the indicated oligonucleotides.
The data in Tables 8a and 8b show that antisense oligonucleotides targeted to GCCR are effective at reducing expression in vivo in a dose-dependent manner. ISIS 345198 (GTCAAAGGTGCTTTGGTCTG; SEQ ID NO: 16) was chosen for further evaluation in structure-activity experiments focusing on gap optimization. This compound is perfectly complementary to mouse, rat, human, monkey, rabbit and guinea pig glucocorticoid receptor RNA.
Example 10 Antisense Inhibition of GCCR mRNA Levels In Vivo—Gap Optimization StudyA series of oligomeric compounds were designed to target GCCR with varying sizes of the deoxynucleotide gap and 2′-MOE wings. Each of the oligonucleotides tested has the same nucleobase sequence (GTCAAAGGTGCTTTGGTCTG, incorporated herein as SEQ ID NO: 16) and therefore targets the same segment of SEQ ID NO: 105 (nucleobases 689 to 709). As shown in Example 7, this oligonucleotide is also perfectly complementary to rat GCCR.
The compounds are shown in Table 9. Plain text indicates a deoxynucleotide, and nucleobases designated with bold, underlined text are 2′-O-(2-methoxyethyl)nucleotides. Internucleoside linkages are phosphorothioate throughout, and all cytosines are 5-methylcytosines.
Indicated in Table 9 is the “motif' of each compound indicative of chemically distinct regions comprising the oligonucleotide.
Nine-week old Sprague-Dawley male rats were treated twice weekly for three weeks with doses of 50, 25, 12.5, and 6.25 mg/kg of the oligonucleotides presented in Table 9. Animals injected with saline alone served as controls. Each treatment group was comprised of four animals.
At the end of the study, animals were sacrificed, and tissues were collected for determination of target reduction and oligonucleotide concentration.
White adipose tissue was analyzed for target reduction using real-time PCR analysis methods described herein. Results are shown in Tables 10a, 10b, and 10c (separate experiments) as the percentage reduction in GCCR mRNA measured after treatment with the indicated doses of the indicated oligonucleotides. Tissues from animals treated with each gap-widened oligonucleotide were assayed for target reduction alongside tissues from animals treated with the 5-10-5 motif oligonucleotide for comparison.
Liver tissue was also analyzed for target reduction using real-time PCR analysis methods described herein. Results are shown in Tables 11a, 11b, and 11c (separate experiments) as the percentage reduction in GCCR mRNA measured after treatment with the indicated doses of the indicated oligonucleotides. Tissues from animals treated with each gap-widened oligonucleotide were assayed for target reduction alongside tissues from animals treated with the 5-10-5 motif oligonucleotide for comparison.
As shown in Tables 11a, 11b, and 11c, all of the gap-widened oligonucleotides tested were effective at reducing GCCR levels in a dose-dependent manner in vivo. In addition, the gap-widened oligonucleotides show a trend toward greater potency than the 5-10-5 gapmer in the liver.
In addition, to determine effects of altering the gap size on pharmacokinetics, oligonucleotide concentration in kidney and liver were determined. Methods to determine oligonucleotide concentration in tissues are known in the art (Geary et al., Anal Biochem, 1999, 274, 241-248). Total oligonucleotide is the sum of all oligonucleotides metabolites detected in the tissue. Shown in Table 12 are the total concentration and the concentration of full length oligonucleotide (in μg/g) in the liver of animals treated with the indicated oligonucleotide at the indicated concentration.
As shown in Table 12, the levels of full-length oligonucleotide in the liver are comparable or reduced for ISIS 372339 and ISIS 377130 as compared to ISIS 345198. Coupled with the target reduction as shown in Table 11, these data show that the enhanced potency of the gap-widened compounds is not due to enhanced accumulation of the compound in the liver. Thus, preferred oligonucleotides of the present invention include gap-widened oligonucleotides that show enhanced or comparable potency with regard to target reduction to the corresponding 5-10-5 gapmer without enhanced accumulation of the compound in a target tissue. In some embodiments, the target tissue is adipose and in some embodiments, the target tissue is liver.
Example 11 Design of “Gap-Widened” Antisense Oligonucleotides Targeting Human GCGRA series of oligomeric compounds were designed to target human GCGR (Genbank accession number: NM—000160.1, incorporated herein as SEQ ID NO: 108), with varying sizes of the deoxynucleotide gap and 2′-MOE wings. Each of the oligonucleotides is 20 nucleobases in length and has the same nucleobase sequence (GCACTTTGTGGTGCCAAGGC, incorporated herein as SEQ ID NO: 93), and therefore targets the same segment of SEQ ID NO: 108 (nucleobases 532 to 551). The compounds are shown in Table 13. Plain text indicates a deoxynucleotide, and nucleotides designated with bold, underlined text are 2′-O-(2-methoxyethyl)nucleotides. Internucleoside linkages are phosphorothioate throughout, and all cytosines are 5-methylcytosines. Indicated in Table 13 is the “motif' of each compound, indicative of chemically distinct regions comprising the oligonucleotide.
The 5-10-5 gapmer, ISIS 310457, was tested for its ability to reduce target mRNA levels in vitro. HepG2 cells were treated with ISIS 310457 using methods as described herein. ISIS 310457 was analyzed for its effect on human glucagon receptor mRNA levels by quantitative real-time PCR and was found to reduce expression of GCGR by about 96%.
Example 12 Design of “Gap-Widened” Antisense Oligonucleotides Targeting Rat GCGRA series of oligomeric compounds were designed to target rat GCGR (Genbank accession number: M96674.1, incorporated herein as SEQ ID NO: 109) with varying sizes of the deoxynucleotide gap and 2′-MOE wings. Each of the oligonucleotides tested has the same nucleobase sequence (GCACTTTGTGGTACCAAGGT, incorporated herein as SEQ ID NO: 94) and therefore targets the same segment of SEQ ID NO: 109 (nucleobases 402 to 421). The segment targeted by the rat oligonucleotides corresponds to the segment of human GCGR targeted by ISIS 310457 (SEQ ID NO: 93). The compounds are shown in Table 14. Plain text indicates a deoxynucleotide, and nucleotides designated with bold, underlined text are 2′-O-(2-methoxyethyl)nucleotides. Internucleoside linkages are phosphorothioate throughout, and all cytosines are 5-methylcytosines. Indicated in Table 14 is the “motif' of each compound indicative of chemically distinct regions comprising the oligonucleotide.
In accordance with the present invention, the oligonucleotides designed to target rat GCGR were tested in vivo. Male Sprague Dawley rats, eight weeks of age, were injected with 50, 25, 12.5, or 6.25 mg/kg of ISIS 356171, ISIS 357368, ISIS 357369, ISIS 357370, ISIS 357371, ISIS 357372, or ISIS 357373 twice weekly for 3 weeks for a total of 6 doses. Saline-injected animals served as a control. Each of the oligonucleotides tested has the same nucleobase sequence (GCACTTTGTGGTACCAAGGT, incorporated herein as SEQ ID NO: 94), and the chemistry and motif of each compound is described above.
After the treatment period, rats were sacrificed and target nucleic acid levels were evaluated in liver. RNA isolation and target mRNA expression level quantitation are performed as described by other examples herein using RIBOGREEN™. RNA from each treatment group was assayed alongside RNA from the group treated with ISIS 356171. Results are presented in Table 15a, 15b, 15c, 15d, 15e, and 15f as a percentage of saline-treated control levels.
As shown in Tables 15a, 15b, 15c, 15d, and 15e the gap-widened antisense oligonucleotides were effective at reducing GCGR levels in vivo in a dose-dependent manner.
In addition, oligonucleotide concentration in kidney and liver were determined. Methods to determine oligonucleotide concentration in tissues are known in the art (Geary et al., Anal. Biochem., 1999, 274, 241-248). Shown in Table 16 are the total oligonucleotide concentration and the concentration of full length oligonucleotide (in μg/g) in the kidney or liver of animals treated with 25 mg/kg of the indicated oligonucleotide. Total oligonucleotide is the sum of all oligonucleotides metabolites detected in the tissue.
As shown in Table 16, the concentrations of the gap-widened oligonucleotides in kidney were generally reduced with respect to those found for ISIS 356171 in these tissues. Taken with the target reduction data shown in Table 15 wherein potency was maintained with ISIS 356371, ISIS 356372, and ISIS 356373 with respect to ISIS 356171, these data suggest that gap-widened oligos, particularly ISIS 356371, ISIS 356372, and ISIS 356373 are, in essence, more effective than ISIS 356171 at reducing target levels in the liver.
Example 14 Effects of Antisense Oligonucleotides Targeting GCGR—In Vivo Study in Cynomolgus MonkeysTo evaluate alterations in tissue distribution, potency, or therapeutic index caused by modification of the antisense oligonucleotide motif in a primate, cynomolgus monkeys were injected with ISIS 310457 (5-10-5 motif) or ISIS 325568 (2-16-2 motif) at doses of 3, 10, or 20 mg/kg per week. These antisense compounds show 100% complementarity to the monkey GCGR target sequence. Animals injected with saline alone served as controls. The duration of the study was 7 weeks, and the animals were dosed three times during the first week, followed by once-weekly dosing for 6 weeks. Each treatment group was comprised of 5 animals. One group treated with 20 mg/kg of ISIS 310457 and one group treated with 20 mg/kg of ISIS 325568 recovered for three weeks after cessation of dosing prior to sacrifice (”20 mg/kg recovery“). Other treatment groups were sacrificed at the end of the study. Liver tissues were collected to assess target reduction.
RNA isolation and target mRNA expression level quantitation were performed as described by other examples herein using RIBOGREEN™. Results are presented in Table 17 as a percentage of saline-treated control levels.
As shown in Table 17, treatment with ISIS 310457 and 325568 caused decreases in GCGR levels at all of the doses tested, and reduction in target levels was still observed in the 20 mg/kg recovery groups. ISIS 325568 caused greater reduction than ISIS 310457 at the 3 mg/kg dose.
In addition, oligonucleotide concentration in kidney and liver were determined. Methods to determine oligonucleotide concentration in tissues are known in the art (Geary et al., Anal Biochem, 1999, 274, 241-248). Shown in Table 18 are the total concentration and the concentration of full length oligonucleotide (in μg/g) in the kidney or liver of animals treated with the indicated oligonucleotide.
As shown in Table 18, the kidney concentration of the 5-10-5 motif oligonucleotide ISIS 310457 is higher than that measured for the 2-16-2 motif oligonucleotide ISIS 325568 at all concentrations tested. Taken with the target reduction data in Table 9 for the 2-16-2 motif oligonucleotide, these data suggest that the gap-widened oligonucleotide is more potent than the corresponding 5-10-5 motif oligonucleotide, providing a more robust lowering of target mRNA levels in the liver without enhanced accumulation of oligonucleotide.
Example 15 Effects of Gap-Widened Oligonucleotides on Reduction of DGAT2 mRNA Levels—In Vitro AnalysisIn accord with the present invention, oligonucleotides were designed to target DGAT2.
Shown in Table 19 is the sequence of each oligonucleotide. Plain text indicates a deoxynucleotide, and nucleotides designated with bold, underlined text are 2′-O-(2-methoxyethyl)nucleotides. Also shown for each oligonucleotide in Table 19 is its motif, the target site on human DGAT2 mRNA (GENBANK® accession number NM—032564.2, incorporated herein as SEQ ID NO: 110), and its cross-species identity. For each species listed, an “X” denotes perfect complementarity to the target sequence for that species, “1 MM” denotes one mismatch to the target sequence for the species, etc.
Each of these oligonucleotides was tested in vitro for their ability to reduce human DGAT2 mRNA levels using real time RT-PCR methods as described herein. In HepG2 and A549 cells, each of the oligonucleotides in Table 19 demonstrated IC50 values of about 20 nM.
Example 16 Effects of Gap-Widened Oligonucleotides on Reduction of DGAT2 mRNA Levels—In Vivo AnalysisThe oligonucleotides described in Table 19, along with ISIS 217357 (ACACACTAGAAGTGAGCTTA, SEQ ID NO: 102), which is targeted to rat DGAT2, the complement of nucleotides 15333000 to 15365000 of GENBANK® accession number NW—047561.1, herein incorporated as SEQ ID NO: 111 were tested for their ability to reduce DGAT2 levels in vivo. Eight week-old male Sprague-Dawley rats were injected with 20 mg/kg of oligonucleotide per week for 2 weeks. Each treatment group was comprised of 6 animals. Animals injected with saline alone served as controls.
At the end of the treatment period, animals were sacrificed and liver and kidney tissues were harvested. To determine effects of altering the gap size on pharmacokinetics, oligonucleotide concentration in kidney and liver were determined. Methods to determine oligonucleotide concentration in tissues are known in the art (Geary et al., Anal Biochem, 1999, 274, 241-248). Total oligonucleotide is the sum of all oligonucleotides metabolites detected in the tissue. Shown in Table 20 are the total concentration and the concentration of full length oligonucleotide (in μg/g) in the liver of animals treated with the indicated oligonucleotide concentration.
As shown in Table 20, kidney concentrations of gap-widened oligonucleotides, particularly ISIS 370727 and ISIS 370747, were generally lower than those of oligonucleotides with a 10-deoxynucleotide gap.
Example 17 Effects of Gap-Widened Oligonucleotides on Reduction of DGAT2 mRNA Levels—In Vivo AnalysisIn another arm of the experiment described in Example 16, eight-week old male Sprague-Dawley rats were treated with the oligonucleotides at doses of 50 mg/kg per week for four weeks. Each treatment group was comprised of 4 animals. At the end of the treatment period, animals were sacrificed and target mRNA levels were determined using real-time RT-PCR as described herein. Results are shown in Table 21 as the average % inhibition for each treatment group.
As shown in Table 21, the gap-widened oligonucleotides targeted to DGAT2 show excellent inhibitory activity in the liver. ISIS 370727 and ISIS 370747, in particular, showed superior ability to reduce target expression. Taken with the distribution of these oligonucleotides in the liver as shown in Table 20, these data suggest that gap-widened oligonucleotides provide excellent to superior target reduction without enhanced accumulation of oligonucleotide in target tissues. In addition, the gap-widened oligonucleotides possess a preferred liver to kidney ratio as compared to the 5-10-5 motif oligonucleotides targeting DGAT2.
Example 18 Effects of Gap-Widened Oligonucleotides on Reduction of CRP mRNA Levels—In Vivo AnalysisMonkey-human cross-species oligonucleotides targeted to C-reactive protein (CRP) were designed to target CRP using sequences known in the art (see US application publication number US2005-0014257, herein incorporated by reference in its entirety). Shown in Table 22 is the sequence of oligonucleotides targeted to CRP tested in cynomologus monkeys. Plain text indicates a deoxynucleotide, and nucleotides designated with bold, underlined text are 2′-O-(2-methoxyethyl)nucleotides. Also shown for each oligonucleotide in Table 22 is its motif.
Methods of assaying for activity of CRP compounds in vivo and in vitro are known in the art (see US application publication number US2005-0014257, herein incorporated by reference). Toxicity profiles of gap-widened oligonucleotides were compared to the 5-10-5 oligonucleotides by treating monkeys with 14 or 40 mg/kg/wk for 4 weeks. Activity was compared in a dose-escalation study with each cycle containing four subcutaneous doses administered (Mon., Wed., Fri., Mon.) in 4 dosing cycles over 8 weeks. Doses were 2, 4 and 10 mg/kg. At 48 hr following the last dose in each treatment cycle, monkeys were challenged with 1 to 2 μg/kg IL-6 (administered subcutaneously) and serum CRP levels were quantified over 36 hours. Serum CRP levels may be measured by ELISA using a commercially available kit (for example, ALerCHEK Inc., Portland, Me.). Animals were sacrificed after the second and fourth cycles and liver CRP mRNA, tissue oligonucleotide concentration, clinical signs, serum chemistry, hematology, body weight, and histology were assessed. With regard to tissue oligonucleotide concentration and histology, the primary difference was 30% lower kidney concentration and fewer histologic changes in the 3-14-3 treated animals. Plasma cytokine and CRP levels were examined but not significantly increased.
Several CRP inhibitors were pharmacologically active, with the greatest reductions in serum CRP (30-66%) and hepatic CRP mRNA (60-85%) observed at both the 4 and 10 mg/kg treatment cycles.
We have surprisingly found that chimeric antisense compounds with gaps at least 11 nucleobases long and wings which are from independently from 1 to 4 nucleobases in length which are 2′-MOE-modified. This enhanced efficacy is not predicted by the rank order potency of these compounds in vitro (cell culture). 2-16-2 and 3-14-3 gapmer compounds as well as 3-10-7 and 7-10-3 gapmer compounds have been shown to be more effective than 5-10-5 chimeras of the equivalent sequence and wing modification. 4-12-4 gapmers are also believed to be a useful embodiment.
DETAILED DESCRIPTION OF EMBODIMENTSNon-limiting examples of 2′-modified nucleosides useful in the compounds of the present invention, include but are not limited to 2′-O-alkyl, 2′-O-alkyl-O-alkyl wherein alkyl is a C1 to C6 alkyl or C1 to C6 alkylene when alkyl is not a terminal substituent. These include 2′-O-methyl, 2′-O-propyl and 2′-O-methoxyethyl nucleosides.
DetailsThe present invention uses 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. 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 compositestructures 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, each of which is herein incorporated by reference in its entirety.
Synthesis of Nucleoside PhosphoramiditesThe following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-ODimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl2′-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N4-benzoyl-5-methylcytidin-3′O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4-benzoyl5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N6benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl)nucleoside amidites, 2′-(Dimethylaminooxyethoxy)nucleoside amidites, 5′-O-tertButyldiphenylsilyl-O2-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,Ndimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-(Aminooxyethoxy)nucleoside amidites, N2-isobutyryl6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-dimethylaminoethoxyethoxy (2′DMAEOE) nucleoside amidites, 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
Oligonucleotide and Oligonucleoside SynthesisThe antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine. Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH4OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference. Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference. Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference. Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference. 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference. Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference. Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference. Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonyl amino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference. Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference. Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.
RNA SynthesisIn general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl. Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized. RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.
Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S2Na2) in DMF. The deprotection solution is washed from the solid supportbound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55 ° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.
The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed, Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product. Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1″8, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).
RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 tl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.
Synthesis of Chimeric OligonucleotidesChimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.
[2′-O-Me]-[2′-deoxy]-[2′-O-Me]Chimeric Phosphorothioate OligonucleotidesChimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH4OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.
[2′-O-(2-Methoxyethyl)]-[2′-deoxy]; -[2′-O-(Methoxyethyl)]Chimeric Phosphorothioate Oligonucleotides[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′0-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites.
[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester]Chimeric Oligonucleotides[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphorothioate]-[2′-O(methoxyethyl)phosphodiester]chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.
Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.
The methods of the present invention are particularly useful in antisense therapeutics. It is not necessary that the antisense target be associated with liver disease per se, since many current antisense targets are expressed to high levels in liver and other organs. In particular, targets associated with metabolic and cardiovascular diseases and conditions are particularly amenable to knockdown in the liver and have been shown in animals and in clinical studies to have therapeutic effects).
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. 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. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
Claims
1. A method of reducing expression of a target RNA in an animal, in need of reducing expression of said target RNA, comprising administering to said animal a gap-widened antisense oligonucleotide 18-24 linked nucleosides in length comprising: wherein the gap region is located between said first wing region and said second wing region and, wherein each nucleoside of said first and second wing region is a 2′modified nucleoside thereby reducing expression of said target RNA in said animal.
- (a) a gap region having 12 to 18contiguous 2′-deoxyribonucleosides; and
- (b) a first wing region having 1 to 4 contiguous nucleosides; and
- (c) a second wing region having 1 to 4 contiguous nucleosides;
2.-3. (canceled)
4. The method of claim 1, wherein the target RNA is associated with a metabolic or a cardiovascular disease or condition.
5. The method of claim 1, wherein the metabolic disease or condition is selected from metabolic syndrome, diabetes, obesity, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, Type 2 diabetes, diet-induced obesity, hyperglycemia, insulin resistance, hepatic steatosis, fatty liver disease, or non-alcoholic steatohepatitis.
6. The method of claim 1, wherein the cardiovascular disease or condition is selected from familial hypercholesterolemia, nonfamilial hypercholesterolemia, mixed dyslipidemia, dysbetalipoproteinemia, atherosclerosis, coronary artery disease, myocardial infarction, hypertension, carotid artery diseases, carotid artery disease, stroke, cerebrovascular disease, peripheral vascular disease, thrombosis, or arterial aneurism.
7. The method of claim 1, wherein the gap-widened antisense oligonucleotide has a wing-gap-wing motif selected from 2-16-2, 3-14-3, 2-14-2, 3-12-3 or 4-12-4.
8. The method of claim 1, wherein the gap-widened antisense oligonucleotide has at least one phosphorothioate internucleotide linkage.
9. The method of claim 6, wherein the gap-widened antisense oligonucleotide has all phosphorothioate internucleotide linkages.
10. The method of claim 1, wherein gap-widened antisense oligonucleotide has at least one 5-methylcytosine.
11.-25. (canceled)
26. A method of modulating gene expression in an animal comprising the step of contacting said animal with the pharmaceutical composition comprising a gap-widened antisense oligoncuelotide 18-24 linked nucleosides in length comprising: wherein the gap region is located between said first wing region and said second wing region and, wherein each nucleoside of said first and second wing region is a 2′ modified nucleoside thereby modulating gene expression in said animal.
- (a) a gap region having 12 to 18 contiguous 2′-deoxyribonucleotides;
- (b) a first wing region having 1 to 4 contiguous nucleosides; and
- (c) a second wing region having 1 to 4 contiguous nucleosides;
27.-28. (canceled)
Type: Application
Filed: Jan 18, 2011
Publication Date: Aug 25, 2011
Applicant: Isis Pharmaceuticals, Inc. (Carlsbad, CA)
Inventors: Brett P. Monia (Encinitas, CA), Andrew M. Siwkowski (Encinitas, CA), Sanjay Bhanot (Carlsbad, CA)
Application Number: 13/008,704
International Classification: A61K 31/7088 (20060101); A61P 9/00 (20060101); A61P 3/00 (20060101); A61P 3/10 (20060101); A61P 3/04 (20060101); A61P 3/06 (20060101); A61P 1/16 (20060101); A61P 9/10 (20060101); A61P 9/12 (20060101); A61P 7/02 (20060101);
