# Patent

## Title
Modification of MyD88 splicing using modified oligonucleotides

## Abstract

Antisense compounds, compositions and methods are provided for modulating the expression of MyD88. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding MyD88. Methods of using these compounds for modulation of MyD88 expression and for treatment of diseases associated with expression of MyD88 are provided.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority to U.S. provisional patent application Ser. No. 60/648,823, filed Jan. 31, 2005, which is herein incorporated by reference in its entirety.


INCORPORATION OF THE SEQUENCE LISTING
A paper copy of the sequence listing and a computer-readable form of the sequence listing, on diskette, containing the file named CORE0057USSEQ.txt, which was created on Jan. 20, 2006, are herein incorporated by reference.
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of MyD88 by altering the splicing of MyD88. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding MyD88 which effect splicing of the gene. Such compounds have been shown to modulate expression and splicing of MyD88 in vitro and in vivo.
BACKGROUND OF THE INVENTION
MyD88 is an adaptor protein involved in interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) induced activation of NF-κB (Burns et al. 1998, J. Biol. Chem. 273:12203-12209; Wesche et al. 1997, Immunity 7:837-847; Medzhitov et al. 1998, Mol. Cell. 2:253-258). Both human and mouse MyD88 proteins are composed of a C-terminal Toll/IL-1R homology (TIR) domain and an N-terminal death domain (DD). These domains are separated by a small intermediate domain (ID). MyD88 links the TIR domain of IL-1R/TLR with the DD of the Ser/Thr kinase, IRAK-1. MyD88 also recruits IRAK-4 via its ID. This enables IRAK-4 to phosphorylate IRAK-1. Phosphorylated IRAK-1 then interacts with TRAF6, which in turn, transmits a signal leading to activation of the IκB kinase complex and JNK (Cao et al. 1996, Nature 383:443-446). This eventually results in the activation of transcription factor NF-κB. As MyD88 is known to play a central role in regulating signaling through IL-1 and Toll-receptors, various strategies have been used to demonstrate that inhibition of MyD88 function results in an alleviation of inflammatory responses. For example, in vitro this has been clearly demonstrated using MyD88 deficient cells (Schnare et al. 2000, Curr. Biol. 10: 1139-1142; Yamamoto et al. 2002, Nature. 420:324-329). In animals, genetic ablation of MyD88 produces a diminution of responses to IL-1β and an overall reduction in models of inflammation (Kawai et al. 1999, Immunity 11:115-122; Bjorkbacka et al. 2004, Nature Med. 10:416-421; Adachi et al. 1998, Immunity 9:143-150). MyD88 deficient mice demonstrate reduced pathology associated with polymicrobial sepsis (Weighardt et al. 2002, J Immunol. 169:2823-2827) and defects in T-cell proliferation and induction of acute phase proteins in response to IL-1β (Adachi et al. 1998, Immunity 9:143-150).
A lipopolysaccharide (LPS)-induced splice variant of MyD88, MyD88S, which has been reported to function as a dominant-negative regulator of IL-1β- and LPS-induced NF-κB activation, has recently been identified (Janssens et al. 2002, Curr. Biol. 12:467-471). The MyD88 gene contains 5 exons (Hardiman et al. 1997, Genomics. 45:332-339). MyD88S is produced as the result of complete excision of exon II from the mature mRNA by alternative splicing. This leads to an in-frame deletion of the complete ID (amino acids 110-154). Although MyD88S still binds IL-1R and IRAK-1, it is defective in its ability to recruit IRAK-4 and induce subsequent IRAK-1 phosphorylation and NF-κB activation (Burns et al. 2003, J. Exp. Med. 197:263-268). The ability of MyD88S to function as a dominant negative regulator of IL-1β and LPS signaling suggests that strategies to modify MyD88 RNA splicing, such that MyD88S is expressed preferentially to the larger MyD88 isoform (referred to herein as MyD88L), would be therapeutically valuable in treating inflammatory diseases associated with excessive IL-1R signaling.
Although variations in alternative splicing are widely recognized as a mechanism to generate molecular diversity and clearly contribute to certain diseases (Maniatis and Tasic, 2002, Nature. 418:236-243; Johnson et al. 2003, Science. 302:2141-2144; Yeo et al. 2004, Proc. Natl. Acad. Sci. U.S.A. 101:15700-15705), the development of pharmacological agents capable of controlling mRNA splicing has remained challenging. An approach that has recently gained significant acceptance is the use of chemically modified antisense oligonucleotides. When appropriately designed and directed to hybridize to RNA sequences adjacent to splice junctions, these have been shown to effectively modulate pre-mRNA splicing (Sazani and Kole, 2003, J. Clin. Invest. 112:481-486; Taylor et al. 1999, Nat. Biotech. 17:1097-1100; Garcia-Blanco et al. 2004, Nat. Biotech. 22:535-546).
U.S. Pre-Grant Publication 2005-0181476 discusses the identification of MyD88S and methods of modulating alternative splicing of MyD88 through RNA inhibition and use of antisense oligonucleotides.
A method of controlling the behavior of a cell through modulation of the processing of an mRNA target by contacting the cell with an antisense compound acting via a non-cleavage event is disclosed in U.S. Pat. No. 6,210,892 and U.S. Pre-Grant Publication 2002-0049173.
Taylor et al. (1999, Nat. Biotech. 17:1097-1100) and Karras et al. (2000, Mol. Pharm. 58:380-387) describe modified oligonucleotides for modulation of pre-mRNA splicing of Bcl-x and interleukin-5 receptor alpha, respectively.
Kole et al. (WO 94/26887 and U.S. Pat. Nos. 5,627,274; 5,916,808; 5,976,879; and 5,665,593) disclose methods of combating aberrant splicing using modified antisense oligonucleotides which do not activate RNase H.
Antisense technology is an effective means for modulating expression of one or more specific gene products and is uniquely useful in a number of therapeutic, diagnostic, and research applications. Provided herein are modified antisense compounds for use in modulation of MyD88 splicing.
SUMMARY OF THE INVENTION
Provided herein are antisense compounds targeting MyD88 for modulation of MyD88 splicing. In one aspect of the invention, the antisense compounds are designed to increase the ratio of MyD88S to MyD88L mRNA or protein. In another aspect of the invention, the antisense compounds increase the ratio of MyD88L to MyD88S mRNA or protein.
In one embodiment, the antisense compounds are targeted to the exon II donor site of a nucleic acid molecule encoding human MyD88. The antisense compounds provided herein are resistant to RNase H degradation. In preferred embodiments the antisense compounds are 12 to 50, 12 to 30, or 15 to 30 nucleobases in length. In one embodiment, the antisense compounds comprise at least one modified nucleotide. In another embodiment, the antisense compounds comprise a modified nucleotide at each position. In yet another embodiment, the antisense compounds are uniformly modified at each position.
In some embodiments of the invention, the modification of the antisense compounds is a sugar modification. Sugar modifications included, but are not limited to, 2′-O-(2-methoxyethyl), 2′-fluoro, locked nucleic acid and ethylene bridged nucleic acid. In one preferred embodiment, the sugar modification is 2′-O-(2-methoxyethyl). In some embodiments, the antisense compounds further comprise phosphorothioate internucleoside linkages.
Antisense compounds contemplated include compounds comprising at least an 8-nucleobase portion of one of the illustrated compounds. Also contemplated are antisense compounds having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with one of the illustrated antisense compounds.
Also provided herein are methods of modulating splicing of human MyD88 in cells or tissues, comprising contacting said cells or tissues with the antisense compounds described herein. In one aspect, the modulation of splicing results in an increase in the ratio of MyD88S to MyD88L mRNA. In another aspect, the modulation of splicing results in an increase in the ratio of MyD88S to MyD88L protein. In some embodiments, the cells or tissues are liver cells or tissues.
Further provided are methods of inhibiting IL-1β signaling and inhibiting toll-like receptor signaling in cells or tissues, comprising contacting said cells or tissues with the antisense compounds provided herein.


DETAILED DESCRIPTION OF THE INVENTION
Antisense technology is an effective means for reducing the expression of one or more specific gene products and is uniquely useful in a number of therapeutic, diagnostic, and research applications. Provided herein are antisense compounds useful for modulating gene expression and associated pathways via antisense mechanisms of action based on target degradation or target occupancy.
The principle behind antisense technology is that an antisense compound, which hybridizes to a target nucleic acid, modulates gene expression activities such as transcription, splicing or translation through one of a number of antisense mechanisms. The sequence specificity of antisense compounds makes them extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes or gene products involved in disease.
Given the role of MyD88 in inflammatory signaling pathways, and thus a variety of inflammatory diseases, therapeutic tools for modulating expression of MyD88 are desirable. Furthermore, the finding that alternative splicing of MyD88, such as occurs following LPS stimulation, results in production of a short form (MyD88S) that acts as a repressor of IL-1 (inflammatory) signaling, provides a rationale for identifying compounds capable of modulating MyD88 splicing. The ideal compound would shift MyD88 splicing such that the ratio of splice products favors MyD88S over MyD88L. Provided herein are antisense compounds that drive splicing of MyD88 toward the short form. The antisense compounds, referred to herein as “splice switching oligonucleotides,” target regions of MyD88 pre-mRNA near the exon II splice acceptor site and splice donor site. The antisense compounds provided herein are modified such that they are resistant to degradation by RNase H and are presumed to function by an occupancy-based mechanism.
As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding MyD88” have been used for convenience to encompass DNA encoding MyD88, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA.
As used herein, “targeting” or “targeted to” refer to the process of designing an oligomeric compound such that the compound hybridizes with a selected nucleic acid molecule.
As used herein, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the context of the present invention, an oligomeric compound is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target nucleic acid sequences.
As used herein, “antisense mechanisms” are all those involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
As used herein, compounds “targeted to the exon II donor site of a nucleic acid molecule encoding MyD88” refers to an antisense compound provided herein which hybridizes with at least a portion of a region of a nucleic acid molecule encoding MyD88, wherein the region consists of the exon II donor site and the 20 nucleotides upstream (5′) of the donor site and the 20 nucleotides downstream (3′) of the donor site of MyD88.
As used herein, compounds “targeted to the exon II acceptor site of a nucleic acid molecule encoding MyD88” refers to an antisense compound provided herein which hybridizes with at least a portion of a region of a nucleic acid molecule encoding MyD88, wherein the region consists of the exon II acceptor site and the 20 nucleotides upstream (5′) of the acceptor site and the 20 nucleotides downstream (3′) of the acceptor site of MyD88.
As used herein, “modulation of splicing” refers to altering the processing of a pre-mRNA transcript such that there is an increase or decrease of one or more splice products, or a change in the ratio of two or more splice products. Modulation of splicing can also refer to altering the processing of a pre-mRNA transcript such that a spliced mRNA molecule contains either a different combination of exons as a result of exon skipping or exon inclusion, a deletion in one or more exons, or additional sequence not normally found in the spliced mRNA (e.g., intron sequence).
As used herein, “splice products” or “splicing products” are the mature mRNA molecules generated from the process of splicing a pre-mRNA. Alternatively spliced pre-mRNAs have at least two different splice products. For example, a first splicing product may contain an additional exon, or portion of an exon, relative to a second splicing product. Splice products of a selected pre-mRNA can be identified by a variety of different techniques well known to those of skill in the art.
As used herein, “splice site” refers to the junction between an exon and an intron in a pre-mRNA (unspliced RNA) molecule (also known as a “splice junction”). A “cryptic splice site” is a splice site that is not typically used but may be used when the usual splice site is blocked or unavailable or when a mutation causes a normally dormant site to become an active splice site. An “aberrant splice site” is a splice site that results from a mutation in the native DNA and mRNA. In the context of the present invention, an oligomeric compound “targeted to a splice site” refers to a compound that hybridizes with at least a portion of a region of nucleic acid encoding a splice site or a compound that hybridizes with an intron or exon in proximity to a splice site, such that splicing of the mRNA is modulated.
As used herein “splice donor site” refers to a splice site found at the 5′ end of an intron, or alternatively, the 3′ end of an exon. Splice donor site is used interchangeably with “5′ splice site.” As used herein “splice acceptor site” refers to a splice site found at the 3′ end of an intron, or alternatively, the 5′ end of an exon. Splice acceptor site is used interchangeably with “3′ splice site.”
As used herein, compounds “resistant to RNase H degradation” are antisense compounds having a least one chemical modification that increases resistance of the compound to RNase H cleavage. Such modifications include, but are not limited to, nucleotides with sugar modifications. As used herein, a nucleotide with a modified sugar includes, but is not limited to, any nucleotide wherein the 2′-deoxyribose sugar has been substituted with a chemically modified sugar moiety. In the context of the present invention, chemically modified sugar moieties include, but are not limited to, 2′-O-(2-methoxyethyl), 2′-fluoro, 2′-dimethylaminooxyethoxy, 2′-dimethylaminoethoxyethoxy, 2′-guanidinium, 2′-O-guanidinium ethyl, 2′-carbamate, 2′-aminooxy, 2′-acetamido, locked nucleic acid (LNA) and ethylene bridged nucleic acid (ENA). Modified compounds resistant to RNase H cleavage are thoroughly described herein and are well know to those of skill in the art.
As used herein, “uniformly modified” refers to antisense compounds having the same chemical modification or modifications at each nucleotide.
In accordance with the present invention are compositions and methods for modulating the expression of MyD88 (also known as MYD88; myeloid differentiation factor; and myeloid differentiation primary response gene 88). Listed in Table 1 are GENBANK® accession numbers of sequences used to design oligomeric compounds targeted to MyD88. Oligomeric compounds of the invention include oligomeric compounds which hybridize with one or more target nucleic acid molecules shown in Table 1, as well as oligomeric compounds which hybridize to other nucleic acid molecules encoding MyD88. The oligomeric compounds may target any region, segment, or site of nucleic acid molecules which encode MyD88. Suitable target regions, segments, and sites include, but are not limited to, the 5′UTR, the start codon, the stop codon, the coding region, the 3′UTR, the 5′ cap region, introns, exons, intron-exon junctions, exon-intron junctions, and exon-exon junctions.







TABLE 1







Gene Target Names and Sequences










Target





Name
Species
Genbank ® #
SEQ ID NO













MyD88
Human
AB026898.1 (nt 86001-91000)
3


MyD88
Human
AL526415.1
10


MyD88
Human
NM_002468.1
11


MyD88
Human
U70451.1
90


MyD88
Mouse
NM_010851.1
91


MyD88
Human
NT_037565.3 (nt 119121-123478)
146


MyD88
Mouse
NT_039482.1 (nt 5061829-5065869)
147









The term “oligomeric compound” refers to a polymeric structure capable of hybridizing to a region of a nucleic acid molecule. This term includes oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and chimeric combinations of these. Oligomeric compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular. Moreover, branched structures are known in the art. An “antisense compound” or “antisense oligomeric compound” refers to an oligomeric compound that is at least partially complementary to the region of a nucleic acid molecule to which it hybridizes and which modulates (increases or decreases) its expression. Consequently, while all antisense compounds can be said to be oligomeric compounds, not all oligomeric compounds are antisense compounds. An “antisense oligonucleotide” is an antisense compound that is a nucleic acid-based oligomer. An antisense oligonucleotide can be chemically modified. Nonlimiting examples of oligomeric compounds include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides and alternate splicers. In one embodiment, the oligomeric compound comprises an antisense strand hybridized to a sense strand. Oligomeric compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops. Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
The oligomeric compounds in accordance with this invention comprise compounds 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 this comprehends antisense 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 one embodiment, the antisense compounds of the invention comprise 13 to 80 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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 one embodiment, the antisense compounds of the invention comprise 12 to 50 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 12 to 30 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases.
In some embodiments, the antisense compounds of the invention comprise 15 to 30 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 20 to 30 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 20 to 24 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 20, 21, 22, 23, or 24 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 16 to 20 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 16, 17, 18, 19 or 20 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 20 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 19 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 18 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 17 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 16 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 15 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 14 nucleobases.
In one embodiment, the antisense compounds of the invention comprise 13 nucleobases.
Antisense compounds 8-80 nucleobases in length, and any length within the range, comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds.
Compounds of the invention include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative 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, or about 13 to about 80, or about 12 to about 50, or about 12 to about 30, or about 15 to about 30, or about 20 to about 30, or about 20 to about 24, or about 16 to about 20 nucleobases). Other compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative 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, or about 13 to about 80, or about 12 to about 50, or about 12 to about 30, or about 15 to about 30, or about 20 to about 30, or about 20 to about 24, or about 16 to about 20 nucleobases). It is also understood that compounds may be represented by oligonucleotide sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of an illustrative compound, and may extend in either or both directions until the oligonucleotide contains about 8 to about 80, or about 13 to about 80, or about 12 to about 50, or about 12 to about 30, or about 15 to about 30, or about 20 to about 30, or about 20 to about 24, or about 16 to about 20 nucleobases.
One having skill in the art armed with the antisense compounds illustrated herein will be able, without undue experimentation, to identify further antisense compounds.
“Hybridization” means the pairing of complementary strands of oligomeric compounds. While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
An oligomeric compound is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric 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.
“Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound 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 “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.
“Complementarity,” as used herein, refers to the capacity for precise pairing between two nucleobases on one or two oligomeric compound strands. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligomeric compound and the further DNA or RNA 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 oligomeric compound and a target nucleic acid.
It is understood in the art that the sequence of an oligomeric 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, mismatch or hairpin structure). The oligomeric compounds of the present invention comprise at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an oligomeric compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an oligomeric compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).
Oligomeric compounds, or a portion thereof, may have a defined percent identity to a SEQ ID NO, or a compound having a specific ISIS number. This identity may be over the entire length of the oligomeric compound, or in a portion of the oligomeric compound (e.g., nucleobases 1-20 of a 27-mer may be compared to a 20-mer to determine percent identity of the oligomeric compound to the SEQ ID NO) It is understood by those skilled in the art that an oligonucleotide need not have an identical sequence to those described herein to function similarly to the oligonucleotides described herein. Shortened (i.e., deleted, and therefore non-identical) versions of oligonucleotides taught herein, or non-identical (i.e., one base replaced with another) versions of the oligonucleotides taught herein fall within the scope of the invention. Percent identity is calculated according to the number of bases that are identical to the SEQ ID NO or compound to which it is being compared. The non-identical bases may be adjacent to each other, dispersed through out the oligonucleotide, or both.
For example, a 16-mer having the same sequence as nucleobases 2-17 of a 20-mer is 80% identical to the 20-mer. Alternatively, a 20-mer containing four nucleobases not identical to the 20-mer is also 80% identical to the 20-mer. A 14-mer having the same sequence as nucleobases 1-14 of an 18-mer is 78% identical to the 18-mer. Such calculations are well within the ability of those skilled in the art.
The percent identity is based on the percent of nucleobases in the original sequence present in a portion of the modified sequence. Therefore, a 30 nucleobase oligonucleotide comprising the full sequence of a 20 nucleobase SEQ ID NO would have a portion of 100% identity with the 20 nucleobase SEQ ID NO while further comprising an additional 10 nucleobase portion. In the context of the invention, the full length of the modified sequence may constitute a single portion.
The oligomeric compounds of the invention also include 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, compounds may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the oligomeric compound. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of MyD88 mRNA.
As used herein, “targeting” or “targeted to” refer to the process of designing an oligomeric compound such that the compound hybridizes with a selected nucleic acid molecule. Targeting an oligomeric compound to a particular target nucleic acid molecule can be a multistep process. The process usually begins with the identification of a target nucleic acid whose expression is to be modulated. As used herein, the terms “target nucleic acid” and “nucleic acid encoding MyD88” encompass DNA encoding MyD88, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. For example, the target nucleic acid can be 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. As disclosed herein, the target nucleic acid encodes MyD88.
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. “Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Target regions may include, for example, a particular exon or intron, or may include only selected nucleobases within an exon or intron which are identified as appropriate target regions. 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 unique nucleobase positions within a target nucleic acid. As used herein, the “target site” of an oligomeric compound is the 5′-most nucleotide of the target nucleic acid to which the compound binds.
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. “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 a protein, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′ UAA, 5′ UAG and 5′ UGA (the corresponding DNA sequences are 5′ TAA, 5′ TAG and 5′ TGA, respectively).
The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with oligomeric compounds of the invention.
The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, one region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.
Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. The 5′ cap region is also a target.
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, resulting in exon-exon junctions at the site where exons are joined. Targeting exon-exon junctions can be useful in situations where aberrant levels of a normal splice product are implicated in disease, or where aberrant levels of an aberrant splice product are implicated in disease. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions can 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 suitable targets. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts” and are also suitable targets. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA. Single-stranded antisense compounds such as oligonucleotide compounds that work via an RNase H mechanism are effective for targeting pre-mRNA. Antisense compounds that function via an occupancy-based mechanism are effective for redirecting splicing as they do not, for example, elicit RNase H cleavage of the mRNA, but rather leave the mRNA intact and promote the yield of desired splice product(s).
It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants.” More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants.” Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants.” If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.
It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Consequently, the types of variants described herein are also suitable target nucleic acids.
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 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. 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.
Specific examples of oligomeric compounds useful of the present invention include oligonucleotides containing modified e.g. non-naturally occurring internucleoside linkages. As defined in this specification, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. 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.
Oligomeric compounds can have one or more modified internucleoside linkages. Modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thiono-alkylphosphonates, thionoalkylphosphotriesters, phosphonoacetate and thiophosphonoacetate (see Sheehan et al., Nucleic Acids Research, 2003, 31(14), 4109-4118 and Dellinger et al., J. Am. Chem. Soc., 2003, 125, 940-950), 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. 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.
N3′-P5′-phosphoramidates have been reported to exhibit both a high affinity towards a complementary RNA strand and nuclease resistance (Gryaznov et al., J. Am. Chem. Soc., 1994, 116, 3143-3144). N3′-P5′-phosphoramidates have been studied with some success in vivo to specifically down regulate the expression of the c-myc gene (Skorski et al., Proc. Natl. Acad. Sci., 1997, 94, 3966-3971; and Faira et al., Nat. Biotechnol., 2001, 19, 40-44).
Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050.
In some embodiments of the invention, oligomeric compounds may have one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular—CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— (known as a methylene(methylimino) or MMI backbone), —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —O—N(CH3)—CH2—CH2— (wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH2—). The MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Amide internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,602,240.
Some oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439.
Oligomeric compounds may also contain one or more substituted sugar moieties. Suitable compounds can comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Also suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Other oligonucleotides comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. One modification includes 2′-methoxyethoxy(2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group. A further modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O-(CH2)2—O—(CH2)2—N(CH3)2, also described in examples hereinbelow.
Other modifications include 2′-methoxy(2′-O—CH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2), 2′-allyl(2′-CH2—CH═CH2), 2′-O-allyl(2′-O—CH2—CH═CH2) and 2′-fluoro(2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. One 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Antisense compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; and, 6,147,200.
The terms used to describe the conformational geometry of homoduplex nucleic acids are “A Form” for RNA and “B Form” for DNA. In general, RNA:RNA duplexes are more stable and have higher melting temperatures (Tm's) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNA biases the sugar toward a C3′ endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. In addition, the 2′ hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleic acids prefer a C2′ endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.). As used herein, B-form geometry is inclusive of both C2′-endo pucker and O4′-endo pucker.
The structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533). Consequently, compounds that favor an A-form geometry can enhance stacking interactions, thereby increasing the relative Tm and potentially enhancing a compound's antisense effect.
In one aspect of the present invention oligomeric compounds include nucleosides synthetically modified to induce a 3′-endo sugar conformation. A nucleoside can incorporate synthetic modifications of the heterocyclic base, the sugar moiety or both to induce a desired 3′-endo sugar conformation. These modified nucleosides are used to mimic RNA-like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3′-endo conformational geometry.
There is an apparent preference for an RNA type duplex (A form helix, predominantly 3′-endo) as a requirement (e.g. trigger) of RNA interference which is supported in part by the fact that duplexes composed of 2′-deoxy-2′-F-nucleosides appears efficient in triggering RNAi response in the C. elegans system. Properties that are enhanced by using more stable 3′-endo nucleosides include but are not limited to: modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage. Also provided herein are oligomeric triggers of RNAi having one or more nucleosides modified in such a way as to favor a C3′-endo type conformation.
Nucleoside conformation is influenced by various factors including substitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2′ position to favor the 3′-endo conformation can be achieved while maintaining the 2′-OH as a recognition element (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3′-endo conformation can be achieved by deletion of the 2′-OH as exemplified by 2′deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3′-endo conformation positioning the electronegative fluorine atom in the axial position. Representative 2′-substituent groups amenable to the present invention that give A-form conformational properties (3′-endo) to the resultant duplexes include 2′-O-alkyl, 2′-O-substituted alkyl and 2′-fluoro substituent groups. Other suitable substituent groups are various alkyl and aryl ethers and thioethers, amines and monoalkyl and dialkyl substituted amines.
Other modifications of the ribose ring, for example substitution at the 4′-position to give 4′-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3′-endo conformation. Along similar lines, triggers of RNAi response might be composed of one or more nucleosides modified in such a way that conformation is locked into a C3′-endo type conformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENATM, Morita et al, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.)
It is further intended that multiple modifications can be made to one or more of the oligomeric compounds of the invention at multiple sites of one or more monomeric subunits (nucleosides are suitable) and or internucleoside linkages to enhance properties such as but not limited to activity in a selected application.
The synthesis of numerous of the modified nucleosides amenable to the present invention are known in the art (see for example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenum press). The conformation of modified nucleosides and their oligomers can be estimated by various methods routine to those skilled in the art such as molecular dynamics calculations, nuclear magnetic resonance spectroscopy and CD measurements.
Another group of oligomeric compounds includes oligonucleotide mimetics. The term “mimetic” as it is applied to oligonucleotides includes oligomeric compounds wherein the furanose ring or the furanose ring and the internucleotide linkage are replaced with novel groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid.
One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA) (Nielsen et al., Science, 1991, 254, 1497-1500). PNAs have favorable hybridization properties, high biological stability and are electrostatically neutral molecules. PNA compounds have been used to correct aberrant splicing in a transgenic mouse model (Sazani et al., Nat. Biotechnol., 2002, 20, 1228-1233). In PNA oligomeric compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases 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 oligomeric compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. PNA compounds can be obtained commercially from Applied Biosystems (Foster City, Calif., USA). Numerous modifications to the basic PNA backbone are known in the art; particularly useful are PNA compounds with one or more amino acids conjugated to one or both termini. For example, 1-8 lysine or arginine residues are useful when conjugated to the end of a PNA molecule.
Another class of oligonucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups have been selected to give a non-ionic oligomeric compound. Morpholino-based oligomeric compounds are non-ionic mimetics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based oligomeric compounds have been studied in zebrafish embryos (see: Genesis, volume 30, issue 3, 2001 and Heasman, J., Dev. Biol., 2002, 243, 209-214). Further studies of morpholino-based oligomeric compounds have also been reported (Nasevicius et al., Nat. Genet., 2000, 26, 216-220; and Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596). Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No. 5,034,506. The morpholino class of oligomeric compounds have been prepared having a variety of different linking groups joining the monomeric subunits. Linking groups can be varied from chiral to achiral, and from charged to neutral. U.S. Pat. No. 5,166,315 discloses linkages including —O—P(═O)(N(CH3)2)—O—; U.S. Pat. No. 5,034,506 discloses achiral intermorpholino linkages; and U.S. Pat. No. 5,185,444 discloses phosphorus containing chiral intermorpholino linkages.
A further class of oligonucleotide mimetic is referred to as cyclohexene nucleic acids (CeNA). In CeNA oligonucleotides, the furanose ring normally present in a DNA or RNA molecule is replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation. Furthermore the incorporation of CeNA into a sequence targeting RNA was stable to serum and able to activate E. coli RNase H resulting in cleavage of the target RNA strand.
A further modification includes bicyclic sugar moieties such as “Locked Nucleic Acids” (LNAs) in which the 2′-hydroxyl group of the ribosyl sugar ring is linked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage to form the bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (—CH2—) group bridging the 2′ oxygen atom and the 4′ carbon atom, for which the term LNA is used for the bicyclic moiety; in the case of an ethylene group in this position, the term ENATM is used (Singh et al., Chem. Commun., 1998, 4, 455-456; ENATM: Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). LNA and other bicyclic sugar analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradation and good solubility properties. LNAs are commercially available from ProLigo (Paris, France and Boulder, Colo., USA).
An isomer of LNA that has also been studied is alpha-L-LNA which has been shown to have superior stability against a 3′-exonuclease. The alpha-L-LNAs were incorporated into antisense gapmers and chimeras that showed potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
Another similar bicyclic sugar moiety that has been prepared and studied has the bridge going from the 3′-hydroxyl group via a single methylene group to the 4′ carbon atom of the sugar ring thereby forming a 3′-C,4′-C-oxymethylene linkage (see U.S. Pat. No. 6,043,060).
LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level. Introduction of 3 LNA monomers (T or A) significantly increased melting points (Tm=+15/+11° C.) toward DNA complements. The universality of LNA-mediated hybridization has been stressed by the formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking of LNA was reflected with regard to the N-type conformational restriction of the monomers and to the secondary structure of the LNA:RNA duplex.
LNAs also form duplexes with complementary DNA, RNA or LNA with high thermal affinities. Circular dichroism (CD) spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination of an LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands. DNALNA chimeras have been shown to efficiently inhibit gene expression when targeted to a variety of regions (5′-untranslated region, region of the start codon or coding region) within the luciferase mRNA (Braasch et al., Nucleic Acids Research, 2002, 30, 5160-5167).
Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638). The authors have demonstrated that LNAs confer several desired properties. LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. LNA/DNA copolymers exhibited potent antisense activity in assay systems as disparate as G-protein-coupled receptor signaling in living rat brain and detection of reporter genes in Escherichia coli. Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has also been accomplished. Further successful in vivo studies involving LNA's have shown knock-down of the rat delta opioid receptor without toxicity (Wahlestedt et al., Proc. Natl. Acad. Sci., 2000, 97, 5633-5638) and in another study showed a blockage of the translation of the large subunit of RNA polymerase II (Fluiter et al., Nucleic Acids Res., 2003, 31, 953-962).
The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
Analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.
Another oligonucleotide mimetic that has been prepared and studied is threose nucleic acid. This oligonucleotide mimetic is based on threose nucleosides instead of ribose nucleosides. Initial interest in (3′,2′)-alpha-L-threose nucleic acid (TNA) was directed to the question of whether a DNA polymerase existed that would copy the TNA. It was found that certain DNA polymerases are able to copy limited stretches of a TNA template (reported in Chemical and Engineering News, 2003, 81, 9). In another study it was determined that TNA is capable of antiparallel Watson-Crick base pairing with complementary DNA, RNA and TNA oligonucleotides (Chaput et al., J. Am. Chem. Soc., 2003, 125, 856-857).
In one study (3′,2′)-alpha-L-threose nucleic acid was prepared and compared to the 2′ and 3′ amidate analogs (Wu et al., Organic Letters, 2002, 4(8), 1279-1282). The amidate analogs were shown to bind to RNA and DNA with comparable strength to that of RNA/DNA.
Further oligonucleotide mimetics have been prepared to include bicyclic and tricyclic nucleoside analogs (see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002; and Renneberg et al., Nucleic acids res., 2002, 30, 2751-2757). These modified nucleoside analogs have been oligomerized using the phosphoramidite approach and the resulting oligomeric compounds containing tricyclic nucleoside analogs have shown increased thermal stabilities (Tm's) when hybridized to DNA, RNA and itself. Oligomeric compounds containing bicyclic nucleoside analogs have shown thermal stabilities approaching that of DNA duplexes.
Another class of oligonucleotide mimetic is referred to as phosphonomonoester nucleic acids which incorporate a phosphorus group in the backbone. This class of oligonucleotide mimetic is reported to have useful physical and biological and pharmacological properties in the areas of inhibiting gene expression (antisense oligonucleotides, sense oligonucleotides and triplex-forming oligonucleotides), as probes for the detection of nucleic acids and as auxiliaries for use in molecular biology. Further oligonucleotide mimetics amenable to the present invention have been prepared wherein a cyclobutyl ring replaces the naturally occurring furanosyl ring.
Oligomeric compounds can 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). A “substitution” is the replacement of an unmodified or natural base with another unmodified or natural base. “Modified” nucleobases mean other synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are known to those skilled in the art as suitable for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently suitable base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. It is understood in the art that modification of the base does not entail such chemical modifications as to produce substitutions in a nucleic acid sequence.
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; 5,681,941; and 5,750,692.
Oligomeric compounds of the present invention can also include polycyclic heterocyclic compounds in place of one or more of the naturally-occurring heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs. Representative cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include 1,3-diazaphenoxazine-2-one (Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one, (Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388). Incorporated into oligonucleotides these base modifications were shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions (also see U.S. Pre-Grant Publications 20030207804 and 20030175906).
Further helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid 1,3-diazaphenoxazine-2-one scaffold (Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). Binding studies demonstrated that a single incorporation could enhance the binding affinity of a model to its complementary target DNA or RNA with a ΔTm of up to 18° C. relative to 5-methyl cytosine (dC5me), which is a high affinity enhancement for a single modification. On the other hand, the gain in helical stability does not compromise the specificity of the oligonucleotides.
Further tricyclic heterocyclic compounds and methods of using them that are amenable to use in the present invention are disclosed in U.S. Pat. Nos. 6,028,183, and 6,007,992.
The enhanced binding affinity of the phenoxazine derivatives together with their uncompromised sequence specificity makes them valuable nucleobase analogs for the development of more potent antisense-based drugs. In fact, promising data have been derived from in vitro experiments demonstrating that heptanucleotides containing phenoxazine substitutions are capable to activate RNase H, enhance cellular uptake and exhibit an increased antisense activity (Lin, K-Y; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). The activity enhancement was even more pronounced in case of G-clamp, as a single substitution was shown to significantly improve the in vitro potency of a 20 mer 2′-deoxyphosphorothioate oligonucleotides (Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518).
Further modified polycyclic heterocyclic compounds useful as heterocyclic bases are disclosed in but 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; 5,434,257; 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; 5,646,269; 5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. Pre-Grant Publication 20030158403.
Another modification of the oligomeric compounds of the invention involves chemically linking to the oligomeric compound one or more moieties or conjugates which enhance the properties of the oligomeric compound, such as to enhance the activity, cellular distribution or cellular uptake of the oligomeric compound. 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. Nos. 6,287,860 and 6,762,169.
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 triethyl-ammonium 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. Oligomeric compounds of the invention may also be conjugated to drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. Pat. No. 6,656,730.
Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.
Oligomeric compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of an oligomeric compound to enhance properties such as for example nuclease stability. Included in stabilizing groups are cap structures. By “cap structure or terminal cap moiety” is meant chemical modifications, which have been incorporated at either terminus of oligonucleotides (see for example Wincott et al., WO 97/26270). These terminal modifications protect the oligomeric compounds having terminal nucleic acid molecules from exonuclease degradation, and can improve delivery and/or localization within a cell. The cap can be present at either the 5′-terminus (5′-cap) or at the 3′-terminus(3′-cap) or can be present on both termini of a single strand, or one or more termini of both strands of a double-stranded compound. This cap structure is not to be confused with the inverted methylguanosine “5′ cap” present at the 5′ end of native mRNA molecules. In non-limiting examples, the 5′-cap includes inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety; 1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al., International PCT publication No. WO 97/26270).
Particularly suitable 3′-cap structures include, for example 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5′-mercapto moieties (for more details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925).
Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an oligomeric compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.
It is not necessary for all positions in a given oligomeric compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even within a single nucleoside within an oligomeric compound.
The present invention also includes oligomeric compounds which are chimeric compounds. “Chimeric” oligomeric compounds or “chimeras,” in the context of this invention, are single- or double-stranded oligomeric compounds, such as oligonucleotides, which contain two or more chemically distinct regions, each comprising at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. Chimeric antisense oligonucleotides are one form of oligomeric compound. These oligonucleotides typically contain at least one region which is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, alteration of charge, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for RNAses or other enzymes. 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 when bound by a DNA-like oligomeric compound, 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 RNase III or RNAseL which cleaves both cellular and viral RNA. Cleavage products of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
Chimeric oligomeric compounds of the invention can be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, oligonucleotide mimetics, or regions or portions thereof. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922.
A “gapmer” is defined as an oligomeric compound, generally an oligonucleotide, having a 2′-deoxyoligonucleotide region flanked by non-deoxyoligonucleotide segments. The central region is referred to as the “gap.” The flanking segments are referred to as “wings.” While not wishing to be bound by theory, the gap of the gapmer presents a substrate recognizable by RNase H when bound to the RNA target whereas the wings do not provide such a substrate but can confer other properties such as contributing to duplex stability or advantageous pharmacokinetic effects. Each wing can be one or more non-deoxyoligonucleotide monomers (if one of the wings has zero non-deoxyoligonucleotide monomers, a “hemimer” is described). In one embodiment, the gapmer is a ten deoxynucleotide gap flanked by five non-deoxynucleotide wings. This is referred to as a 5-10-5 gapmer. Other configurations are readily recognized by those skilled in the art. In one embodiment the wings comprise 2′-MOE modified nucleotides. In another embodiment the gapmer has a phosphorothioate backbone. In another embodiment the gapmer has 2′-MOE wings and a phosphorothioate backbone. Other suitable modifications are readily recognizable by those skilled in the art.
Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
Oligomeric compounds of the present invention can 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.
The following precursor compounds, including amidites and their intermediates can be prepared by methods routine to those skilled in the art; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N4-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidine penultimate intermediate, (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methylcytidin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N6-benzoyladenosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), (5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4-isobutyrylguanosin-3′-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxy-ethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-((2-phthalimidoxy)ethyl)-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-((2-formadoximinooxy)ethyl)-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(N,N dimethylaminooxyethyl)-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite), 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-((2-cyanoethyl)-N,N-diisopropylphosphoramidite), 2′-dimethylaminoethoxyethoxy(2′-DMAEOE) nucleoside amidites, 2′-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
The preparation of such precursor compounds for oligonucleotide synthesis are routine in the art and disclosed in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743.
2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites can be purchased from commercial sources (e.g. Chemgenes, Needham, Mass. or Glen Research, Inc. Sterling, Va.). Other 2′-O-alkoxy substituted nucleoside amidites can be prepared as described in U.S. Pat. No. 5,506,351.
Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C) nucleotides can be synthesized routinely according to published methods (Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham, Mass.).
2′-fluoro oligonucleotides can be synthesized routinely as described (Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841) and U.S. Pat. No. 5,670,633.
2′-O-Methoxyethyl-substituted nucleoside amidites can be prepared routinely as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.
Aminooxyethyl and dimethylaminooxyethyl amidites can be prepared routinely as per the methods of U.S. Pat. No. 6,127,533.
Phosphorothioate-containing oligonucleotides (P═S) can be synthesized by methods routine to those skilled in the art (see, for example, Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press). Phosphinate oligonucleotides can be prepared as described in U.S. Pat. No. 5,508,270.
Alkyl phosphonate oligonucleotides can be prepared as described in U.S. Pat. No. 4,469,863.
3′-Deoxy-3′-methylene phosphonate oligonucleotides can be prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.
Phosphoramidite oligonucleotides can be prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
Alkylphosphonothioate oligonucleotides can be 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 can be prepared as described in U.S. Pat. No. 5,476,925.
Phosphotriester oligonucleotides can be prepared as described in U.S. Pat. No. 5,023,243.
Borano phosphate oligonucleotides can be prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198.
4′-thio-containing oligonucleotides can be synthesized as described in U.S. Pat. No. 5,639,873.
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═O or P═S linkages can be 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 can be prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564.
Ethylene oxide linked oligonucleosides can be prepared as described in U.S. Pat. No. 5,223,618.
Peptide nucleic acids (PNAs) can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, 5,719,262, 6,559,279 and 6,762,281.
Oligomeric compounds incorporating at least one 2′-O-protected nucleoside by methods routine in the art. After incorporation and appropriate deprotection the 2′-O-protected nucleoside will be converted to a ribonucleoside at the position of incorporation. The number and position of the 2-ribonucleoside units in the final oligomeric compound can vary from one at any site or the strategy can be used to prepare up to a full 2′-OH modified oligomeric compound.
The main RNA synthesis strategies that are presently being used commercially include 5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS), 5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP), 2′-O-[(triisopropylsilyl)oxy]methyl(2′-O—CH2—O—Si(iPr)3 (TOM), and the 5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). Some companies currently offering RNA products include Pierce Nucleic Acid Technologies (Milwaukee, Wis.), Dharmacon Research Inc. (a subsidiary of Fisher Scientific, Lafayette, Colo.), and Integrated DNA Technologies, Inc. (Coralville, Iowa). One company, Princeton Separations, markets an RNA synthesis activator advertised to reduce coupling times especially with TOM and TBDMS chemistries. Such an activator would also be amenable to the oligomeric compounds of the present invention.
All of the aforementioned RNA synthesis strategies are amenable to the oligomeric compounds of the present invention. Strategies that would be a hybrid of the above e.g. using a 5′-protecting group from one strategy with a 2′-O-protecting from another strategy is also contemplated herein.
(2′-O-Me)-(2′-deoxy)-(2′-O-Me) Chimeric Phosphorothioate Oligonucleotides
Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments can be routinely synthesized by one skilled in the art, using, for example, an Applied Biosystems automated DNA synthesizer Model 394. Oligonucleotides can be synthesized using an automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for the 2′-O-alkyl portion. In one nonlimiting example, the standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH4OH) for 12-16 hr at 55° C. The deprotected oligonucleotide is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo) and analyzed by methods routine in the art.
(2′-O-(2-Methoxyethyl))-(2′-deoxy)-(2′-O-(2-Methoxyethyl)) Chimeric Phosphorothioate Oligonucleotides
(2′-O-(2-methoxyethyl))-(2′-deoxy)-(-2′-O-(2-methoxyethyl)) chimeric phosphorothioate oligonucleotides can be 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
(2′-O-(2-methoxyethyl phosphodiester)-(2′-deoxy phosphorothioate)-(2′-O-(methoxyethyl)phosphodiester) chimeric oligonucleotides can be prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.
Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides can be synthesized according to U.S. Pat. No. 5,623,065.
Methods of oligonucleotide purification and analysis are known to those skilled in the art. Analysis methods include capillary electrophoresis (CE) and electrospray-mass spectroscopy. Such synthesis and analysis methods can be performed in multi-well plates.
Modulation of expression of a target nucleic acid can be achieved through alteration of any number of nucleic acid (DNA or RNA) functions. “Modulation” means a perturbation of function, for example, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in expression. As another example, modulation of expression can include perturbing splice site selection of pre-mRNA processing. “Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. These structures include the products of transcription and translation. “Modulation of expression” means the perturbation of such functions. The functions of DNA to be modulated 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 modulated can include translocation functions, which include, but are not limited to, 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, and translation of protein from the RNA. RNA processing functions that can be modulated include, but are not limited to, splicing of the RNA to yield one or more RNA species, capping of the RNA, 3′ maturation of the RNA and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. Modulation of expression can result in the increased level of one or more nucleic acid species or the decreased level of one or more nucleic acid species, either temporally or by net steady state level. One result of such interference with target nucleic acid function is modulation of the expression of MyD88. Thus, in one embodiment modulation of expression can mean increase or decrease in target RNA or protein levels. In another embodiment modulation of expression can mean an increase or decrease of one or more RNA splice products, or a change in the ratio of two or more splice products.
Modulation of MyD88 expression can be assayed in a variety of ways known in the art. MyD88 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA by methods known in the art. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.
Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISMTM 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
Levels of a protein encoded by MyD88 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to a protein encoded by MyD88 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997. Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.
Once one or more target regions, segments or sites have been identified, oligomeric compounds are designed which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. The oligomeric compounds of the present invention can be targeted to features of a target nucleobase sequence.
The locations on the target nucleic acid to which active oligomeric compounds hybridize are hereinbelow referred to as “validated target segments.” As used herein the term “validated target segment” is defined as at least an 8-nucleobase portion of a target region to which an active oligomeric 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.
Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of a validated target segment (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, or about 13 to about 80, or about 12 to about 50, or about 12 to about 30, or about 15 to about 30, or about 20 to about 30, or about 20 to about 24, or about 16 to about 20 nucleobases). Similarly validated target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of a validated target segment (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, or about 13 to about 80, or about 12 to about 50, or about 12 to about 30, or about 15 to about 30, or about 20 to about 30, or about 20 to about 24, or about 16 to about 20 nucleobases). It is also understood that a validated oligomeric target segment can be represented by DNA or RNA sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of a validated target segment, and can extend in either or both directions until the oligonucleotide contains about 8 to about 80, or about 13 to about 80, or about 12 to about 50, or about 12 to about 30, or about 15 to about 30, or about 20 to about 30, or about 20 to about 24, or about 16 to about 20 nucleobases.
In another embodiment, the validated target segments identified herein can be employed in a screen for additional compounds that modulate the expression of MyD88. “Modulators” are those compounds that modulate the expression of MyD88 and which comprise at least an 8-nucleobase portion which is complementary to a validated target segment. The screening method comprises the steps of contacting a validated target segment of a nucleic acid molecule encoding MyD88 with one or more candidate modulators, and selecting for one or more candidate modulators which perturb the expression of a nucleic acid molecule encoding MyD88. Once it is shown that the candidate modulator or modulators are capable of modulating the expression of a nucleic acid molecule encoding MyD88, the modulator can then be employed in further investigative studies of the function of MyD88, or for use as a research, diagnostic, or therapeutic agent. The validated target segments can also be combined with a second strand as disclosed herein to form stabilized double-stranded (duplexed) oligonucleotides for use as a research, diagnostic, or therapeutic agent.
The oligomeric compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense compounds, which are able to inhibit gene expression with specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
For use in kits and diagnostics, the oligomeric compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
As one nonlimiting example, expression patterns within cells or tissues treated with one or more compounds or compositions of the present invention are compared to control cells or tissues not treated with compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.
Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).
Compounds of the invention can be used to modulate the expression of MyD88 in an animal, such as a human. In one non-limiting embodiment, the methods comprise the step of administering to said animal an effective amount of an antisense compound that inhibits expression of MyD88. In one embodiment, the antisense compounds of the present invention effectively inhibit the levels or function of MyD88 RNA. Because reduction in MyD88 mRNA levels can lead to alteration in MyD88 protein products of expression as well, such resultant alterations can also be measured. Antisense compounds of the present invention that effectively inhibit the levels or function of MyD88 RNA or protein products of expression are considered active antisense compounds. In one embodiment, the antisense compounds of the invention inhibit the expression of MyD88 causing a reduction of RNA 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%.
In another non-limiting embodiment, the methods comprise the step of administering to said animal an effective amount of an antisense compound that modulates splicing of MyD88 pre-mRNA.
For example, the reduction of the expression of MyD88 mRNA products can be measured in a bodily fluid, tissue or organ of the animal. Bodily fluids include, but are not limited to, blood (serum or plasma), lymphatic fluid, cerebrospinal fluid, semen, urine, synovial fluid and saliva and can be obtained by methods routine to those skilled in the art. Tissues or organs include, but are not limited to, blood (e.g., hematopoietic cells, such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34+ cells CD4+ cells), lymphocytes and other blood lineage cells, skin, bone marrow, spleen, thymus, lymph node, brain, spinal cord, heart, skeletal muscle, liver, pancreas, prostate, kidney, lung, oral mucosa, esophagus, stomach, ilium, small intestine, colon, bladder, cervix, ovary, testis, mammary gland, adrenal gland, and adipose (white and brown). Samples of tissues or organs can be routinely obtained by biopsy. In some alternative situations, samples of tissues or organs can be recovered from an animal after death.
The cells contained within said fluids, tissues or organs being analyzed can contain a nucleic acid molecule encoding MyD88 protein and/or the MyD88-encoded protein itself. For example, fluids, tissues or organs procured from an animal can be evaluated for expression levels of the target mRNA or protein. mRNA levels can be measured or evaluated by real-time PCR, Northern blot, in situ hybridization or DNA array analysis. Protein levels can be measured or evaluated by ELISA, immunoblotting, quantitative protein assays, protein activity assays (for example, caspase activity assays) immunohistochemistry or immunocytochemistry. Furthermore, the effects of treatment can be assessed by measuring biomarkers associated with the target gene expression in the aforementioned fluids, tissues or organs, collected from an animal contacted with one or more compounds of the invention, by routine clinical methods known in the art. These biomarkers include but are not limited to: glucose, cholesterol, lipoproteins, triglycerides, free fatty acids and other markers of glucose and lipid metabolism; liver transaminases, bilirubin, albumin, blood urea nitrogen, creatine and other markers of kidney and liver function; interleukins, tumor necrosis factors, intracellular adhesion molecules, C-reactive protein and other markers of inflammation; testosterone, estrogen and other hormones; tumor markers; vitamins, minerals and electrolytes.
The compounds of the present invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. In one aspect, the compounds of the present invention inhibit the expression of MyD88. The compounds of the invention can also be used in the manufacture of a medicament for the treatment of diseases and disorders related to MyD88 expression.
Methods whereby bodily fluids, organs or tissues are contacted with an effective amount of one or more of the antisense compounds or compositions of the invention are also contemplated. Bodily fluids, organs or tissues can be contacted with one or more of the compounds of the invention resulting in modulation of MyD88 expression in the cells of bodily fluids, organs or tissues. An effective amount can be determined by monitoring the modulatory effect of the antisense compound or compounds or compositions on target nucleic acids or their products by methods routine to the skilled artisan. Further contemplated are ex vivo methods of treatment whereby cells or tissues are isolated from a subject, contacted with an effective amount of the antisense compound or compounds or compositions and reintroduced into the subject by routine methods known to those skilled in the art.
The oligomeric compounds of the present invention comprise any pharmaceutically acceptable salts, esters, or salts of such esters, or any other functional chemical equivalent which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the oligomeric compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
The term “prodrug” indicates a therapeutic agent that is prepared in an inactive or less active form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE ((S-acetyl-2-thioethyl)phosphate) derivatives according to the methods disclosed in WO 93/24510 or WO 94/26764.
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.
The oligomeric compounds of the invention may also be formulated with active or inert ingredients, or a combination of both, for delivery via parenteral and non-parenteral routes of administration. Compositions and methods of preparing formulations are well known to those skilled in the art.
While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds of 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.
Example 1
Cell Culture and Treatment with Oligomeric Compounds
The effect of oligomeric compounds on target nucleic acid expression was tested in A549, T24 or b.END cells. The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (Manassas, Va.). A549 cells are routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum, 100 units per ml penicillin, and 100 micrograms per ml streptomycin (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 5000 cells/well for use in oligomeric compound transfection experiments.
The transitional cell bladder carcinoma cell line T24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T24 cells are routinely cultured in complete McCoy's 5A basal media (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 μg/mL (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for treatment with the compound of the invention.
The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany). b.END cells are routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 3000 cells/well for use in oligomeric compound transfection experiments.
RAW264.7 cells were obtained from the American Type Tissue Culture Collection, Rockville, Md.) and 293FT cells were obtained from Invitrogen Corp. (Carlsbad, Calif.). The cells were cultivated in DMEM supplemented with 10% fetal bovine serum.
When cells reach appropriate confluency, they are treated with oligonucleotide using LipofectinTM as described. When cells reached 65-75% confluency, they were treated with oligonucleotide. Oligonucleotide was mixed with LIPOFECTINTM Invitrogen Life Technologies, Carlsbad, Calif.) in Opti-MEMTM-1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired concentration of oligonucleotide and a LIPOFECTINTM concentration of 2.5 or 3 μg/Ml per 100 Nm oligonucleotide. This transfection mixture was incubated at room temperature for approximately 0.5 hours. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEMTM-1 and then treated with 130 μL of the transfection mixture. Cells grown in 24-well plates or other standard tissue culture plates are treated similarly, using appropriate volumes of medium and oligonucleotide. Cells are treated and data are obtained in duplicate or triplicate. After approximately 4-7 hours of treatment at 37° C., the medium containing the transfection mixture was replaced with fresh culture medium. Cells were harvested 16-24 hours after oligonucleotide treatment.
The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) 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 H-ras 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 25 nM to 300 nM when the antisense oligonucleotide is transfected using a liposome reagent and 1 μM to 40 μM when the antisense oligonucleotide is transfected by electroporation.
Example 2
Analysis of Oligonucleotide Inhibition of MyD88 Expression
Antisense modulation of MyD88 expression can be assayed in a variety of ways known in the art. For example, MyD88 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. Methods of RNA isolation are taught in, for example, Ausubel, F. M et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISMTM 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
Protein levels of MyD88 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to MyD88 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.
Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.
Example 3
Poly(A)+ mRNA Isolation
Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.
Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.
Example 4
Total RNA Isolation
Total RNA was isolated using an RNEASY 96TM kit and buffers purchased from Qiagen Inc. (Valencia Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96TM well plate attached to a QIAVACTM manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96TM plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96TM plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAVACTM manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVACTM manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.
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 5
Real-Time Quantitative PCR Analysis of MyD88 mRNA Levels
Quantitation of MyD88 mRNA levels was determined by real-time quantitative PCR using the ABI PRISMTM 7700 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., JOE, FAM, or VIC, obtained from either Operon Technologies Inc. (Alameda, Calif.) or PE-Applied Biosystems (Foster City, Calif.)) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc. or PE-Applied Biosystems) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISMTM 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.
PCR reagents were obtained from PE-Applied Biosystems. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1× TAQMANTM buffer A, 5.5 mM MgCl2, 300 μM each of dATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLDTM, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the AMPLITAQ GOLDTM, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreenTM RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreenTM are taught in Jones, L. J., et al, Analytical Biochemistry, 1998, 265, 368-374.
In this assay, 175 μL of RiboGreenTM working reagent (RiboGreenTM reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.
Probes and primers to human MyD88 were designed to hybridize to a human MyD88 sequence, using published sequence information (a truncated version containing bases 86001 to 91000 of GenBank accession number AB026898.1, incorporated herein as SEQ ID NO: 3). For human MyD88 the PCR primers were:
forward primer: GGGCTGCTTTTCATTTCCAC (SEQ ID NO: 4)
reverse primer: AGGCCAGGATCCCTTCTCAT (SEQ ID NO: 5) and the PCR probe was: FAM-AGGATGCCTGTGGTCATGCTCTCAGC-TAMRA
(SEQ ID NO: 6) where FAM (PE-Applied Biosystems) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems) is the quencher dye. For human GAPDH the PCR primers were:
forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 7)
reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where JOE (PE-Applied Biosystems) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems) is the quencher dye.
Example 6
Northern Blot Analysis of MyD88 mRNA Levels
Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOLTM (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBONDTM-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N. J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALNKERTM UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYBTM hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.
To detect human MyD88, a human MyD88 specific probe was prepared by PCR using the forward primer GGGCTGCTTTTCATTTCCAC (SEQ ID NO: 4) and the reverse primer AGGCCAGGATCCCTTCTCAT (SEQ ID NO: 5). 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.).
Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGERTM and IMAGEQUANTTM Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.
Example 7
Antisense Inhibition of Human MyD88 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap
In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human MyD88 RNA, using published sequences (residues 86001-91000 from GenBank accession number AB026898.1, incorporated herein as SEQ ID NO: 3, GenBank accession number AL526415.1, incorporated herein as SEQ ID NO: 10, and GenBank accession number NM—002468.1, incorporated herein as SEQ ID NO: 11). The oligonucleotides are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human MyD88 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”.







TABLE 2







Inhibition of human MyD88 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
INHIBITION
NO
















190933
5′UTR
3
9
gtcgcattgtctgccagcgc
0
12


 


190935
5′UTR
3
14
gtcgggtcgcattgtctgcc
16
13


 


190937
5′UTR
3
31
cctggagcctcagcgcggtc
59
14


 


190939
5′UTR
3
36
gcggtcctggagcctcagcg
55
15


 


190941
Start
3
45
ccatggcgggcggtcctgga
42
16



Codon







 


190943
Start
3
53
tcctgcagccatggcgggcg
43
17



Codon







 


190945
Start
3
60
cgggacctcctgcagccatg
81
18



Codon







 


190947
Coding
3
122
catgttgagagcagccaggg
27
19


 


190949
Coding
3
128
cactcgcatgttgagagcag
65
20


 


190951
Coding
3
156
cgttcaagaacagagacagg
10
21


 


190953
Coding
3
164
tgtccgcacgttcaagaaca
35
22


 


190955
Coding
3
214
tactcaaagtccatctcctc
0
23


 


190957
Coding
3
220
tccaagtactcaaagtccat
53
24


 


190959
Coding
3
230
ttgccggatctccaagtact
81
25


 


190961
Coding
3
238
gtctccagttgccggatctc
87
26


 


190963
Coding
3
269
gtccagcagcctgccagtgg
75
27


 


190965
Coding
3
308
cagtcggcctacagaggcgc
65
28


 


190967
Coding
3
317
cagctcgagcagtcggccta
54
29


 


190969
Coding
3
323
ggtaagcagctcgagcagtc
30
30


 


190971
Coding
3
332
gcccagcttggtaagcagct
84
31


 


190973
Coding
3
338
gtcgcggcccagcttggtaa
45
32


 


190975
Coding
3
371
ctcaatgctgggtcccagct
69
33


 


190977
Coding
3
382
tggcaatcctcctcaatgct
41
34


 


190979
Coding
3
401
ctgcttcaagatatactttt
76
35


 


190981
Coding
3
416
agcctcctcctgctgctgct
79
36


 


190983
Coding
3
425
aggcttctcagcctcctcct
84
37


 


190985
Coding
3
436
gccacctgtaaaggcttctc
80
38


 


190987
Coding
3
451
ctgctgtctacagcggccac
57
39


 


190989
Coding
3
466
gctgtccgtgggacactgct
84
40


 


190991
Coding
3
473
cagctctgctgtccgtggga
57
41


 


190993
Coding
3
494
atcaagtgtggtgatgcccg
56
42


 


190995
Coding
3
520
cgctcaggcatatgccccag
52
43


 


190997
Coding
3
544
caatagcagatgaaggcatc
66
44


 


190998
Coding
3
578
gatcatctcctgcacaaact
77
45


 


190999
Coding
3
583
tgccggatcatctcctgcac
23
46


 


191000
Coding
3
588
ccagttgccggatcatctcc
16
47


 


191001
Coding
3
604
cgatagtttgtctgttccag
71
48


 


191002
Coding
3
610
ttcagtcgatagtttgtctg
32
49


 


191003
Coding
3
627
ggtcagacacacacaacttc
52
50


 


191004
Coding
3
636
ggacatcgcggtcagacaca
45
51


 


191005
Coding
3
660
tagaccagacacaggtgcca
81
52


 


191006
Coding
3
670
tcactagcaatagaccagac
44
53


 


191007
Coding
3
678
cgatgagctcactagcaata
66
54


 


191008
Coding
3
688
cacctcttttcgatgagctc
62
55


 


191009
Coding
3
697
atccggcggcacctcttttc
58
56


 


191010
Coding
3
718
tcatcagagacaaccaccac
0
57


 


191011
Coding
3
724
aggtaatcatcagagacaac
48
58


 


191012
Coding
3
729
tctgcaggtaatcatcagag
83
59


 


191013
Coding
3
734
cttgctctgcaggtaatcat
84
60


 


191014
Coding
3
745
aagtcacattccttgctctg
75
61


 


191015
Coding
3
750
tctggaagtcacattccttg
91
62


 


191016
Coding
3
757
aatttggtctggaagtcaca
27
63


 


191017
Coding
3
762
gtgcaaatttggtctggaag
79
64


 


191018
Coding
3
771
agaggctgagtgcaaatttg
53
65


 


191019
Coding
3
778
cctggagagaggctgagtgc
75
66


 


191020
Coding
3
784
tgggcacctggagagaggct
80
67


 


191021
Coding
3
795
gtcgcttctgatgggcacct
79
68


 


191022
Coding
3
822
tcattgccttgtacttgatg
81
69


 


191023
Coding
3
856
gtgatgaacctcaggatgct
74
70


 


191024
Coding
3
868
tagtcgcagacagtgatgaa
37
71


 


191025
Coding
3
873
tggtgtagtcgcagacagtg
78
72


 


191026
Coding
3
900
tccagaaccaagatttggtg
88
73


 


191027
Coding
3
907
aggcgagtccagaaccaaga
69
74


 


191028
Coding
3
912
tggcaaggcgagtccagaac
84
75


 


191029
Coding
3
918
aggccttggcaaggcgagtc
64
76


 


191030
Coding
3
924
gggacaaggccttggcaagg
45
77


 


191031
5′UTR
2
2
gggtgcccacctctaccctt
0
78


 


191032
5′UTR
2
33
tctggagccccgagcaaaag
0
79


 


191033
5′UTR
2
38
tacaatctggagccccgagc
0
80


 


191034
5′UTR
2
47
gccctgccctacaatctgga
0
81


 


191035
Exon 1:
1
753
acgtcctcaccaatgctggg
51
82



Intron 1







 


191036
Intron 1
1
952
tccgcctatccggacctttc
36
83


 


191037
Intron 1
1
1037
aaacagcccagatgccccca
29
84


 


191038
Intron 1
1
1058
cctgtcctgttgctctactt
52
85


 


191039
Exon 3
1
2293
tctagccaacctcttttcga
35
86


 


191040
Exon 4:
1
2573
ttgagcttacctggagagag
20
87



Intron 4







 


191041
Intron 4
1
2694
gctcaggtgtgcagagatct
64
88


 


191042
Intron 4:
1
2856
tgatgggcacctgcaagcca
47
89



Exon 5









As shown in Table 2, SEQ ID NOs 14, 15, 16, 17, 18, 20, 24, 25, 26, 27, 28, 29, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 48, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 72, 73, 74, 75, 76, 77, 82, 85, 88 and 89 demonstrated at least 42% inhibition of human MyD88 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.
Example 8
Western Blot Analysis of MyD88 Protein Levels
Whole cell extracts were prepared by lysing cells in RIPA buffer (1×PBS, 1% NP40, 0.1% deoxyolate, 0.1% SDS) containing complete protease inhibitor mix (Boehringer Mannheim). Protein concentration of the cell extracts was measured by Bradford assay (BioRad # 500-0201). Equal amounts of protein (10-20 μg) were resolved on a NuPAGE Novex 10% Bis-Tris gel in MES running buffer (Invitrogen) and transferred to PVDF membranes (Invitrogen). The membranes were blocked for 1 hour in TBS containing 0.05% Tween-20 (TBST) and 5% milk powder. After overnight incubation at 4° C. with a 1:500 dilution of a rabbit polyclonal MyD88 antibody (Abcam, ab2064 or eBioscience, 14-6223), the membranes were washed in PBS containing 0.05% Tween-20 and incubated with a 1:5000 dilution of goat anti-rabbit horseradish peroxidase conjugated antibody in blocking buffer. Membranes were washed and developed using Enhanced Chemiluminescent (ECL) detection system (Amersham). Subsequently membranes were blocked for 2 hours at room temperature in TBST plus 5% milk powder. After incubation at room temperature with a 1:5000 dilution of a mouse monoclonal tubulin antibody (Sigma #T-5168), the membranes were washed in PBS containing 0.1% Tween-20 and incubated with a 1:5000 dilution of goat anti-mouse horseradish peroxidase conjugated antibody in blocking buffer and developed as detailed above. Blots were quantitated by laser scanning densitometry.
Example 9
Analysis of RNA for Studies of MyD88 Splicing Modulation
Total RNA was harvested at the indicated times following the initiation of transfection using an RNeasy mini prep kit (Qiagen, Valencia, Calif.) according to the manufacturers protocol. For Northern blots, RNA was separated on 1.2% agarose gels containing 1.1% formaldehyde, then transferred to HyBond membranes (Amersham). Blots were hybridized with [32P]dCTP random prime labeled cDNA probes specific for MyD88 exon II (bases 360-494 of Genbank Accession No. U70451, incorporated herein as SEQ ID NO: 90) or MyD88 exon V (bases 1138-1377 of Genbank Accession No. U70451) for 2 hours in Rapid-hyb solution (Amersham). Blots were washed with 2×SSC containing 0.1% SDS at room temperature, followed by 0.1×SSC containing 0.1% SDS at 60° C. Polysomes were isolated from cells as detailed by Laroia et al. (1999, Science 284:499-502). PolyA RNA was purified using a Dynabeads mRNA DIRECT kit according to the manufacturers protocol (Dyna1 Biotech # 610-11).
Quantitative RT-PCR (qRT-PCR) was performed by methods well known to those of skill in the art (Winer et al. 1999, Anal. Biochem. 270:41-49). Briefly, 200 ng of total RNA was analyzed in a final volume of 50 μl containing 200 nM gene specific PCR primers, 0.2 mM of each dNTP, 75 nM fluorescently labeled oligonucleotide probe, 1× RT-PCR buffer, 5 mM MgCl2, 2U Platinum Taq DNA Polymerase (Invitrogen), and 8U ribonuclease inhibitor. Reverse transcription was performed for 30 minutes at 48° C. followed by PCR, which included 40 thermal cycles of 30 s at 94° C. and 1 minute at 60° C., using an ABI Prism 7700 Sequence Detector (Foster City, Calif.). Probes and primers to human and mouse MyD88 were designed to hybridize to a human MyD88 sequence (Genbank Accession No. U70451, incorporated herein as SEQ ID NO: 90) or a mouse MyD88 sequence (Genbank Accession No. NM—010851, incorporated herein as SEQ ID NO: 91), using published sequence information. Primers and probes were also designed for hybridization with human ICAM-1 (Genbank Accession No. J03132), human IL-8 (Genbank Accession No. M28130), mouse TNF-α (Genbank Accession No. M13049) and mouse SAA-1 (Genbank Accession No. NM—009117). All quantitative RT-PCR data was normalized to expression of non-target gene human C-raf kinase (Genbank Accession No. X03484), or mouse C-raf kinase (Genbank Accession No. AB057663). The sequences and target genes of all primers and probes are shown in Table 3.







TABLE 3







Primers and probes for quantitative RT-PCR









SEQ











Target

Sequence

ID


Name
Species
Description
Sequence (5′ to 3′)
NO














MyD88
Human
Forward
CAGAGGAGGATTGCCAAAAG
92


exon II

primer




 


MyD88
Human
Reverse
GGGGTCATCAAGTGTGGTG
93


exon II

primer




 


MyD88
Human
Probe
GCAGTGTCCCCACGGACAGCA
94


exon II






 


MyD88
Human
Forward
TGCCCTGAAGACTGTTCTGA
95


exon V

primer




 


MyD88
Human
Reverse
ACTGGTTCCATGCAGGACAT
96


exon V

primer




 


MyD88
Human
Probe
TGTCTGCCTGTCCATGTACTTC
97


exon V






 


MyD88
Mouse
Forward
CACTCGAGTTTGTTGGATG
98


exon II

primer




 


MyD88
Mouse
Reverse
CCACCTGTAAAGGCTTCTCG
99


exon II

primer




 


MyD88
Mouse
Probe
GCTCGTAGAGCTGCTGGCCTTG
100


exon II






 


MyD88
Mouse
Forward
GATGGTGGTGGTTGTTTCTG
101


exon V

primer




 


MyD88
Mouse
Reverse
CTTGGTGCAAGGGTTGGTAT
102


exon V

primer




 


MyD88
Mouse
Probe
TCAGCCTGTCTCCAGGTGTCCA
103


exon V






 


ICAM-1
Human
Forward
CATAGAGACCCCGTTGCCTAAA
104




primer




 


ICAM-1
Human
Reverse
TGGCTATCTTCTTGCACATTGC
105




primer




 


ICAM-1
Human
Probe
CTCCTGCCTGGGAACAACCGGAA
106


 


IL-8
Human
Forward
GAAGGAACCATCTCACTGTGTGTAA
107




primer




 


IL-8
Human
Reverse
AAATCAGGAAGGCTGCCAAGA
108




primer




 


IL-8
Human
Probe
CATGACTTCCAAGCTGGCCGTGG
109


 


TNF-α
Mouse
Forward
TCTCTTCAAGGGACAAGGCTG
110




primer




 


TNF-α
Mouse
Reverse
GATAGCAAATCGGCTGACGG
111




primer




 


TNF-α
Mouse
Probe
CCCGACTACGTGCTCCTCAGCCAC
112


 


SAA-1
Mouse
Forward
GCTGACCAGGAAGCCAACAG
113




primer




 


SAA-1
Mouse
Reverse
CAGGCAGTCCAGGAGGTCTG
114




primer




 


SAA-1
Mouse
Probe
CATGGCCGCAGTGGCAAAGACC
115


 


C-raf
Human
Forward
AGCTTGGAAGACGATCAGCAA
116




primer




 


C-raf
Human
Reverse
AAACTGCTGAACTATTGTAGGAGAGATG
117




primer




 


C-raf
Human
Probe
AGATGCCGTGTTTGATGGCTCCAGC
118


 


C-raf
Mouse
Forward
TTGTTCAGCAGTTTGGCTATCAG
119




primer




 


C-raf
Mouse
Reverse
AAACCCGGATAGTATTGCTTGTCT
120




primer




 


C-raf
Mouse
Probe
CAGATGATGGCAAGCTCACGGATTCTTCT
121









For standard RT-PCR reactions, 5 μg of total RNA was reverse transcribed in the presence of oligo dT using SuperScriptII reverse transcriptase according to the manufacturer's protocol (Invitrogen). Following a one hour incubation at 42° C., the cDNA was diluted by the addition of 80 μl of water. Three μl of the diluted cDNA was combined with 15 μl HotStarTaq mix (Qiagen) and 2.5 μl each of 10 μM forward and reverse PCR primer in a final volume of 30 μl. The PCR reaction was the cycled 30 seconds at 94° C., 30 seconds at 72° C. and 2 minutes at 60° C. with 35 repetitions. Primers used for standard RT-PCR reactions are shown in Table 4.







TABLE 4







Primers for standard RT-PCR









SEQ











Target

Sequence

ID


Name
Species
Description
Sequence (5′ to 3′)
NO














MyD88
Human
Forward
CGGCAACTGGAGACACAAG
122




primer




 


MyD88
Human
Reverse
TCTGGAAGTCACATTCCTTGC
123




primer




 


MyD88
Mouse
Forward
CACTCGCAGTTTGTTGGATG
124




primer




 


MyD88
Mouse
Reverse
TCTGGAAGTCACATTCCTTGC
123




primer









The expected PCR product for MyD88L is 525 bp. MyD88 exon II is 135 bp in length, therefore the expected length for the MyD88S PCR fragment is 390 bp. Products were visualized by electrophoresis on 2% agarose gels stained with ethidium bromide.
Example 10
NF-κB Luciferase Assays
293FT cells were seeded in 24-well tissue culture plates at 40,000 cells/well. The following day, cells were transfected with oligonucleotide in the presence of Lipofectin Reagent as described in previous examples herein. Following an overnight incubation, pNFκB-Luc (BD Biosciences) and pRL-CMV (Promega) plasmids and were introduced into the cells using SuperFect Reagent (Qiagen) according to the manufacturer's protocol. The following morning cells were stimulated with 30 ng/ml IL-1β or 15 ng/ml TNF-α for 4 hours then harvested in 120 μl of Passive Lysis Buffer (Promega). 30 μl of lysate was added to each well of a black 96 well plate, followed by Photinus and Renilla luciferase activity measured using a Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Luminescence was measured using a Packard TopCount. pNFκB-Luc activity was normalized to pRL-CMV activity.
Example 11
RAW264.7 Activation Assays
RAW264.7 cells were suspended at 107 cells/mL in DMEM+10% FCS. 90 μl of the cell suspension was transferred to a 90 μl electroporation cuvette to which ASOs were added at indicated concentrations. Cells were pulsed at 90V for 6 mS using a BTX electroporator, then transferred to 1 ml media and incubated 48 hours. Cells were then stimulated for 6 hours with 10 μg/mL LPS or 5 μM CpG oligonucleotide ISIS 12449 (ACCGATAACGTTGCCGGTGACG; SEQ ID NO: 125) for 4 hours. NF-κB activity was determined by accessing TNF-α mRNA levels in treated cells by quantitative RT-PCR as described in previous examples herein. All values were normalized to expression of C-raf kinase, a non-target gene.
Example 12
Animal Studies
Twelve-week old male C57 BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, Me.) and maintained in compliance with Isis Institutional Animal Care and Use Committee Guidelines in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility. Mice were injected intraperitoneally with 10-50 mg/kg of oligonucleotide in 200 μL of saline solution, every 2-3 days for a total of 4 to 9 doses. Where indicated, mice were challenged with 0.1 μg of recombinant mouse IL-1β (R&D Systems, Minneapolis, Minn.) in 200 μL of saline by intravenous injection twenty-four hours after last dose of oligonucleotide. Liver and spleen were collected from mice under anesthesia at 2 hours after IL-1β challenge or 24 hour after the final oligonucleotide dose and samples prepared for RNA and western blot analyses.
Example 13
Design and Screening of Modified Oligomeric Compounds for Modulation of MyD88 Splicing
MyD88 mRNA has been shown to undergo alternative splicing in response to such signals as LPS stimulation, resulting in production of the short form of MyD88 (MyD88S). The short form, which results from skipping of exon II, acts as a dominant negative inhibitor of IL-1R signaling. Altering the ratio of MyD88 splice products to increase the level of MyD88S would function to decrease the cellular inflammatory response.
To modulate splicing of MyD88 pre-mRNA, a series of antisense compounds was designed to hybridize with either human MyD88 (Genbank Accession No. NT—037565.3; SEQ ID NO: 146) or mouse MyD88 (Genbank Accession No. NT—039482.1; SEQ ID NO: 147). Shown in Table 5 and Table 6 are antisense oligonucleotides (splice switching oligonucleotides) targeting either the exon II acceptor site or exon II donor site of MyD88. Exon II of human MyD88 encompasses nucleotides 1224-1358 of SEQ ID NO: 146 and exon II of mouse MyD88 encompasses nucleotides 1331-1465 of SEQ ID NO: 147. Each compound is 20 nucleobases in length and uniformly modified with 2′-O-(2-methoxyethyl) nucleotides at each position. The internucleoside (backbone) linkages are phosphorothioate throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. Bold residues indicate those complementary to MyD88 intronic sequence. The start site for each oligonucleotide (5′-most nucleotide of the target sequence to which the oligonucleotide binds) is shown relative to the exon II splice junction.







TABLE 5







Human MyD88 splice switching oligonucleotides









SEQ












Splice Site
Start

ID


ISIS #
Target
Site
Sequence (5′ to 3′)
NO














337840
Acceptor
−20
CTGTGGGGAAGAGACAGAGT
126


 


337843
Acceptor
−15
CTCCTCTGTGGGGAAGAGAC
127


 


337842
Acceptor
−10
CAATCCTCCTCTGTGGGGAA
128


 


337844
Acceptor
−5
TTTGGCAATCCTCCTCTGTG
129


 


337841
Acceptor
0
ATACTTTTGGCAATCCTCCT
130


 


337845
Donor
−20
CCAGGGGGTCATCAAGTGTG
131


 


337848
Donor
−15
CTTACCCAGGGGGTCATCAA
132


 


337847
Donor
−10
GGACCCTTACCCAGGGGGTC
133


 


337849
Donor
−5
GTATTGGACCCTTACCCAGG
134


 


337846
Donor
0
GAACAGTATTGGACCCTTAC
135
















TABLE 6







Mouse MyD88 splice switching oligonucleotides









SEQ












Splice Site
Start

ID


ISIS #
Target
Site
Sequence (5′ to 3′)
NO














337850
Acceptor
−20
CTGTGGAGAAGAGAAGGGTG
136


 


337853
Acceptor
−15
CTCCTCTGTGGAGAAGAGAA
137


 


337852
Acceptor
−10
CAGTCCTCCTCTGTGGAGAA
138


 


337854
Acceptor
−5
TCTGGCAGTCCTCCTCTGTG
139


 


337851
Acceptor
0
GTATTTCTGGCAGTCCTCCT
140


 


337856
Donor
−20
CTAGGGGGTCATCAAGGGTG
141


 


337859
Donor
−15
CTTACCTAGGGGGTCATCAA
142


 


337857
Donor
−10
GGGCCCTTACCTAGGGGGTC
143


 


337858
Donor
−5
GTACTGGGCCCTTACCTAGG
144


 


337855
Donor
0
GCACAGTACTGGGCCCTTAC
145









To determine whether the MyD88 splice switching oligonucleotides are capable of modulating splicing MyD88, A549 cells were treated with 0, 25, 50, 100 or 200 nM of the human MyD88 antisense oligonucleotides shown in Table 5. After an overnight incubation, cells were harvested and total RNA isolated and subjected to RT-PCR. The PCR primers employed were designed with complementarity to sequence flanking exon II such that a 525 bp fragment would be produced from the MyD88L message while the MyD88S mRNA, lacking exon II, would yield a PCR fragment 135 bp shorter. The results of the transfection experiments showed untreated A549 cells primarily expressed the long form of MyD88. Antisense oligonucleotides targeting the exon II acceptor site (ISIS 337840-337844) had only a slight effect on splice site selection, with the long form being predominant. However, treatment of cells with antisense oligonucleotides targeted to the exon II donor site (ISIS 337845-337849) resulted in a noticeable shift from the long to the short form of MyD88. Antisense oligonucleotides were also evaluated in a number of other cell lines (e.g., T24, 293FT, T47D and MCF-7) with similar results, demonstrating that the effectiveness of splice switching antisense oligonucleotides is not cell line dependent.
To further characterize antisense oligonucleotide efficacy, 293FT cells were treated with the most active antisense oligonucleotides at concentrations ranging from 50 to 200 nM. Total RNA was analyzed for expression of MyD88 by RT-PCR. The most potent oligonucleotide, ISIS 337846, caused an approximately 50% shift to the short form of MyD88 at a dose of 100 nM and greater than 80% shift at 200 nM. The potency of ISIS 337846 was also confirmed by quantitative RT-PCR. For qRT-PCR two probes were used. The sequence of one probe was designed to be complementary to exon II sequence only. This qRT-PCR probe can therefore recognize only the long form of MyD88 since exon II is not present in MyD88S. A second probe was designed with complementarity to exon V sequence. This probe can recognize both long and short form of MyD88. When MyD88 message was measured using the exon V probe the amount of MyD88 message measured was not significantly reduced by treatment with ISIS 337846. Conversely, when MyD88 message was measured using the exon II probe, a dose dependant reduction in mRNA levels was observed with an IC50 of approximately 100 nM, in close agreement with the potency estimated by standard RT-PCR. Since the reduction in target mRNA was observed with the probe that recognizes only the long form of the message, but not the probe that recognizes both the short and long form, it can be concluded that overall MyD88 message is not being reduced by oligonucleotide treatment. Rather, the ratios of the long and short forms of the message are being altered.
In order to further characterize the MyD88 message, Northern blots were performed using polysome associated RNA or polyA+ RNA isolated from ISIS 337846 transfected or mock-transfected A549 cells as described in previous examples herein. The blots were probed with a radiolabeled exon II or exon V specific DNA fragment. Treatment with ISIS 337846 had no effect on RNA levels when the exon V probe was utilized. In contrast, the signal detected by the exon II probe, which can only detect the long form of Myd88, was significantly reduced by ISIS 337846 as compared to the untreated control. These data support the conclusion that ISIS 337846 alters splicing of the Myd88 message, reducing the abundance of the long form and increasing the abundance of the short form. Since these blots were prepared with polysomal or polyA+ RNA rather than total RNA, it can also be implied that the short isoform is exported from the nucleus and is polysome associated, and is therefore being actively translated.
To confirm that the short isoform of the protein is expressed in oligonucleotide treated cells, Western blot analysis was performed. MCF-7 cells were treated at a concentration of 100 nM with ISIS 337846 or ISIS 337840, an oligonucleotide that promotes very little alternative splicing of the target RNA. RNA was isolated for RT-PCR and cell extracts were prepared for analysis of MyD88 protein expression by Western blot 48 hours post-oligonucleotide treatment. Full-length MyD88 was found to migrate at 35 kD in accordance with the predicted size of the full-length protein. While the control ASO had no effect on MyD88 protein expression, a truncated version of the protein migrating at the expected size of the MyD8*S product ( ̃25 kD) was observed in cells treated with ISIS 337846. The relative ratios of the short to the long form of the MyD88 protein are approximately equal to those observed for the RNA isoforms as determined by RT-PCR of RNA isolated from the same cells.
Taken together, these studies demonstrate that MyD88 splicing switching oligonucleotides are capable of altering the ratio of MyD88S and MyD88L mRNA and protein without an overall reduction in total MyD88 mRNA and protein. While not wishing to be bound by theory, splice switching oligonucleotides may function by blocking use of the splice site to which they are targeted. Thus, splice switching oligonucleotides targeting MyD88 exon II splice sites induce skipping of exon II.
Example 14
Effect of ISIS 337846 on IL-1β Signaling
The effect of ISIS 337846 treatment on the expression of IL-1β inducible genes was assessed. ICAM-1 is an adhesion molecule expressed at low levels in resting endothelial cells that is markedly up-regulated in response to IL-1β and other cytokines (Springer, 1990, Nature. 346:425-434). IL-8 is a member of the chemokine gene superfamily, members of which promote the pro-inflammatory phenotype of macrophages, vascular smooth muscle cells and endothelial cells (Koch et al. 1992, Science. 258:1798-1801). Up-regulation of IL-8 in response to IL-1β stimulation has also been documented (Medzhitov et al. 1997, Nature. 388:394-397). T24 cells were treated at a concentration of 200 nM with ISIS 337846, ISIS 337840 or a negative control oligonucleotide. Cells were then stimulated for 4 hours with IL-1β at 0.1 ng/mL 44 hours post-transfection. Total RNA was harvested and quantitative RT-PCR performed using primer/probe sets for either ICAM-1 or IL-8. Both ICAM-1 and IL-8 message were strongly up-regulated by IL-1β. Treatment of the cells with either the non-target control oligonucleotide or with ISIS 337840, which induces little MyD88S expression, did not effect IL-1β signaling. However, treatment of cells with ISIS 337846 reduced IL-1β if dependant up-regulation of both genes.
The ability of ISIS 337846 to inhibit activation of a NF-κB luciferase reporter plasmid, pNFκB-Luc, was also examined. 293FT cells were transfected with antisense oligonucleotide at a concentration of 200 nM. The following day, pNFκB-Luc was introduced into the cells. After an overnight incubation, the cells were stimulated with IL-1β or TNF-α for 4 hours. Cells were harvested and assayed for luciferase activity. The results were normalized to a co-transfected Renilla luciferase reporter. Stimulation of cells with both TNF-α and IL-1β resulted in a marked increase in luciferase activity. Treatment of cells with control or ISIS 337840 had little effect on either IL-1β or TNF-α signaling. TNF-α signaling was also not affected in cells treated with ISIS 337846 in agreement with previous reports that TNF-α mediated induction of NF-κB is independent of MyD88 (Burns et al. 1998, J. Biol. Chem. 273:12203-12209). However, luciferase expression was significantly reduced in IL-1β stimulated cells treated with ISIS 337846.
These results support the idea that modulating the MyD88 splice product ratio to increase levels of MyD88S significantly reduces IL-1β signaling. These results are in accordance with previous studies showing MyD88S acts as a dominant-negative regulator of IL-1β-induced NF-κB activation.
Example 15
Comparison of Splice Switching, RNase H-Dependent and siRNA Oligomeric Compounds for Modulation of MyD88 Splicing
The splice switching antisense oligonucleotide, ISIS 337846, was compared with a published MyD88 siRNA (Oshiumi et al. 2003, Nature Immunol. 4:161-167) and a highly active RNAse H-dependent antisense oligonucleotide targeting MyD88, ISIS 191015 (SEQ ID NO: 62) to determine if one antisense mechanism was substantially more effective than others. T47D cells were treated with oligonucleotide at 200 nM in the presence of Lipofectin reagent as described in previous Examples herein. After 48 hours, cells were harvested and RNA was isolated. MyD88 mRNA levels were measured by qRT-PCR using the exon II and exon V specific probes as described above. With RNase H-dependent or siRNA antisense treatment, reduction of MyD88 message was observed with either the exon II or exon V probe. In contrast, treatment with the splice switching antisense oligonucleotide, ISIS 337846, resulted in reduction of MyD88 mRNA when the exon II probe was employed, but showed no significant reduction of MyD88 message when the exon V probe was utilized.
These results were confirmed by Northern blot analysis. Each type of antisense compound reduced signal detected via the exon II probe to the same extent. In contrast, the signal detected by the exon V probe was significantly reduced by the RNase H-dependent and siRNA antisense compounds, but not by the splicing switching antisense oligonucleotide ISIS 337846. This confirms that ISIS 337846 works not by cleavage and degradation of the target message as RNase H-dependent and siRNA antisense compounds do, but by forcing the use of a specific alternative splice site.
Western blots prepared with lysates from the same cells were also performed. Treatment with the RNase H-dependent or siRNA antisense compounds resulted in significant reduction in MyD88 protein as compared to the untreated control. The splice switching oligonucleotide also resulted in a reduction of full length MyD88 protein; however, in contrast to the siRNA and RNase H-dependent oligonucleotide treated cells, MyD88S protein was also observed following treatment with ISIS 337846.
The overall potencies of the siRNA, RNase H-dependent, and splice switching antisense compounds were next compared. T24 cells were treated with antisense oligonucleotides at 25, 50, 100 and 200 nM. The following day RNA was isolated and mRNA levels accessed by qRT-PCR using the MyD88 exon II specific probe. The results indicate that all antisense compounds exhibit similar potencies with IC50s in the 50-100 nM range.
Splicing, RNase H-dependent and siRNA antisense compounds were also evaluated for their ability to inhibit IL-1β-dependant NF-κB activation. 293FT cells were treated as described above with splicing, RNase H-dependent, and siRNA oligomeric compounds at 50, 100 and 200 nM. The following day the reporter plasmid pNFκB-luc was introduced into the cells. Activation of luciferase by IL-1β and TNF-α was assayed after an overnight incubation. The antisense compounds exhibited similar abilities to reduce IL-1β signaling and diminish luciferase levels, approximately 50% at the 50 nM dose. As expected, there was no effect of the antisense compound treatments on TNF-α signaling even at the highest dose.
Example 16
Inhibition of Toll-Like Receptor Signaling in Mouse Cells using MyD88 Antisense Compounds
To determine whether treatment of mouse cells with MyD88 splice switching oligonucleotide inhibits Toll-like receptor (TLR) signaling, RAW 264.7 cells were treated with the mouse compounds targeting exon II of MyD88, shown in Table 6 above. Cells were electroporated with antisense compound at a concentration of 10 μM and total RNA was isolated after 48 h. RNA was subjected to RT-PCR using primers flanking exon II, as described in previous Examples herein. The results demonstrated that treatment with ISIS 337856 led to a significant alteration in the ratio of MyD88 splice products with the short form predominating.
The effect of MyD88 antisense oligonucleotide administration on TLR9 signaling was next determined. TLR9 is involved in the recognition of specific unmethylated CpG-ODN sequences that distinguish bacterial DNA from mammalian DNA. The MyD88-dependent signaling pathway is common to many TLRs as MyD88 deficient mice are unable to produce inflammatory cytokines in response to most TLR ligands (Adachi et al. 1998, Immunity. 9:143-150; Medzhitov et al. 1997, Nature. 388:394-397). RAW 264.7 cells, which have been shown to express active TLR9 (An et al. 2002, Immunol. Lett. 81:165-169; Rhee et al. 2000, J. Biol. Chem. 275:34035-34040), were electroporated with 10 μM of ISIS 337856 (a splice switching oligonucleotide), ISIS 337853 (a non-active control oligonucleotide), or ISIS 191015 (an RNase H-dependent oligonucleotide), which is complementary to both mouse and human MyD88 mRNA. After 48 hours cells were treated with a previously identified CpG oligonucleotide TLR9 activator (Henry et al. 2000, J. Pharm. Exp. Ther. 292:468-479) at a concentration of 5 μM. After 4 h, RNA was harvested and analyzed for expression of TNF-α mRNA by qRT-PCR. TLR9 activation resulted in a 3- to 4-fold increase in TNF-α mRNA. In cells treated with either the splice switching or RNase H-dependent oligonucleotide, CpG signaling, as measured by TNF-α induction, was reduced by greater than 50 percent, while the control antisense oligonucleotide had no effect.
These results indicate that compounds which reduce expression of either total MyD88 or MyD88L specifically are capable of reducing TLR signaling.
Example 17
Inhibition of IL-1β Signaling in Mouse Liver Following Treatment with MyD88 Antisense Compounds
The ability of modified antisense compounds to regulate MyD88 splicing in mice was next evaluated. Twelve-week old male C57 BL/6 mice were treated twice weekly for two weeks at doses of 10, 25, and 50 mg/kg. 24 hours after the final antisense oligonucleotide treatment, liver RNA was isolated and subjected to RT-PCR using primers bracketing exon II of MyD88. At the 25 mg/kg dose there was an approximately equal distribution of MyD88L and MyD88S. At 50 mg/kg the majority of the MyD88 RNA present is the short form. At both doses there is a general reduction in the total overall amount of MyD88 message. This observation was confirmed using quantitative RT-PCR. Liver RNA from the same experiment was analyzed by TaqMan RT-PCR using a primer/probe set specific to exon V of mouse MyD88. A corresponding loss in the MyD88 message was observed as the ASO dose was increased. Since this primer/probe set recognizes both MyD88 isoforms, the reduction cannot be attributed solely to oligonucleotide directed switching of splicing to the short form.
Lysates prepared from the livers of treated animals were also analyzed by Western blot. In agreement with the reduction observed at the RNA level, MyD88 protein levels were reduced at all treatment doses as compared to the saline treated control mice. Therefore, in vivo, the splicing ASO results in an overall reduction in MyD88 RNA and protein not observed in vitro. However, in contrast to cell culture experiments, no short form of the MyD88 protein was observed in vivo.
Activity of the splicing ASO was evaluated in other tissues. Treatment of mice with ISIS 337856 at a dose of 50 mg/Kg twice weekly for four weeks resulted in an approximately 60% reduction in MyD88 message in liver as assessed by quantitative RT-PCR using the exon V primer/probe set 24 hours after the final treatment. In intestine, the splice switching oligonucleotide reduced MyD88 message levels to 70% of the saline treated control. In adipose tissue, the splice switching oligonucleotide appeared to be even more active, reducing mRNA levels by almost 90%. Western blots performed using lysates from the same tissues showed that MyD88 protein was reduced in intestine and adipose tissue as well as liver in the ISIS 337856 treated animals, however, as in the liver, no short form of the protein was observed in the Western blots from either tissue.
To determine if antisense oligonucleotide mediated modulation of MyD88 splicing could disrupt IL-1β signaling in vivo, mice were dosed with 50 mg/kg of ISIS 337856 or a control oligonucleotide, 3 times weekly for three weeks. 24 hours after the final dose animals were challenged with 0.1 μg of IL-1β. After 2 hours, livers were taken and expression of MyD88 and serum amyloid A (SAA-1) RNA analyzed. Although a shift to MyD88S RNA was observed, overall expression of both isoforms of MyD88 was again found to be reduced by ISIS 337856 as determined by RT-PCR using primers flanking exon II. Liver SAA-1 levels were analyzed by quantitative RT-PCR. In agreement with a previous study demonstrating up-regulation of the SAA-1 gene in the liver of IL-1β treated mice (Adachi et al. 1998, Immunity. 9:143-150), SAA-1 mRNA was strongly up-regulated by IL-1β administration in saline treated mice. However, the IL-1β-mediated increase in SAA-1 expression was completely blocked mice treated with ISIS 337856. In contrast, the control oligonucleotide did not significantly effect IL-1β induction of SAA-1 mRNA expression.
These results demonstrate that splice switching oligonucleotides are active in vivo. Although overall MyD88 mRNA and protein expression was reduced, an increase in the ratio of MyD88S to MyD88L also was demonstrated by treatment with splice switching oligonucleotides in vivo.

## Claims

1. A compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides and having a nucleobase sequence at least 80% complementary to the exon II donor site of a nucleic acid molecule encoding human MyD88 set forth in nucleotides 1338 to 1378 of SEQ ID NO: 146 as measured over the entirety of said modified oligonucleotide, and wherein each nucleoside of the modified oligonucleotide comprises a sugar modification.

2. The compound of claim 1 wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides.

3. The compound of claim 1 wherein the modified oligonucleotide consists of 15 to 30 linked nucleosides.

4. The compound of claim 1, wherein, each nucleoside of the modified oligonucleotide comprises the same sugar modification.

5. The compound of claim 4 wherein the sugar modification is selected from the group consisting of 2′-O-methoxyethyl, 2′-fluoro, locked nucleic acid and ethylene bridged nucleic acid.

6. The compound of claim 5 wherein the sugar modification is 2′-O-methoxyethyl.

7. The compound of claim 6, wherein each internucleoside linkage of said modified oligonucleotide is a phosphorothioate internucleoside linkage.

8. The compound of claim 7, wherein said compound is single-stranded and not hybridized to a second strand in a duplex.

9. The compound of claim 1, wherein said compound is single-stranded and not hybridized to a second strand in a duplex.

10. A compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides and having a nucleobase sequence at least 80% complementary to the exon II donor site of a nucleic acid molecule encoding human MyD88 as set forth in SEQ ID NO: 146 as measured over the entirety of said modified oligonucleotide, wherein each nucleoside of the modified oligonucleotide comprises a sugar modification, and wherein the nucleobase sequence of the modified oligonucleotide has 100% identity to SEQ ID NO: 135 over the length of the modified oligonucleotide.

11. The compound of claim 10, wherein said compound is single-stranded and not hybridized to a second strand in a duplex.

12. A compound comprising a modified oligonucleotide consisting of 15 to 30 linked nucleosides and having a nucleobase sequence at least 80% complementary to the exon II donor site of a nucleic acid molecule encoding human MyD88 as set forth in SEQ ID NO: 146 as measured over the entirety of said modified oligonucleotide, wherein each nucleoside of the modified oligonucleotide comprises a sugar modification, and wherein the nucleobase sequence of the modified oligonucleotide has at least 80% identity to SEQ ID NO: 135 over the length of the modified oligonucleotide.

13. The compound of claim 12, wherein said compound is single-stranded and not hybridized to a second strand in a duplex.

14. The compound of claim 12, wherein the nucleobase sequence of the modified oligonucleotide has at least 90% identity to SEQ ID NO: 135 over the length of the modified oligonucleotide.

15. The compound of claim 12, wherein the nucleobase sequence of the modified oligonucleotide has at least 95% identity to SEQ ID NO: 135 over the length of the modified oligonucleotide.

16. A compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides and having a nucleobase sequence at least 90% complementary to the exon II donor site of a nucleic acid molecule encoding human MyD88 set forth in nucleotides 1338 to 1378 of SEQ ID NO: 146 as measured over the entirety of said modified oligonucleotide, wherein said nucleobase sequence comprises at least 8 consecutive nucleobases from the nucleobase sequence of SEQ ID NO:135, and wherein each nucleoside of the modified oligonucleotide comprises a sugar modification.

17. The compound of claim 16, wherein said nucleobase sequence is at least 95% complementary to the exon II donor site of a nucleic acid molecule encoding human MyD88 set forth in nucleotides 1338 to 1378 of SEQ ID NO: 146 as measured over the entirety of said modified oligonucleotide.

18. The compound of claim 16, wherein said compound is single-stranded and not hybridized to a second strand in a duplex.

19. The compound of claim 16, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides.

20. The compound of claim 16 wherein the modified oligonucleotide consists of 15 to 30 linked nucleosides.

21. The compound of claim 16, wherein each nucleoside of the modified oligonucleotide comprises the same sugar modification.

22. The compound of claim 21, wherein the sugar modification is selected from the group consisting of 2′-O-methoxyethyl, 2′-fluoro, locked nucleic acid and ethylene bridged nucleic acid.

23. The compound of claim 22, wherein the sugar modification is 2′-O-methoxyethyl.

24. The compound of claim 23, wherein each internucleoside linkage of said modified oligonucleotide is a phosphorothioate internucleoside linkage.

25. The compound of claim 24, wherein said compound is single-stranded and not hybridized to a second strand in a duplex.

26. A compound comprising a modified oligonucleotide consisting of 12 to 50 linked nucleosides and having a nucleobase sequence that is 100% complementary to the exon II donor site of a nucleic acid molecule encoding human MyD88 as set forth in nucleotides 1338 to 1378 of SEQ ID NO: 146 as measured over the entirety of said modified oligonucleotide, wherein said nucleobase sequence comprises at least 8 consecutive nucleobases from the nucleobase sequence of SEQ ID NO: 135, and wherein each nucleoside of the modified oligonucleotide comprises a sugar modification.

27. The compound of claim 26, wherein said compound is single-stranded and not hybridized to a second strand in a duplex.