Easy To Use Patents Search & Patent Lawyer Directory

At Patents you can conduct a Patent Search, File a Patent Application, find a Patent Attorney, or search available technology through our Patent Exchange. Patents are available using simple keyword or date criteria. If you are looking to hire a patent attorney, you've come to the right place. Protect your idea and hire a patent lawyer.


Search All Patents:



  This Patent May Be For Sale or Lease. Contact Us

  Is This Your Patent? Claim This Patent Now.



Register or Login To Download This Patent As A PDF




United States Patent Application 20180273949
Kind Code A1
Brown-Driver; Vickie L. ;   et al. September 27, 2018

MODULATION OF TRANSTHYRETIN EXPRESSION

Abstract

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


Inventors: Brown-Driver; Vickie L.; (Solana Beach, CA) ; Jain; Ravi; (Fremont, CA)
Applicant:
Name City State Country Type

Ionis Pharmaceuticals, Inc.

Carlsbad

CA

US
Assignee: Ionis Pharmaceuticals, Inc.
Carlsbad
CA

Family ID: 1000003377558
Appl. No.: 15/803484
Filed: November 3, 2017


Related U.S. Patent Documents

Application NumberFiling DatePatent Number
15092476Apr 6, 2016
15803484
14614647Feb 5, 2015
15092476
11097928Apr 1, 2005
14614647
60559863Apr 5, 2004

Current U.S. Class: 1/1
Current CPC Class: C12N 15/113 20130101; C12N 2310/11 20130101; C12Q 2600/158 20130101; C12Q 2600/136 20130101; C12N 2310/321 20130101; Y10T 436/143333 20150115; C12N 2310/346 20130101; C12N 2310/341 20130101; C12N 2310/3341 20130101; C12Q 1/6883 20130101; C12N 2310/315 20130101; C12N 15/1136 20130101
International Class: C12N 15/113 20060101 C12N015/113; C12Q 1/6883 20060101 C12Q001/6883

Claims



1-18. (canceled)

19. An antisense compound comprising a modified oligonucleotide 20 to 25 nucleobases in length, wherein the modified oligonucleotide has at least a 13-nucleobase portion of SEQ ID NO: 75 and is at least 95% complementary to SEQ ID NO: 4, and wherein the modified oligonucleotide comprises at least one modified sugar moiety.

20. The antisense compound of claim 19, wherein each "T" of SEQ ID NO: 75 is a uracil.

21. The antisense compound of claim 19, wherein at least one modified sugar moiety comprises a 2'-O--CH.sub.3 modification.

22. The antisense compound of claim 20, wherein at least one modified sugar moiety comprises a 2'-O--CH.sub.3 modification.

23. The antisense compound of claim 19, wherein: the modified oligonucleotide is 21 nucleobases in length; the modified oligonucleotide has at least a 13-nucleobase portion of SEQ ID NO: 75 and is at least 95% complementary to SEQ ID NO: 4, wherein each "T" of SEQ ID NO: 75 is a uracil; and the modified oligonucleotide comprises at least one 2'-O--CH.sub.3 modified sugar moiety.

24. The antisense compound of claim 19, wherein the antisense compound is single-stranded.

25. The antisense compound of claim 20, wherein the antisense compound is single-stranded.

26. The antisense compound of claim 21, wherein the antisense compound is single-stranded.

27. The antisense compound of claim 22, wherein the antisense compound is single-stranded.

28. The antisense compound of claim 23, wherein the antisense compound is single-stranded.

29. A compound comprising a nanoparticle and the antisense compound of claim 19.

30. A compound comprising a nanoparticle and the antisense compound of claim 23.

31. A compound comprising a nanoparticle and the antisense compound of claim 28.

32. A compound comprising a liposome and the antisense compound of claim 19.

33. A compound comprising a liposome and the antisense compound of claim 23.

34. A compound comprising a liposome and the antisense compound of claim 28.

35. A method of inhibiting the expression of human transthyretin in a cell or tissue comprising contacting said cell or tissue with the antisense compound of claim 19, thereby inhibiting expression of human transthyretin in the cell or tissue.

36. A method of inhibiting the expression of human transthyretin in a cell or tissue comprising contacting said cell or tissue with the antisense compound of claim 23, thereby inhibiting expression of human transthyretin in the cell or tissue.

37. A method of inhibiting the expression of human transthyretin in a cell or tissue comprising contacting said cell or tissue with the antisense compound of claim 28, thereby inhibiting expression of human transthyretin in the cell or tissue.

38. A method of inhibiting the expression of human transthyretin in a cell or tissue comprising contacting said cell or tissue with the compound of claim 29, thereby inhibiting expression of human transthyretin in the cell or tissue.

39. A method of inhibiting the expression of human transthyretin in a cell or tissue comprising contacting said cell or tissue with the compound of claim 30, thereby inhibiting expression of human transthyretin in the cell or tissue.

40. A method of inhibiting the expression of human transthyretin in a cell or tissue comprising contacting said cell or tissue with the compound of claim 31, thereby inhibiting expression of human transthyretin in the cell or tissue.

41. A method of inhibiting the expression of human transthyretin in a cell or tissue comprising contacting said cell or tissue with the compound of claim 32, thereby inhibiting expression of human transthyretin in the cell or tissue.

42. A method of inhibiting the expression of human transthyretin in a cell or tissue comprising contacting said cell or tissue with the compound of claim 33, thereby inhibiting expression of human transthyretin in the cell or tissue.

43. A method of inhibiting the expression of human transthyretin in a cell or tissue comprising contacting said cell or tissue with the compound of claim 34, thereby inhibiting expression of human transthyretin in the cell or tissue.
Description



SEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled RTS0531USC7SEQ_ST25.txt, created Nov. 3, 2017, which is 38 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention provides compositions and methods for modulating the expression of transthyretin. In particular, this invention relates to antisense compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding transthyretin. Such compounds are shown herein to modulate the expression of transthyretin.

BACKGROUND OF THE INVENTION

[0003] Steroid hormones, thyroid hormones, retinoids, and vitamin D are small hydrophobic molecules that serve as important signaling molecules throughout the body. Although all of these molecules are insoluble in water, they are made soluble for transport in the bloodstream and other extracellular fluids by binding to specific carrier proteins, from which they dissociate before entering a target cell. One such carrier protein is transthyretin.

[0004] Transthyretin (also known as TTR; TTR, prealbumin; prealbumin, thyroxine; PALB; TBPA; HST2651; amyloidosis 1, included; dysprealbuminemic euthyroidal hyperthyroxinemia, included; hyperthytoxinemia, dysprealbuminemic, included; hyperthytoxinemia, dystransthyretinemic, included; amyloid polyneuropathy, multiple forms, included; senile systemic amyloidosis, included) is a homotetrameric transport protein found in the extracellular fluids of vertebrates (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300).

[0005] Transthyretin was first identified as the major thyroid hormone carrier in the cerebrospinal fluid (CSF) and in the serum (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300; Seibert, J. Biol. Chem, 1942, 143, 29-38). Transthyretin was cloned from adult human cDNA libraries and the gene was subsequently mapped to chromosome region 18q11.2-q12.1 (Mita et al., Biochem Biophys Res Commun, 1984, 124, 558-564; Sparkes et al., Hum Genet, 1987, 75, 151-154; Whitehead et al., Mol Biol Med, 1984, 2, 411-423).

[0006] The liver and the choroid plexus are the primary sites of transthyretin synthesis in humans (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300).

[0007] Transthyretin that is synthesized in the liver is secreted into the blood, whereas transthyretin originating in the choroid plexus is destined for the CSF. In the choroid plexus, transthyretin synthesis represents about 20% of total local protein synthesis and as much as 25% of the total CSF protein (Dickson et al., J Biol Chem, 1986, 261, 3475-3478). Transthyretin synthesis has also been identified in the yolk sac of developing rats(Soprano et al., Proc Natl Acad Sci USA, 1986, 83, 7330-7334); the retina, ciliar body and optic nerve regions of bovine and rat eyes (Martone et al., Biochem Biophys Res Commun, 1988, 151, 905-912; Ong et al., Biochemistry, 1994, 33, 1835-1842); human and porcine pancreatic islets (Jacobsson et al., J Histochem Cytochem, 1989, 37, 31-37) and, in minor amounts, in the stomach, heart, skeletal muscle, and spleen of rats (Soprano et al., J Biol Chem, 1985, 260, 11793-11798).

[0008] It is currently believed that transthyretin serves as a hormone reservoir. As demand for thyroid hormone increases, transthyretin increases the transport and release of hormone to targets such as brain, kidney, and cardiac tissues, thereby ensuring a uniform hormone distribution within the cells in each of these tissues (Palha, Clin Chem Lab Med, 2002, 40, 1292-1300). Transthyretin transports the thyroid hormones triiodothyronine (T.sub.3) and thyroxine (T.sub.4) as well as the retinol/retinol-binding protein complex. A mouse strain deficient in transthyretin is viable and fertile, yet exhibits significantly depressed levels of serum retinol, retinol-binding protein, and thyroid hormone, confirming transthyretin's role in maintaining normal levels of these metabolites in circulating plasma (Episkopou et al., Proc Natl Acad Sci USA, 1993, 90, 2375-2379). In addition to serving as a transport protein, transthyretin has been reported to have a variety of other functions, including: inhibiting interleukin-1 production in monocytes and endothelial cells (Borish et al., Inflammation, 1992, 16, 471-484); involvement in the metabolism of the environmental pollutant polyhalogenated biphenyl (Brouwer and van den Berg, Toxicol Appl Pharmacol, 1986, 85, 301-312); and binding pterins (Ernstrom et al., FEBS Lett, 1995, 360, 177-182). Furthermore, in recent years a link between transthyretin and lipoprotein biology has become increasingly apparent. A fraction of plasma transthyretin circulates in high density lipoproteins (HDL) through binding to apolipoprotein A-1 (Sousa et al., J Biol Chem, 2000, 275, 38176-38181), and transthyretin has been shown to proteolytically process apolipoprotein A-1 (Liz et al., J Biol Chem, 2004). Furthermore, transthyretin reabsorption by the kidneys is mediated by the lipoprotein receptor megalin (Sousa et al., J Biol Chem, 2000, 275, 38176-38181). This reabsorption serves as a means for preventing hormone loss in urine. Finally, the major site of degradation for both transthyretin and lipoproteins is the liver. There is considerable evidence that hepatic uptake of both transthyretin and lipoproteins is mediated by an as yet unidentified lipoprotein receptor, suggesting a shared degradation pathway (Sousa and Saraiva, J Biol Chem, 2001, 276, 14420-14425).

[0009] Transthyretin is associated with both local and systemic amyloidosis, a disorder characterized by extracellular systemic deposition of mutated or wild-type transthyretin as amyloid fibrils (Cornwell et al., Biochem Biophys Res Commun, 1988, 154, 648-653; Saraiva et al., J Clin Invest, 1984, 74, 104-119; Yazaki et al., Muscle Nerve, 2003, 28, 438-442), leading to organ dysfunction and death. Senile systemic amyloidosis is a sporadic disorder resulting from the extracellular deposition of wild-type transthyretin fibrils in cardiac and other tissues. Over 80 mutations in transthyretin are associated with familial amyloidotic polyneuropathy and cardiomyopathy. In most of these cases, inheritance is autosomal dominant (Reixach et al., Proc Natl Acad Sci USA, 2004, 101, 2817-2822). Jiang et al (Jiang et al., Proc Natl Acad Sci USA, 2001, 98, 14943-14948) demonstrated that the variant with a valine to isoleucine mutation at amino acid 122 (Val122Ile), which is among the most common amyloidogenic mutations worldwide, increases the velocity of rate-limiting tetramer dissociation, thereby resulting in accelerated amyloidogenesis. This finding suggests the possibility that treatments for transthyretin-related amyloidoses may include small molecules that stabilize the tetrameric form (Adamski-Werner et al., J Med Chem, 2004, 47, 355-374; Altland and Winter, Neurogenetics, 1999, 2, 183-188). Small molecule stabilizers were also shown to be of use in preventing the formation of amyloid fibrils of the wildtype transthyretin (Reixach et al., Proc Natl Acad Sci USA, 2004, 101, 2817-2822). Other common transthyretin mutations associated with amyloidosis include Val30Met and Glu61Lys. In vitro studies have shown success using ribozymes to specifically target and inhibit the expression of the Glu61Lys and Val30Met variants (Propsting et al., Biochem Biophys Res Commun, 1999, 260, 313-317; Tanaka et al., J Neurol Sci, 2001, 183, 79-84). Single-stranded oligonucleotides have been used both in vitro and in vivo to correct single-base mutation (Val30Met) in transthyretin to the wild-type form through targeted recombination (Nakamura et al., Gene Ther, 2004). The success of this therapy was limited, however, with gene conversion rates of 11% in vitro and 9% in vivo. These levels are not sufficient for suppression of the variant transthyretin in clinical terms (Nakamura et al., Gene Ther, 2004).

[0010] Thus liver transplantation is currently the only available therapy for familial amyloidotic polyneuropathy. However, this therapy is associated with several problems, and does not address conditions which are caused by transthyretin variant production in tissues other than liver (Yazaki et al., Muscle Nerve, 2003, 28, 438-442). Consequently, there remains an unmet need for agents capable of effectively modulating transthyretin expression (Nakamura et al., Gene Ther, 2004; Tanaka et al., J Neurol Sci, 2001, 183, 79-84).

[0011] The PCT publication WO200259621 and the US pre-grant publication 20020160394 claim pharmaceutical compositions for treating obesity, comprising an antisense oligonucleotide that hybridizes to a polynucleotide encoding transthyretin and reduces expression of the polynucleotide. Also claimed is the use of said oligonucleotide in the manufacture of a medicament for treating obesity (Wu, 2002).

[0012] The U.S. Pat. No. 5,744,368 discloses a primer of 22 nucleotides in length targeted to Exon 4 of transthyretin (Goldgaber et al., 1998).

[0013] 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 transthyretin expression. The present invention provides compositions and methods for modulating transthyretin expression.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to antisense compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding transthyretin, and which modulate the expression of transthyretin. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of transthyretin and methods of modulating the expression of transthyretin 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 transthyretin are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

A. Overview of the Invention

[0015] The present invention employs antisense compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding transthyretin. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding transthyretin. As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding transthyretin" have been used for convenience to encompass DNA encoding transthyretin, 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.

[0016] 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 transthyretin. 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] "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.

[0021] 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. 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).

[0022] 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, 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

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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).

[0027] 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 transthyretin mRNA.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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 to 50 nucleobases in length, inclusive as detailed above.

[0032] 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 to 30 nucleobases in length, inclusive as detailed above.

[0033] Particularly preferred compounds are oligonucleotides from about 13 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0034] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0035] 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.

[0036] 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

[0037] "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 transthyretin.

[0038] 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.

[0039] 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 transthyretin, 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).

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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, 641-650, or any combination thereof.

[0053] Oligomeric compounds targeted to nucleobases 3880-3899 of SEQ ID NO: 11, or to nucleobases 6-25, 59-78, 91-119, 126-152, 170-189, 197-216, 217-236, 232-251, 250-269, 264-297, 323-361, 425-469, 460-532, 532-619 of SEQ ID NO: 4 are also suitable embodiments.

D. Screening and Target Validation

[0054] In a further embodiment, the "preferred target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of transthyretin. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding transthyretin 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 transthyretin 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 transthyretin. 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 transthyretin, the modulator may then be employed in further investigative studies of the function of transthyretin, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0055] 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.

[0056] 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).

[0057] 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 transthyretin and a disease state, phenotype, or condition. These methods include detecting or modulating transthyretin comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of transthyretin 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

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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).

[0062] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding transthyretin. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective transthyretin 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 transthyretin and in the amplification of said nucleic acid molecules for detection or for use in further studies of transthyretin. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding transthyretin 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 transthyretin in a sample may also be prepared.

[0063] 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.

[0064] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of transthyretin 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 transthyretin inhibitor. The transthyretin inhibitors of the present invention effectively inhibit the activity of the transthyretin protein or inhibit the expression of the transthyretin protein. In one embodiment, the activity or expression of transthyretin in an animal is inhibited by about 10%. Preferably, the activity or expression of transthyretin in an animal is inhibited by about 30%. More preferably, the activity or expression of transthyretin in an animal is inhibited by 50% or more. Thus, the oligomeric antisense compounds modulate expression of transthyretin 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%.

[0065] For example, the reduction of the expression of transthyretin 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 transthyretin protein and/or the transthyretin protein itself.

[0066] 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.

F. Modifications

[0067] 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)

[0068] 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.

[0069] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriaminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphos-phonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0070] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S.: 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.

[0071] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts.

[0072] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S.: 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

[0073] 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.: 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.

[0074] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH.sub.2--NH--O--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--O--CH.sub.2-[known as a methylene (methylimino) or MMI backbone], --CH.sub.2--O--N(CH.sub.3)--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and --O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native phosphodiester backbone is represented as --O--P--O--CH.sub.2-] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified Sugars

[0075] 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-Co-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3].sub.2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly-alkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2, also described in examples hereinbelow.

[0076] Other preferred modifications include 2'-methoxy (2'-O--CH.sub.3), 2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl (2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl (2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. 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.: 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.

[0077] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (--CH.sub.2--).sub.n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

[0078] 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.ident.C--CH.sub.3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-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.degree. C. and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0079] 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

[0080] 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-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. 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, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0081] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S.: 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

[0082] 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.

[0083] 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.

[0084] 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.: 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

[0085] 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.: 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.

[0086] 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.

[0087] 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. Sodium salts have been shown to be suitable forms of oligonucleotide drugs.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 .mu.m in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug 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.

[0093] 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.

[0094] 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. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0095] The pharmaceutical formulations and compositions of the present invention may also include surfactants. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0096] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0097] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0098] 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).

[0099] 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.

[0100] 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), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0101] 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.

[0102] 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.

[0103] 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

[0104] 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. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC.sub.50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or at desired intervals. 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.

[0105] 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--Synthesis of Nucleoside Phosphoramidites

[0106] Synthesis of nucleoside phorsphoramidates, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743, both of which are incorporated herein by reference.

Example 2--Oligonucleotide and Oligonucleoside Synthesis

[0107] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0108] Oligonucleotides: Unsubstituted and substituted phosphodiester (P.dbd.O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0109] Phosphorothioates (P.dbd.S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH.sub.4OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0110] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863. 3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050. Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. 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). 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925. Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243. Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198. Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P.dbd.O or P.dbd.S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289. Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618. All patents and applications are incorporated herein by reference.

Example 3--RNA Synthesis

[0111] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand, 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0112] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 .mu.l of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 .mu.l of 5.times. annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90.degree. C., then 1 hour at 37.degree. C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid, or for diagnostic or therapeutic purposes.

Example 4--Synthesis of Chimeric Compounds

[0113] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers".

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

[0114] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3' wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at 55.degree. C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed 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

[0115] [2'-O-(2-methoxyethyl)]--[2'-deoxy]--[-2'-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2'-O-methyl chimeric oligonucleotide, with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites.

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

[0116] [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.

[0117] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5--Design and Screening of Duplexed Antisense Compounds Targeting Transthyretin

[0118] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target transthyretin. The nucleobase sequence of the antisense strand of the duplex comprises at least an 8-nucleobase portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.

[0119] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 134) and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:

TABLE-US-00001 cgagaggcggacgggaccgTT Antisense Strand (SEQ ID NO: 135) ||||||||||||||||||| TTgctctccgcctgccctggc Complement (SEQ ID NO: 136)

[0120] In another embodiment, a duplex comprising an antisense strand having the same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 134) may be prepared with blunt ends (no single stranded overhang) as shown:

TABLE-US-00002 cgagaggcggacgggaccg Antisense Strand (SEQ ID NO: 134) ||||||||||||||||||| gctctccgcctgccctggc Complement (SEQ ID NO: 137)

[0121] 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.

[0122] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5.times. solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90.degree. C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37.degree. C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (-20.degree. C.) and freeze-thawed up to 5 times. Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate transthyretin expression.

[0123] When cells reach 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 .mu.L OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 .mu.L of OPTI-MEM-1 containing 12 .mu.g/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6--Oligonucleotide Isolation

[0124] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55.degree. C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH.sub.4OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis 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 7--Oligonucleotide Synthesis--96 Well Plate Format

[0125] 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 using methods known to those skilled in the art.

Example 8--Oligonucleotide Analysis--96-Well Plate Format

[0126] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy using methods known to those skilled in the art.

Example 9--Cell Culture and Oligonucleotide Treatment

[0127] 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. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR. 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. All cell types were cultured under standard conditions, using methods known to those skilled in the art.

[0128] T-24 Cells:

[0129] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). 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.

[0130] A549 Cells:

[0131] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).

[0132] NHDF Cells:

[0133] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.).

[0134] HEK Cells:

[0135] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.).

[0136] HepG2 Cells:

[0137] The human hepatoblastoma cell line HepG2 was obtained from the American Type Culture Collection (Manassas, Va.). Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 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.

[0138] Treatment with Antisense Compounds:

[0139] When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 .mu.L, OPTI-MEM.TM.-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 .mu.L, of OPTI-MEM.TM.-1 containing 3.75 .mu.g/mL LIPOFECTIN.TM. (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37.degree. C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0140] 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-(2-methoxyethyl) gapmers (2'-O-(2-methoxyethyl) nucleotides 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-(2-methoxyethyl) gapmers (2'-O-(2-methoxyethyl) nucleotides shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10--Analysis of Oligonucleotide Inhibition of Transthyretin Expression

[0141] Antisense modulation of transthyretin expression can be assayed in a variety of ways known in the art. For example, transthyretin mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM.TM. 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0142] Protein levels of transthyretin 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 transthyretin can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11--Design of Phenotypic Assays for the Use of Transthyretin Inhibitors

[0143] Phenotypic assays-Once transthyretin 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 transthyretin in health and disease. Phenotypic assay can be purchased from any one of several commercial vendors.

Example 12--RNA Isolation

[0144] Poly(A)+ mRNA Isolation.

[0145] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 .mu.L of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 .mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70.degree. C., was added to each well, the plate was incubated on a 90.degree. C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0146] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions

[0147] Total RNA was isolated using an RNEASY 96.TM. kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures.

[0148] 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 13--Real-Time Quantitative PCR Analysis of Transthyretin mRNA Levels

[0149] Quantitation of transthyretin mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM.TM. 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM.TM. Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0150] 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.

[0151] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 .mu.L PCR cocktail (2.5.times. PCR buffer minus MgCl.sub.2, 6.6 mM MgCl.sub.2, 375 .mu.M each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5 Units MuLV reverse transcriptase, and 2.5.times. ROX dye) to 96-well plates containing 30 .mu.L total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48.degree. C. Following a 10 minute incubation at 95.degree. C. to activate the PLATINUM.RTM. Taq, 40 cycles of a two-step PCR protocol were carried out: 95.degree. C. for 15 seconds (denaturation) followed by 60.degree. C. for 1.5 minutes (annealing/extension).

[0152] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen.TM. RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen.TM. are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0153] In this assay, 170 .mu.L of RiboGreen.TM. working reagent (RiboGreen.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0154] Probes and primers to human transthyretin were designed to hybridize to a human transthyretin sequence, using published sequence information (GenBank accession number BCO20791.1, incorporated herein as SEQ ID NO: 4). For human transthyretin the PCR primers were:

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

Example 14--Northern Blot Analysis of Transthyretin mRNA Levels

[0155] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM. (TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER.TM. UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB.TM. hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0156] To detect human transthyretin, a human transthyretin specific probe was prepared by PCR using the forward primer CCCTGCTGAGCCCCTACTC (SEQ ID NO: 5) and the reverse primer TCCCTCATTCCTTGGGATTG (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.).

[0157] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER.TM. and IMAGEQUANT.TM. Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15--Antisense Inhibition of Human Transthyretin Expression by Chimeric Phosphorothioate Oligonucleotides Having 2'-MOE Wings and a Deoxy Gap

[0158] In accordance with the present invention, a series of antisense compounds was designed to target different regions of the human transthyretin RNA, using published sequences (GenBank accession number BC020791.1, incorporated herein as SEQ ID NO: 4, and nucleotides 2009236 to 2017289 of the sequence with GenBank accession number NT_010966.10, incorporated herein as SEQ ID NO: 11). 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 often 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) 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 (P.dbd.S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human transthyretin mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which HepG2 cells were treated with 50 nM of the antisense oligonucleotides of the present invention. The positive control ISIS 18078 (SEQ ID NO: 2) was used for this assay. If present, "N.D." indicates "no data".

TABLE-US-00003 TABLE 1 Inhibition of human transthyretin mRNA levels by chimeric phosphorothioate oligonucleotides having 2'-MOE wings and a deoxy gap TARGET SEQ SEQ TARGET % ID ISIS # REGION ID NO SITE SEQUENCE INHIB NO 304237 Exon 1: 11 596 aaacactcaccgtagggcca 6 12 Intron 1 junction 304238 Intron 1: 11 1520 caccggtgccctgggtgtag 0 13 Exon 2 junction 304239 Intron 2 11 1718 tgagcctctctctaccaagt 0 14 304240 Exon 3: 11 3880 gtatactcacctctgcatgc 33 15 Intron 3 junction 304241 Intron 3 11 4039 ttctcagagtgttgtgaatt 0 16 304242 Intron 3 11 6252 actctgcataaatacatttt 0 17 304243 Intron 3 11 6967 tcttgttttgcaaattcacg 0 18 304244 Intron 3 11 7192 tgaataccacctatgagaga 0 19 304245 5'UTR 4 6 ctgccaagaatgagtggact 33 20 304246 Start Codon 4 18 tgagaagccatcctgccaag 6 21 304247 Start Codon 4 25 cagacgatgagaagccatcc 2 22 304248 Coding 4 30 aggagcagacgatgagaagc 10 23 304249 Coding 4 59 acacaaataccagtccagca 33 24 304250 Coding 4 60 gacacaaataccagtccagc 0 25 304251 Coding 4 66 gcctcagacacaaataccag 14 26 304252 Coding 4 75 gtagggccagcctcagacac 3 27 304253 Coding 4 86 caccggtgcccgtagggcca 16 28 304254 Coding 4 91 ggattcaccggtgcccgtag 32 29 304255 Coding 4 100 aggacacttggattcaccgg 47 30 304256 Coding 4 105 atcagaggacacttggattc 0 31 304257 Coding 4 110 tgaccatcagaggacacttg 21 32 304258 Coding 4 114 actttgaccatcagaggaca 16 33 304259 Coding 4 126 acagcatctagaactttgac 33 34 304260 Coding 4 133 gcctcggacagcatctagaa 34 35 304261 Coding 4 146 tgatggcaggactgcctcgg 16 36 304262 Coding 4 170 ttctgaacacatgcacggcc 41 37 304263 Coding 4 185 tgtcatcagcagcctttctg 8 38 304264 Coding 4 197 atggctcccaggtgtcatca 34 39 304265 Coding 4 203 aggcaaatggctcccaggtg 15 40 304266 Coding 4 210 ttcccagaggcaaatggctc 0 41 304267 Coding 4 217 actggttttcccagaggcaa 56 42 304268 Coding 4 222 gactcactggttttcccaga 0 43 304269 Coding 4 232 cagctctccagactcactgg 44 44 304270 Coding 4 239 gcccatgcagctctccagac 14 45 304271 Coding 4 244 tgtgagcccatgcagctctc 3 46 304272 Coding 4 250 ctcagttgtgagcccatgca 36 47 304273 Coding 4 257 attcctcctcagttgtgagc 10 48 304274 Coding 4 264 tctacaaattcctcctcagt 34 49 304275 Coding 4 278 ctttgtatatcccttctaca 43 50 304276 Coding 4 298 agatttggtgtctatttcca 1 51 304277 Coding 4 314 caagtgccttccagtaagat 14 52 304278 Coding 4 323 gggagatgccaagtgccttc 53 53 304279 Coding 4 342 tctgcatgctcatggaatgg 42 54 304280 Coding 4 353 tgaataccacctctgcatgc 7 55 304281 Coding 4 360 ttggctgtgaataccacctc 5 56 304282 Coding 4 369 ccggagtcgttggctgtgaa 16 57 304283 Coding 4 401 tcagcagggcggcaatggtg 1 58 304284 Coding 4 425 ccgtggtggaataggagtag 63 59 304285 Coding 4 427 agccgtggtggaataggagt 53 60 304286 Coding 4 431 cgacagccgtggtggaatag 56 61 304287 Coding 4 438 ttggtgacgacagccgtggt 92 62 304288 Coding 4 440 gattggtgacgacagccgtg 70 63 304289 Coding 4 442 gggattggtgacgacagccg 73 64 304290 Coding 4 443 tgggattggtgacgacagcc 83 65 304291 Coding 4 449 attccttgggattggtgacg 45 66 304292 Stop Codon 4 450 cattccttgggattggtgac 27 67 304293 Stop Codon 4 451 tcattccttgggattggtga 20 68 304294 Stop Codon 4 460 agaagtccctcattccttgg 37 69 304295 3'UTR 4 472 gtccactggaggagaagtcc 47 70 304296 3'UTR 4 481 gtccttcaggtccactggag 86 71 304297 3'UTR 4 489 catccctcgtccttcaggtc 76 72 304298 3'UTR 4 501 tacatgaaatcccatccctc 52 73 304299 3'UTR 4 507 cttggttacatgaaatccca 78 74 304300 3'UTR 4 513 aatactcttggttacatgaa 52 75 304301 3'UTR 4 526 ttagtaaaaatggaatactc 20 76 304302 3'UTR 4 532 actgctttagtaaaaatgga 57 77 304303 3'UTR 4 539 tgaaaacactgctttagtaa 54 78 304304 3'UTR 4 546 tatgaggtgaaaacactgct 48 79 304305 3'UTR 4 551 tagcatatgaggtgaaaaca 68 80 304306 3'UTR 4 559 ttctaacatagcatatgagg 72 81 304307 3'UTR 4 564 tggacttctaacatagcata 79 82 304308 3'UTR 4 572 tctctgcctggacttctaac 75 83 304309 3'UTR 4 578 ttattgtctctgcctggact 83 84 304310 3'UTR 4 595 cctttcacaggaatgtttta 46 85 304311 3'UTR 4 597 tgcctttcacaggaatgttt 79 86 304312 3'UTR 4 598 gtgcctttcacaggaatgtt 80 87 304313 3'UTR 4 600 aagtgcctttcacaggaatg 68 88 304314 3'UTR 4 604 tgaaaagtgcctttcacagg 8 89

[0159] As shown in Table 1, SEQ ID NOs 15, 20, 24, 29, 30, 34, 35, 37, 39, 42, 44, 47, 49, 50, 53, 54, 59, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 73, 74, 75, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 and 88 demonstrated at least 27% inhibition of human transthyretin expression in this assay and are therefore preferred. More preferred are SEQ ID NOs 84, 87, and 86. The target regions to which these preferred sequences are complementary are herein referred to as "preferred target segments" and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 2. These sequences are shown to contain thymine (T) but one of skill in the art will appreciate that thymine (T) is generally replaced by uracil (U) in RNA sequences. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. "Target site" indicates the first (5'-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 2 is the species in which each of the preferred target segments was found.

TABLE-US-00004 TABLE 2 Sequence and position of preferred target segments identified in transthyretin. REV TARGET COMP SEQ SITE SEQ ID TARGET OF SEQ ID ID NO SITE SEQUENCE ID ACTIVE IN NO 220029 11 3880 gcatgcagaggtgagtatac 15 H. sapiens 90 220034 4 6 agtccactcattcttggcag 20 H. sapiens 91 220038 4 59 tgctggactggtatttgtgt 24 H. sapiens 92 220043 4 91 ctacgggcaccggtgaatcc 29 H. sapiens 93 220044 4 100 ccggtgaatccaagtgtcct 30 H. sapiens 94 220048 4 126 gtcaaagttctagatgctgt 34 H. sapiens 95 220049 4 133 ttctagatgctgtccgaggc 35 H. sapiens 96 220051 4 170 ggccgtgcatgtgttcagaa 37 H. sapiens 97 220053 4 197 tgatgacacctgggagccat 39 H. sapiens 98 220056 4 217 ttgcctctgggaaaaccagt 42 H. sapiens 99 220058 4 232 ccagtgagtctggagagctg 44 H. sapiens 100 220061 4 250 tgcatgggctcacaactgag 47 H. sapiens 101 220063 4 264 actgaggaggaatttgtaga 49 H. sapiens 102 220064 4 278 tgtagaagggatatacaaag 50 H. sapiens 103 220067 4 323 gaaggcacttggcatctccc 53 H. sapiens 104 220068 4 342 ccattccatgagcatgcaga 54 H. sapiens 105 220073 4 425 ctactcctattccaccacgg 59 H. sapiens 106 220074 4 427 actcctattccaccacggct 60 H. sapiens 107 220075 4 431 ctattccaccacggctgtcg 61 H. sapiens 108 220076 4 438 accacggctgtcgtcaccaa 62 H. sapiens 109 220077 4 440 cacggctgtcgtcaccaatc 63 H. sapiens 110 220078 4 442 cggctgtcgtcaccaatccc 64 H. sapiens 111 220079 4 443 ggctgtcgtcaccaatccca 65 H. sapiens 112 220080 4 449 cgtcaccaatcccaaggaat 66 H. sapiens 113 220081 4 450 gtcaccaatcccaaggaatg 67 H. sapiens 114 220083 4 460 ccaaggaatgagggacttct 69 H. sapiens 115 220084 4 472 ggacttctcctccagtggac 70 H. sapiens 116 220085 4 481 ctccagtggacctgaaggac 71 H. sapiens 117 220086 4 489 gacctgaaggacgagggatg 72 H. sapiens 118 220087 4 501 gagggatgggatttcatgta 73 H. sapiens 119 220088 4 507 tgggatttcatgtaaccaag 74 H. sapiens 120 220089 4 513 ttcatgtaaccaagagtatt 75 H. sapiens 121 220091 4 532 tccatttttactaaagcagt 77 H. sapiens 122 220092 4 539 ttactaaagcagtgttttca 78 H. sapiens 123 220093 4 546 agcagtgttttcacctcata 79 H. sapiens 124 220094 4 551 tgttttcacctcatatgcta 80 H. sapiens 125 220095 4 559 cctcatatgctatgttagaa 81 H. sapiens 126 220096 4 564 tatgctatgttagaagtcca 82 H. sapiens 127 220097 4 572 gttagaagtccaggcagaga 83 H. sapiens 128 220098 4 578 agtccaggcagagacaataa 84 H. sapiens 129 220099 4 595 taaaacattcctgtgaaagg 85 H. sapiens 130 220100 4 597 aaacattcctgtgaaaggca 86 H. sapiens 131 220101 4 598 aacattcctgtgaaaggcac 87 H. sapiens 132 220102 4 600 cattcctgtgaaaggcactt 88 H. sapiens 133

[0160] As these "preferred target segments" have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of transthyretin.

[0161] 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 16--Western Blot Analysis of Transthyretin Protein Levels

[0162] 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 transthyretin is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER.TM. (Molecular Dynamics, Sunnyvale Calif.).

[0163] All of the applications, patents and references cited are hereby incorporated herein by reference.

[0164] It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present invention without departing from the spirit or scope of the invention. Thus it is intended that the present invention cover modifications and variations of thi invention. The invention is limited only by the claims below.

Sequence CWU 1

1

137120DNAArtificial sequenceSynthetic oligonucleotide 1tccgtcatcg ctcctcaggg 20220DNAArtificial sequenceSynthetic oligonucleotide 2gtgcgcgcga gcccgaaatc 20320DNAArtificial sequenceSynthetic oligonucleotide 3atgcattctg cccccaagga 204650DNAHomo sapiens 4acagaagtcc actcattctt ggcaggatgg cttctcatcg tctgctcctc ctctgccttg 60ctggactggt atttgtgtct gaggctggcc ctacgggcac cggtgaatcc aagtgtcctc 120tgatggtcaa agttctagat gctgtccgag gcagtcctgc catcaatgtg gccgtgcatg 180tgttcagaaa ggctgctgat gacacctggg agccatttgc ctctgggaaa accagtgagt 240ctggagagct gcatgggctc acaactgagg aggaatttgt agaagggata tacaaagtgg 300aaatagacac caaatcttac tggaaggcac ttggcatctc cccattccat gagcatgcag 360aggtggtatt cacagccaac gactccggcc cccgccgcta caccattgcc gccctgctga 420gcccctactc ctattccacc acggctgtcg tcaccaatcc caaggaatga gggacttctc 480ctccagtgga cctgaaggac gagggatggg atttcatgta accaagagta ttccattttt 540actaaagcag tgttttcacc tcatatgcta tgttagaagt ccaggcagag acaataaaac 600attcctgtga aaggcacttt tcattccaaa aaaaaaaaaa aaaaaaaaaa 650519DNAArtificial sequencePrimer 5ccctgctgag cccctactc 19620DNAArtificial sequencePrimer 6tccctcattc cttgggattg 20721DNAArtificial sequenceProbe 7attccaccac ggctgtcgtc a 21819DNAArtificial sequencePrimer 8gaaggtgaag gtcggagtc 19920DNAArtificial sequencePrimer 9gaagatggtg atgggatttc 201020DNAArtificial sequenceProbe 10caagcttccc gttctcagcc 20118054DNAHomo sapiens 11ttgttgaccc atggatccat caagtgcaaa cattttctaa tgcactatat ttaagcctgt 60gcagctagat gtcattcaac atgaaataca ttattacaac ttgcatctgt ctaaaatctt 120gcatctaaaa tgagagacaa aaaatctata aaaatggaaa acatgcatag aaatatgtga 180gggaggaaaa aattaccccc aagaatgtta gtgcacgcag tcacacaggg agaagactat 240ttttgttttg ttttgattgt tttgttttgt tttggttgtt ttgttttggt gacctaactg 300gtcaaatgac ctattaagaa tatttcatag aacgaatgtt ccgatgctct aatctctcta 360gacaaggttc atatttgtat gggttactta ttctctcttt gttgactaag tcaataatca 420gaatcagcag gtttgcagtc agattggcag ggataagcag cctagctcag gagaagtgag 480tataaaagcc ccaggctggg agcagccatc acagaagtcc actcattctt ggcaggatgg 540cttctcatcg tctgctcctc ctctgccttg ctggactggt atttgtgtct gaggctggcc 600ctacggtgag tgtttctgtg acatcccatt cctacattta agattcacgc taaatgaagt 660agaagtgact ccttccagct ttgccaacca gcttttatta ctagggcaag ggtacccagc 720atctattttt aatataatta attcaaactt caaaaagaat gaagttccac tgagcttact 780gagctgggac ttgaactctg agcattctac ctcattgctt tggtgcatta ggtttgtaat 840atctggtacc tctgtttcct cagatagatg atagaaataa agatatgata ttaaggaagc 900tgttaatact gaattttcag aaaagtatcc ctccataaaa tgtatttggg ggacaaactg 960caggagatta tattctggcc ctatagttat tcaaaacgta tttattgatt aatctttaaa 1020aggcttagtg aacaatattc tagtcagata tctaattctt aaatcctcta gaagaattaa 1080ctaatactat aaaatgggtc tggatgtagt tctgacatta ttttataaca actggtaaga 1140gggagtgact atagcaacaa ctaaaatgat ctcaggaaaa cctgtttggc cctatgtatg 1200gtacattaca tcttttcagt aattccactc aaatggagac ttttaacaaa gcaactgttc 1260tcaggggacc tattttctcc cttaaaattc attatacaca tccctggttg atagcagtgt 1320gtctggaggc agaaaccatt cttgctttgg aaacaattac gtctgtgtta tactgagtag 1380ggaagctcat taattgtcga cacttacgtt cctgataatg ggatcagtgt gtaattcttg 1440tttcgctcca gatttctaat accacaaaga ataaatcctt tcactctgat caattttgtt 1500aacttctcac gtgtcttctc tacacccagg gcaccggtga atccaagtgt cctctgatgg 1560tcaaagttct agatgctgtc cgaggcagtc ctgccatcaa tgtggccgtg catgtgttca 1620gaaaggctgc tgatgacacc tgggagccat ttgcctctgg gtaagttgcc aaagaaccct 1680cccacaggac ttggttttat cttcccgttt gcccctcact tggtagagag aggctcacat 1740catctgctaa agaatttaca agtagattga aaaacgtagg cagaggtcaa gtatgccctc 1800tgaaggatgc cctctttttg ttttgcttag ctaggaagtg accaggaacc tgagcatcat 1860ttaggggcag acagtagaga aaagaaggaa tcagaactcc tctcctctag ctgtggtttg 1920caaccctttt gggtcacaga acactttatg taggtgatga aaagtaaaca ttctatgccc 1980agaaaaaatg cacagataca cacacataca aaatcatata tgtgatttta ggagtttcac 2040agattccctg gtgtccctgg gtaacaccaa agctaagtgt ccttgtctta gaattttagg 2100aaaaggtata atgtgtatta acccattaac aaaaggaaag gaattcagaa atattattaa 2160ccaggcatct gtctgtagtt aatatggatc acccaaaacc caaggctttt gcctaatgaa 2220cactttgggg cacctactgt gtgcaaggct gggggctgtc aagctcagtt aaaaaaaaaa 2280agatagaaga gatggatcca tgaggcaaag tacagcccca ggctaatccc acgatcaccc 2340gacttcatgt ccaagagtgg cttctcacct tcattagcca gttcacaatt ttcatggagt 2400ttttctacct gcactagcaa aaacttcaag gaaaatacat attaataaat ctaagcaaag 2460tgaccagaag acagagcaat caggagaccc tttgcatcca gcagaagagg aactgctaag 2520tatttacatc tccacagaga agaatttctg ttgggtttta attgaacccc aagaaccaca 2580tgattcttca accattattg ggaagatcat tttcttaggt ctggttttaa ctggcttttt 2640atttgggaat tcatttatgt ttatataaaa tgccaagcat aacatgaaaa gtggttacag 2700gactattcta agggagagac agaatggaca ccaaaaatat tccaatgttc ttgtgaatct 2760tttccttgca ccaggacaaa aaaaaaaaga agtgaaaaga agaaaggagg aggggcataa 2820tcagagtcag taaagacaac tgctattttt atctatcgta gctgttgcag tcaaatggga 2880agcaatttcc aacattcaac tatggagctg gtacttacat ggaaatagaa gttgcctagt 2940gtttgttgct ggcaaagagt tatcagagag gttaaatata taaaagggaa aagagtcaga 3000tacaggttct tcttcctact ttaggttttc cactgtgtgt gcaaatgata ctccctggtg 3060gtgtgcagat gcctcaaagc tatcctcaca ccacaaggga gaggagcgag atcctgctgt 3120cctggagaag tgcagagtta gaacagctgt ggccacttgc atccaatcat caatcttgaa 3180tcacagggac tctttcttaa gtaaacatta tacctggccg ggcacggtgg ctcacgcctg 3240taatcccagc actttgggat gccaaagtgg gcatatcatc tgaggtcagg agttcaagac 3300cagcctggcc aacatggcaa aactccgtct ttatgaaaaa tacaaaaatt agccaggcat 3360ggtggcaggc gcctgtaatc ccagctaatt gggaggctga ggctggagaa tcccttgaat 3420ctaggaggca gaggttgcag tgagctgaga tcgtgccatt gcactccagc ctgggtgaca 3480agagtaaaac tctgtctcaa aaaaaaaaaa ttatacctac attctcttct tatcagagaa 3540aaaaatctac agtgagcttt tcaaaaagtt tttacaaact ttttgccatt taatttcagt 3600taggagtttt ccctacttct gacttagttg aggggaaatg ttcataacat gtttataaca 3660tgtttatgtg tgttagttgg tgggggtgta ttactttgcc atgccatttg tttcctccat 3720gcgtaactta atccagactt tcacacctta taggaaaacc agtgagtctg gagagctgca 3780tgggctcaca actgaggagg aatttgtaga agggatatac aaagtggaaa tagacaccaa 3840atcttactgg aaggcacttg gcatctcccc attccatgag catgcagagg tgagtataca 3900gaccttcgag ggttgttttg gttttggttt ttgcttttgg cattccagga aatgcacagt 3960tttactcagt gtaccacaga aatgtcctaa ggaaggtgat gaatgaccaa aggttccctt 4020tcctattata caagaaaaaa ttcacaacac tctgagaagc aaatttcttt ttgactttga 4080tgaaaatcca cttagtaaca tgacttgaac ttacatgaaa ctactcatag tctattcatt 4140ccactttata tgaatattga tgtatctgct gttgaaataa tagtttatga ggcagccctc 4200cagaccccac gtagagtgta tgtaacaaga gatgcaccat tttatttctc gaaaacccgt 4260aacattcttc attccaaaac acatctggct tctcggaggt ctggacaagt gattcttggc 4320aacacatacc tatagagaca ataaaatcaa agtaataatg gcaacacaat agataacatt 4380taccaagcat acaccatgtg gcagacacaa ttataagtgt tttccatatt taacctactt 4440aatcctcagg aataagccac tgaggtcagt cctattatta tccccatctt atagatgaag 4500aaaatgaggc accaggaagt caaataactt gtcaaaggtc acaagactag gaaatacaca 4560agtagaaatg tttacaatta aggcccaggc tgggtttgcc ctcagttctg ctatgcctcg 4620cattatgccc caggaaactt tttcccttgt gaaagccaag cttaaaaaaa gaaaagccac 4680atttgtaacg tgctctgttc ccctgcctat ggtgaggatc ttcaaacagt tatacatgga 4740cccagtcccc ctgccttctc cttaatttct taagtcattt gaaacagatg gctgtcatgg 4800aaatagaatc cagacatgtt ggtcagagtt aaagatcaac taattccatc aaaaatagct 4860cggcatgaaa gggaactatt ctctggctta gtcatggatg agactttcaa ttgctataaa 4920gtggttcctt tattagacaa tgttaccagg gaaacaacag gggtttgttt gacttctggg 4980gcccacaagt caacaagaga gccccatcta ccaaggagca tgtccctgac tacccctcag 5040ccagcagcaa gacatggacc ccagtcaggg caggagcagg gtttcggcgg cgcccagcac 5100aagacattgc ccctagagtc tcagccccta ccctcgagta atagatctgc ctacctgaga 5160ctgttgtttg cccaagagct gggtctcagc ctgatgggaa ccatataaaa aggttcactg 5220acatactgcc cacatgttgt tctctttcat tagatcttag cttccttgtc tgctcttcat 5280tcttgcagta ttcattcaac aaacattaaa aaaaaaaaaa agcattctat gtgtggaaca 5340ctctgctaga tgctgtggat ttagaaatga aaatacatcc cgacccttgg aatggaaggg 5400aaaggactga agtaagacag attaagcagg accgtcagcc cagcttgaag cccagataaa 5460tacggagaac aagagagagc gagtagtgag agatgagtcc caatgcctca ctttggtgac 5520gggtgcgtgg tgggcttcat gcagcttctt ctgataaatg cctccttcag aactggtcaa 5580ctctaccttg gccagtgacc caggtggtca tagtagattt accaagggaa aatggaaact 5640tttattagga gctcttaggc ctcttcactt catggatttt tttttccttt ttttttgaga 5700tggagttttg ccctgtcacc caggctggaa tgcagtggtg caatctcagc tcactgcaac 5760ctccgcctcc caggttcaag caattctcct gcctcagcct cccgagtagc tgggactaca 5820ggtgtgcgcc accacaccag gctaattttt gtattttttg taaagacagg ttttcaccac 5880gttggccagg ctggtctgaa ctccagacct caggtgattc acctgtctca gcctcccaaa 5940gtgctgggat tacaggtgtg agccaccgtg cccggctact tcatggattt ttgattacag 6000attatgcctc ttacaatttt taagaagaat caagtgggct gaaggtcaat gtcaccataa 6060gacaaaagac atttttatta gttgattcta gggaattggc cttaagggga gccctttctt 6120cctaagagat tcttaggtga ttctcacttc ctcttgcccc agtattattt ttgtttttgg 6180tatggctcac tcagatcctt ttttcctcct atccctaagt aatccgggtt tctttttccc 6240atatttagaa caaaatgtat ttatgcagag tgtgtccaaa cctcaaccca aggcctgtat 6300acaaaataaa tcaaattaaa cacatcttta ctgtcttcta cctctttcct gacctcaata 6360tatcccaact tgcctcactc tgagaaccaa ggctgtccca gcacctgagt cgcagatatt 6420ctactgattt gacagaactg tgtgactatc tggaacagca ttttgatcca caatttgccc 6480agttacaaag cttaaatgag ctctagtgca tgcatatata tttcaaaatt ccaccatgat 6540cttccacact ctgtattgta aatagagccc tgtaatgctt ttacttcgta tttcattgct 6600tgttatacat aaaaatatac ttttcttctt catgttagaa aatgcaaaga ataggagggt 6660gggggaatct ctgggcttgg agacaggaga cttgccttcc tactatggtt ccatcagaat 6720gtagactggg acaatacaat aattcaagtc tggtttgctc atctgtaaat tgggaagaat 6780gtttccagct ccagaatgct aaatctctaa gtctgtggtt ggcagccact attgcagcag 6840ctcttcaatg actcaatgca gttttgcatt ctccctacct tttttttcta aaaccaataa 6900aatagataca gcctttaggc tttctgggat ttcccttagt caagctaggg tcatcctgac 6960tttcggcgtg aatttgcaaa acaagacctg actctgtact cctgctctaa ggactgtgca 7020tggttccaaa ggcttagctt gccagcatat ttgagctttt tccttctgtt caaactgttc 7080caaaatataa aagaataaaa ttaattaagt tggcactgga cttccggtgg tcagtcatgt 7140gtgtcatctg tcacgttttt cgggctctgg tggaaatgga tctgtctgtc ttctctcata 7200ggtggtattc acagccaacg actccggccc ccgccgctac accattgccg ccctgctgag 7260cccctactcc tattccacca cggctgtcgt caccaatccc aaggaatgag ggacttctcc 7320tccagtggac ctgaaggacg agggatggga tttcatgtaa ccaagagtat tccattttta 7380ctaaagcagt gttttcacct catatgctat gttagaagtc caggcagaga caataaaaca 7440ttcctgtgaa aggcactttt cattccactt taacttgatt ttttaaattc ccttattgtc 7500ccttccaaaa aaaagagaat caaaatttta caaagaatca aaggaattct agaaagtatc 7560tgggcagaac gctaggagag atccaaattt ccattgtctt gcaagcaaag cacgtattaa 7620atatgatctg cagccattaa aaagacacat tctgtaaatg agagagcctt attttcctgt 7680aaccttcagc aaatagcaaa agacacattc caagggccca cttctttact gtgggcattt 7740cttttttttt ctttttttct tttttccttt tttgagacaa agtctcactc tgttgcccag 7800gctagaatgc agtggtgtaa tctcagctca ctgcaacctc tgcttcctgg gttcaagcga 7860ttctcctgcc tcagcctccc aagtaactgg gattacaggc gcatgccacc acgcctagct 7920catttttgta tttttagtag agatgggatt ttgccatgtt ggctaggctg gtctacgaac 7980tcctgacctc aggtgatcca cctgcctcag cctcccaaag tgctgggatt acaggcatga 8040gccactacac ccgg 80541220DNAArtificial sequenceSynthetic oligonucleotide 12aaacactcac cgtagggcca 201320DNAArtificial sequenceSynthetic oligonucleotide 13caccggtgcc ctgggtgtag 201420DNAArtificial sequenceSynthetic oligonucleotide 14tgagcctctc tctaccaagt 201520DNAArtificial sequenceSynthetic oligonucleotide 15gtatactcac ctctgcatgc 201620DNAArtificial sequenceSynthetic oligonucleotide 16ttctcagagt gttgtgaatt 201720DNAArtificial sequenceSynthetic oligonucleotide 17actctgcata aatacatttt 201820DNAArtificial sequenceSynthetic oligonucleotide 18tcttgttttg caaattcacg 201920DNAArtificial sequenceSynthetic oligonucleotide 19tgaataccac ctatgagaga 202020DNAArtificial sequenceSynthetic oligonucleotide 20ctgccaagaa tgagtggact 202120DNAArtificial sequenceSynthetic oligonucleotide 21tgagaagcca tcctgccaag 202220DNAArtificial sequenceSynthetic oligonucleotide 22cagacgatga gaagccatcc 202320DNAArtificial sequenceSynthetic oligonucleotide 23aggagcagac gatgagaagc 202420DNAArtificial sequenceSynthetic oligonucleotide 24acacaaatac cagtccagca 202520DNAArtificial sequenceSynthetic oligonucleotide 25gacacaaata ccagtccagc 202620DNAArtificial sequenceSynthetic oligonucleotide 26gcctcagaca caaataccag 202720DNAArtificial sequenceSynthetic oligonucleotide 27gtagggccag cctcagacac 202820DNAArtificial sequenceSynthetic oligonucleotide 28caccggtgcc cgtagggcca 202920DNAArtificial sequenceSynthetic oligonucleotide 29ggattcaccg gtgcccgtag 203020DNAArtificial sequenceSynthetic oligonucleotide 30aggacacttg gattcaccgg 203120DNAArtificial sequenceSynthetic oligonucleotide 31atcagaggac acttggattc 203220DNAArtificial sequenceSynthetic oligonucleotide 32tgaccatcag aggacacttg 203320DNAArtificial sequenceSynthetic oligonucleotide 33actttgacca tcagaggaca 203420DNAArtificial sequenceSynthetic oligonucleotide 34acagcatcta gaactttgac 203520DNAArtificial sequenceSynthetic oligonucleotide 35gcctcggaca gcatctagaa 203620DNAArtificial sequenceSynthetic oligonucleotide 36tgatggcagg actgcctcgg 203720DNAArtificial sequenceSynthetic oligonucleotide 37ttctgaacac atgcacggcc 203820DNAArtificial sequenceSynthetic oligonucleotide 38tgtcatcagc agcctttctg 203920DNAArtificial sequenceSynthetic oligonucleotide 39atggctccca ggtgtcatca 204020DNAArtificial sequenceSynthetic oligonucleotide 40aggcaaatgg ctcccaggtg 204120DNAArtificial sequenceSynthetic oligonucleotide 41ttcccagagg caaatggctc 204220DNAArtificial sequenceSynthetic oligonucleotide 42actggttttc ccagaggcaa 204320DNAArtificial sequenceSynthetic oligonucleotide 43gactcactgg ttttcccaga 204420DNAArtificial sequenceSynthetic oligonucleotide 44cagctctcca gactcactgg 204520DNAArtificial sequenceSynthetic oligonucleotide 45gcccatgcag ctctccagac 204620DNAArtificial sequenceSynthetic oligonucleotide 46tgtgagccca tgcagctctc 204720DNAArtificial sequenceSynthetic oligonucleotide 47ctcagttgtg agcccatgca 204820DNAArtificial sequenceSynthetic oligonucleotide 48attcctcctc agttgtgagc 204920DNAArtificial sequenceSynthetic oligonucleotide 49tctacaaatt cctcctcagt 205020DNAArtificial sequenceSynthetic oligonucleotide 50ctttgtatat cccttctaca 205120DNAArtificial sequenceSynthetic oligonucleotide 51agatttggtg tctatttcca 205220DNAArtificial sequenceSynthetic oligonucleotide 52caagtgcctt ccagtaagat 205320DNAArtificial sequenceSynthetic oligonucleotide 53gggagatgcc aagtgccttc 205420DNAArtificial sequenceSynthetic oligonucleotide 54tctgcatgct catggaatgg 205520DNAArtificial sequenceSynthetic oligonucleotide 55tgaataccac ctctgcatgc 205620DNAArtificial sequenceSynthetic oligonucleotide 56ttggctgtga ataccacctc

205720DNAArtificial sequenceSynthetic oligonucleotide 57ccggagtcgt tggctgtgaa 205820DNAArtificial sequenceSynthetic oligonucleotide 58tcagcagggc ggcaatggtg 205920DNAArtificial sequenceSynthetic oligonucleotide 59ccgtggtgga ataggagtag 206020DNAArtificial sequenceSynthetic oligonucleotide 60agccgtggtg gaataggagt 206120DNAArtificial sequenceSynthetic oligonucleotide 61cgacagccgt ggtggaatag 206220DNAArtificial sequenceSynthetic oligonucleotide 62ttggtgacga cagccgtggt 206320DNAArtificial sequenceSynthetic oligonucleotide 63gattggtgac gacagccgtg 206420DNAArtificial sequenceSynthetic oligonucleotide 64gggattggtg acgacagccg 206520DNAArtificial sequenceSynthetic oligonucleotide 65tgggattggt gacgacagcc 206620DNAArtificial sequenceSynthetic oligonucleotide 66attccttggg attggtgacg 206720DNAArtificial sequenceSynthetic oligonucleotide 67cattccttgg gattggtgac 206820DNAArtificial sequenceSynthetic oligonucleotide 68tcattccttg ggattggtga 206920DNAArtificial sequenceSynthetic oligonucleotide 69agaagtccct cattccttgg 207020DNAArtificial sequenceSynthetic oligonucleotide 70gtccactgga ggagaagtcc 207120DNAArtificial sequenceSynthetic oligonucleotide 71gtccttcagg tccactggag 207220DNAArtificial sequenceSynthetic oligonucleotide 72catccctcgt ccttcaggtc 207320DNAArtificial sequenceSynthetic oligonucleotide 73tacatgaaat cccatccctc 207420DNAArtificial sequenceSynthetic oligonucleotide 74cttggttaca tgaaatccca 207520DNAArtificial sequenceSynthetic oligonucleotide 75aatactcttg gttacatgaa 207620DNAArtificial sequenceSynthetic oligonucleotide 76ttagtaaaaa tggaatactc 207720DNAArtificial sequenceSynthetic oligonucleotide 77actgctttag taaaaatgga 207820DNAArtificial sequenceSynthetic oligonucleotide 78tgaaaacact gctttagtaa 207920DNAArtificial sequenceSynthetic oligonucleotide 79tatgaggtga aaacactgct 208020DNAArtificial sequenceSynthetic oligonucleotide 80tagcatatga ggtgaaaaca 208120DNAArtificial sequenceSynthetic oligonucleotide 81ttctaacata gcatatgagg 208220DNAArtificial sequenceSynthetic oligonucleotide 82tggacttcta acatagcata 208320DNAArtificial sequenceSynthetic oligonucleotide 83tctctgcctg gacttctaac 208420DNAArtificial sequenceSynthetic oligonucleotide 84ttattgtctc tgcctggact 208520DNAArtificial sequenceSynthetic oligonucleotide 85cctttcacag gaatgtttta 208620DNAArtificial sequenceSynthetic oligonucleotide 86tgcctttcac aggaatgttt 208720DNAArtificial sequenceSynthetic oligonucleotide 87gtgcctttca caggaatgtt 208820DNAArtificial sequenceSynthetic oligonucleotide 88aagtgccttt cacaggaatg 208920DNAArtificial sequenceSynthetic oligonucleotide 89tgaaaagtgc ctttcacagg 209020DNAArtificial sequenceSynthetic oligonucleotide 90gcatgcagag gtgagtatac 209120DNAArtificial sequenceSynthetic oligonucleotide 91agtccactca ttcttggcag 209220DNAArtificial sequenceSynthetic oligonucleotide 92tgctggactg gtatttgtgt 209320DNAArtificial sequenceSynthetic oligonucleotide 93ctacgggcac cggtgaatcc 209420DNAArtificial sequenceSynthetic oligonucleotide 94ccggtgaatc caagtgtcct 209520DNAArtificial sequenceSynthetic oligonucleotide 95gtcaaagttc tagatgctgt 209620DNAArtificial sequenceSynthetic oligonucleotide 96ttctagatgc tgtccgaggc 209720DNAArtificial sequenceSynthetic oligonucleotide 97ggccgtgcat gtgttcagaa 209820DNAArtificial sequenceSynthetic oligonucleotide 98tgatgacacc tgggagccat 209920DNAArtificial sequenceSynthetic oligonucleotide 99ttgcctctgg gaaaaccagt 2010020DNAArtificial sequenceSynthetic oligonucleotide 100ccagtgagtc tggagagctg 2010120DNAArtificial sequenceSynthetic oligonucleotide 101tgcatgggct cacaactgag 2010220DNAArtificial sequenceSynthetic oligonucleotide 102actgaggagg aatttgtaga 2010320DNAArtificial sequenceSynthetic oligonucleotide 103tgtagaaggg atatacaaag 2010420DNAArtificial sequenceSynthetic oligonucleotide 104gaaggcactt ggcatctccc 2010520DNAArtificial sequenceSynthetic oligonucleotide 105ccattccatg agcatgcaga 2010620DNAArtificial sequenceSynthetic oligonucleotide 106ctactcctat tccaccacgg 2010720DNAArtificial sequenceSynthetic oligonucleotide 107actcctattc caccacggct 2010820DNAArtificial sequenceSynthetic oligonucleotide 108ctattccacc acggctgtcg 2010920DNAArtificial sequenceSynthetic oligonucleotide 109accacggctg tcgtcaccaa 2011020DNAArtificial sequenceSynthetic oligonucleotide 110cacggctgtc gtcaccaatc 2011120DNAArtificial sequenceSynthetic oligonucleotide 111cggctgtcgt caccaatccc 2011220DNAArtificial sequenceSynthetic oligonucleotide 112ggctgtcgtc accaatccca 2011320DNAArtificial sequenceSynthetic oligonucleotide 113cgtcaccaat cccaaggaat 2011420DNAArtificial sequenceSynthetic oligonucleotide 114gtcaccaatc ccaaggaatg 2011520DNAArtificial sequenceSynthetic oligonucleotide 115ccaaggaatg agggacttct 2011620DNAArtificial sequenceSynthetic oligonucleotide 116ggacttctcc tccagtggac 2011720DNAArtificial sequenceSynthetic oligonucleotide 117ctccagtgga cctgaaggac 2011820DNAArtificial sequenceSynthetic oligonucleotide 118gacctgaagg acgagggatg 2011920DNAArtificial sequenceSynthetic oligonucleotide 119gagggatggg atttcatgta 2012020DNAArtificial sequenceSynthetic oligonucleotide 120tgggatttca tgtaaccaag 2012120DNAArtificial sequenceSynthetic oligonucleotide 121ttcatgtaac caagagtatt 2012220DNAArtificial sequenceSynthetic oligonucleotide 122tccattttta ctaaagcagt 2012320DNAArtificial sequenceSynthetic oligonucleotide 123ttactaaagc agtgttttca 2012420DNAArtificial sequenceSynthetic oligonucleotide 124agcagtgttt tcacctcata 2012520DNAArtificial sequenceSynthetic oligonucleotide 125tgttttcacc tcatatgcta 2012620DNAArtificial sequenceSynthetic oligonucleotide 126cctcatatgc tatgttagaa 2012720DNAArtificial sequenceSynthetic oligonucleotide 127tatgctatgt tagaagtcca 2012820DNAArtificial sequenceSynthetic oligonucleotide 128gttagaagtc caggcagaga 2012920DNAArtificial sequenceSynthetic oligonucleotide 129agtccaggca gagacaataa 2013020DNAArtificial sequenceSynthetic oligonucleotide 130taaaacattc ctgtgaaagg 2013120DNAArtificial sequenceSynthetic oligonucleotide 131aaacattcct gtgaaaggca 2013220DNAArtificial sequenceSynthetic oligonucleotide 132aacattcctg tgaaaggcac 2013320DNAArtificial sequenceSynthetic oligonucleotide 133cattcctgtg aaaggcactt 2013419DNAArtificial sequenceSynthetic oligonucleotide 134cgagaggcgg acgggaccg 1913521DNAArtificial sequenceSynthetic oligonucleotide 135cgagaggcgg acgggaccgt t 2113621DNAArtificial sequenceSynthetic oligonucleotide 136cggtcccgtc cgcctctcgt t 2113719DNAArtificial sequenceSynthetic oligonucleotide 137cggtcccgtc cgcctctcg 19

* * * * *

File A Patent Application

  • Protect your idea -- Don't let someone else file first. Learn more.

  • 3 Easy Steps -- Complete Form, application Review, and File. See our process.

  • Attorney Review -- Have your application reviewed by a Patent Attorney. See what's included.