# Patent

## Title
Compositions and their uses directed to gemin genes

## Abstract

Disclosed herein are compounds, compositions and methods for modulating the expression of a Gemin Gene. Also provided are methods of target validation. Also provided are uses of disclosed compounds and compositions in the manufacture of a medicament for treatment of diseases and disorders.

## Background
REFERENCE TO RELATED APPLICATIONS
This application is a divisional application under 35 U.S.C. §121 of U.S. application Ser. No. 11/226,884, filed Sep. 13, 2005, now U.S. Pat. No. 7,759,479, which claims benefit of U.S. provisional application 60/609,711, filed Sep. 13, 2004, the entire contents of each is being expressly incorporated herein by reference.


SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled RTS0491USC1SEQ.txt, created on May 26, 2010 which is 261 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
Disclosed herein are compounds, compositions and methods for modulating the expression of a Gemin gene in a cell, tissue or animal.
BACKGROUND OF THE INVENTION
Targeting disease-causing gene sequences was first suggested more than thirty years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), and antisense activity was demonstrated in cell culture more than a decade later (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75, 280-284). One advantage of antisense technology in the treatment of a disease or condition that stems from a disease-causing gene is that it is a direct genetic approach that has the ability to modulate (increase or decrease) the expression of specific disease-causing genes. Another advantage is that validation of a therapeutic target using antisense compounds results in direct and immediate discovery of the drug candidate; the antisense compound is the potential therapeutic agent.
Generally, the principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates gene expression activities or function, such as transcription or translation. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi generally refers to antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of targeted endogenous mRNA levels. Regardless of the specific mechanism, this sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of malignancies and other diseases.
Antisense compounds have been employed as therapeutic agents in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs are being safely and effectively administered to humans in numerous clinical trials. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently used in the treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients. A New Drug Application (NDA) for GenasenseTM (oblimersen sodium; developed by Genta, Inc., Berkeley Heights, N.J.), an antisense compound which targets the Bc1-2 mRNA overexpressed in many cancers, was accepted by the FDA. Many other antisense compounds are in clinical trials, including those targeting c-myc (NeuGene® AVI-4126, AVI BioPharma, Ridgefield Park, N.J.), TNF-alpha (ISIS 104838, developed by Isis Pharmaceuticals, Inc.), VLA4 (ATL1102, Antisense Therapeutics Ltd., Toorak, Victoria, Australia) and DNA methyltransferase (MG98, developed by MGI Pharma, Bloomington, Minn.), to name a few.
New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience. Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the resistance to degradation result in slower clearance from the body, allowing for less frequent dosing. Different types of chemical modifications can be combined in one compound to further optimize the compound's efficacy.
Much of the information regarding the biogenesis of the small nuclear ribonucleoproteins (snRNPs) came from studies of spinal muscular atrophy (SMA). SMA, a motor neuron degenerative disease that results from deletions or mutations in the Survival of Motor Neurons (SMN) gene, is an autosomal recessive disease that is the leading hereditary cause of infant mortality (Gubitz et al., Exp. Cell. Res., 2004, 296, 51-56). The SMN protein is present in the cytoplasm and nucleus, where it is enriched within discrete bodies called Gems (for “Gemini of Cajal bodies”) which are related to and often associated with Cajal bodies (Gubitz et al., Exp. Cell. Res., 2004, 296, 51-56; Liu and Dreyfuss, Embo J., 1996, 15, 3555-3565; Yong et al., Trends Cell Biol., 2004, 14, 226-232). Cajal bodies are known to contain high levels of factors involved in the transcription and processing of many types of nuclear RNAs, including snRNPs, nucleolar ribonucleoproteins (snoRNPs), and the three eukaryotic RNA polymerases, and are most likely sites of assembly and modification of the nuclear transcription and RNA machinery (Gubitz et al., Exp. Cell. Res., 2004, 296, 51-56).
The snRNP particles are components of the spliceosome, the eukaryotic pre-mRNA splicing machinery. Each major snRNP contains a small nuclear RNA (snRNA) as well as a common set of Sm proteins and a set of proteins specific to the particular snRNA. The common Sm proteins are arranged into a core on a uridine-rich sequence in the cytoplasm after nuclear export of the nacent snRNAs. Proper assembly of the core is required for subsequent import of the snRNPs into the nucleus. As compared to other RNP complexes, such as the small nucleolar RNPs which are assembled in the nucleus where they function, the assembly of snRNPs appears to be strictly regulated and complex (Yong et al., Trends Cell Biol., 2004, 14, 226-232).
The SMN protein oligomerizes with a group of proteins named the Gemins. The Gemins include Gemin2, Gemin3, a DEAD/H box helicase, Gemin4, Gemin5, Gemin6, and Gemin7. The Gemins colocalize with SMN in gems and are present in the cytoplasm and nucleoplasm (Gubitz et al., Exp. Cell. Res., 2004, 296, 51-56). It appears that individual Gemins of the SMN complex interact with distinct sets of Sm proteins indicating that multiple contacts are likely to be important for the function of the SMN complex in snRNP core assembly (Baccon et al., J. Biol. Chem., 2002, 277, 31957-31962). The SMN complex was found to function as a specificity factor for the assembly of spliceosomal snRNP, ensuring that Sm cores are only formed on the correct RNA molecules (Pellizzoni et al., Science, 2002, 298, 1775-1779).
Gemin2 (also known as SIP1, SMP-interacting protein 1, and survival of motor neuron protein interacting protein 1) was isolated in a yeast two-hybrid screen of a HeLa cell library using SMN as bait. These proteins were tightly associated and colocalized to Gems, prompting a search for other protein components of the complex. The SMN/Gemin2 pair was found to interact directly with several of the snRNP Sm core proteins (Liu et al., Cell, 1997, 90, 1013-1021). Further implicating the pair in snRNP biogenesis, the SMN/Gemin2 complex associated with spliceosomal snRNAs U1 and U5 in the cytoplasm of Xenopus oocytes, and antibodies against Gemin2 inhibited Sm core assembly of the spliceosomal snRNPs U1, U2, U4, and U5 and their transport to the nucleus (Fischer et al., Cell, 1997, 90, 1023-1029).
Together with Gemin2, Gemin3 and Gemin4 (also known as GIP1 or gem-associated protein 4) were also found to complex with SMN and snRNP proteins (Charroux et al., J. Cell Biol., 1999, 147, 1181-1194; Charroux et al., J. Cell Biol., 2000, 148, 1177-1186). Gemin3 was the only protein of the SMN complex that bound specifically to GST-Gemin4, suggesting that the presence of Gemin4 in the complex is a result of its direct interaction with Gemin3, but not with SMN. The direct and avid interaction of Gemin4 with the DEAD/H box-containing helicase protein Gemin3 may indicate that they function together. Gemin4 also interacts with several of the core Sm proteins, and co-localizes with SMN to gems. Gemin4 localizes to the nucleolus, potentially indicating additional functions in ribosome biogenesis (Charroux et al., J. Cell Biol., 2000, 148, 1177-1186).
Gemin4 proteins are found predominantly in the SMN complex, however, a less abundant Gemin3-Gemin4 complex has also been found (Charroux et al., J. Cell Biol., 2000, 148, 1177-1186; Mourelatos et al., Genes & Development, 2002, 16, 720-728). Immunoprecipitation studies showed that Gemin3 and Gemin4 are associated in a complex with eIF2c2, a member of the large Argonaute family of proteins, members of which have been implicated in RNA interference (RNAi) mechanisms and developmental regulation by short temporal RNAs (stRNAs). The complex, a miRNP, also contained RNAs about 22 nucleotides in length, corresponding to microRNAs (miRNAs). 40 miRNAs were captured in these studies (Mourelatos et al., Genes & Development, 2002, 16, 720-728). Monoclonal antibodies to either Gemin3 or Gemin4 immunoprecipitated let-7-programmed RNA-induced silencing complex (RISC) activity, leading to the notion that the Gemin4-containing miRNP may be the human RISC which can carry out both target cleavage in the RNAi pathway and translational control in the miRNA pathway (Hutvagner and Zamore, Science, 2002, 297, 2056-2060).
In a yeast-two hybrid assay, Gemin4 was found to interact with galectin-1 and galectin-3, nuclear-localized proteins that were shown to be required factors for splicing in a cell-free assay. This interaction is thought to be functionally relevant in the splicing pathway (Park et al., Nucleic Acids Res., 2001, 29, 3595-3602).
Gemin5 (also known as DKFZP586M1824 protein; gem-associated protein 5) was found by coimmunoprecipitation of proteins that associate with SMN in vivo. Like SMN, Gemin5 is localized in the cytoplasm, nucleoplasm, and is highly enriched in the nuclear gems. It binds to SMN by direct protein-protein interaction in vitro and interacts with several of the snRNP core Sm proteins. The Gemin5 protein is predicted to contain up to 13 WD repeats in its amino-terminal half, and a coiled-coil near its carboxyl terminus. Because both WD repeats and coiled-coil motifs are protein-protein interaction domains, Gemin5 may serve as a structural platform for protein assembly (Gubitz et al., J. Biol. Chem., 2002, 277, 5631-5636).
Extracts from stable cell lines expressing epitope-tagged SMN or Gemin2 proteins were analyzed by immunoprecipitation with an antibody recognizing the epitope. Both tagged-proteins were isolated with the SMN complex which was found to contain additional previously unidentified proteins including Gemin6 (also known as GEM-associated protein 6 or hypothetical protein FLJ23459). Database searches revealed that Gemin6 is not significantly homologous to other proteins and contains no common motifs which may be indicative of function. Like the other members of the SMN complex, Gemin6 was shown to interact with several Sm proteins (Baccon et al., J. Biol. Chem., 2002, 277, 31957-31962). The Gemin6 localization is similar to the other components of the SMN complex, but direct interaction of Gemin6 with SMN, Gemin2, Gemin3, Gemin4 or Gemin5 was not detectable (Pellizzoni et al., J. Biol. Chem., 2002, 277, 7540-7545).
However, Gemin7 (also known as hypothetical protein FLJ13956), also identified using the epitope-tagged system to purify SMN complexes, was shown to bind directly to Gemin6 and SMN in vitro, therefore it likely mediates the association of Gemin6 with the SMN complex. Like Gemin6, Gemin7 does not contain any known motifs that may suggest possible functions, but it does interact with a subset of the Sm proteins. Like the other complex members, Gemin7 colocalizes with SMN in gems (Baccon et al., J. Biol. Chem., 2002, 277, 31957-31962).
Beyond interactions with Sm proteins and snRNAs, the SMN complex interacts directly with several protein targets that are components of RNPs which function in various aspects of RNA metabolism. Among these substrates are the Sm-like (Lsm) proteins of the snRNPs, also essential components of the splicing machinery (Friesen and Dreyfuss, J. Biol. Chem., 2000, 275, 26370-26375). Fibrillarin and GAR1, components of small nucleolar RNPs (snoRNPs), also interact with SMN. Fibrillarin is a marker for Box C/D snoRNPs, the class that is necessary for cleavage and site-specific methylation of rRNA. Box H/ACA snoRNPs contain GAR1 and guide the pseudouridylation of rRNA (Pellizzoni et al., Curr. Biol., 2001, 11, 1079-1088). Thus, the SMN complex also appears to be involved in snoRNP biogenesis (Jones et al., J Biol. Chem., 2001, 276, 38645-38651; Pellizzoni et al., Curr. Biol., 2001, 11, 1079-1088). Additional SMN complex substrates are hnRNP U and Q, RNA helicase A, and Epstein-Barr virus nuclear antigen 2 (EBNA2), coilin, and nucleolin (Barth et al., J. Virol., 2003, 77, 5008-5013; Gubitz et al., Exp. Cell. Res., 2004, 296, 51-56; Liu and Dreyfuss, Embo J., 1996, 15, 3555-3565; Mourelatos et al., Genes & Development, 2002, 16, 720-728; Yong et al., Trends Cell Biol., 2004, 14, 226-232). Because most SMN complex substrates are components of various RNP complexes involved in RNA processing, the SMN complex may take part in many aspects of cellular RNA metabolism (Gubitz et al., Exp. Cell. Res., 2004, 296, 51-56).
Disclosed in U.S. Pat. No. 6,646,113 is an antisense isolated nucleic acid complementary to the nucleic acid encoding a human Survival of Motor Neuron-Interacting Protein 1, wherein said nucleic acid encodes a protein that differs from the amino acid sequence disclosed by a mutation that inhibits binding of the Survival of Motor Neuron protein, and further wherein said mutation is selected from the group consisting of a deletion of the carboxyl terminal 89 amino acids relative to the amino acid sequence disclosed therein and a deletion of the carboxyl terminal 162 amino acids relative to the amino acid sequence disclosed therein. Also disclosed are antisense oligomers of between about 10 to about 30, and more preferrably about 15 nucleotides (Dreyfuss et al., 2003).
U.S. pregrant publication 20030228617 discloses a kit comprising a plurality of oligonucleotide primers and instructions for employing the plurality of oligonucleotide primers to determine the expression level of at least one of the genes represented in a group of sequences, said group including a nucleic acid sequence of a partial cDNA and a full-length cDNA corresponding to the human survival of motor neuron protein interacting protein 1 (SIP1) gene (Aune and Olsen, 2003).
The role of Gemin2, Gemin4, Gemin5, Gemin6, and Gemin7 in RNA metabolism makes these attractive targets for therapeutic and investigative strategies aimed at antisense technology. Consequently, there remains a need for agents capable of effectively modulating Gemin2, Gemin4, Gemin5, Gemin6, and Gemin7 function.
Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications.
Consequently, there remains a long-felt need for agents that specifically regulate gene expression via antisense mechanisms. Disclosed herein are antisense compounds useful for modulating gene expression pathways, including those relying on mechanisms of action such as RNaseH, RNAi and dsRNA enzymes, as well as other antisense mechanisms based on target degradation or target occupancy. One having skill in the art, once armed with this disclosure will be able, without undue experimentation, to identify, prepare and exploit antisense compounds for these uses.
SUMMARY OF THE INVENTION
Provided herein are oligomeric compounds, especially antisense nucleic acid and nucleic acid-like oligomers, e.g., antisense oligonucleotides, which are targeted to a nucleic acid encoding a Gemin Gene, and which modulate the expression of a Gemin Gene. Gemin Genes disclosed herein include Gemin2, Gemin4, Gemin5, Gemin6, Gemin7. Also disclosed are pharmaceutical compositions including said oligomeric compounds, and methods of using said oligomeric compounds to modulate expression of a Gemin Gene.
In one embodiment, the invention provides oligomeric compounds of from, e.g., 13 to 30 nucleobases in length targeted to a nucleic acid molecule encoding a Gemin Gene, e.g., Gemin2 (SEQ ID NO: 7), wherein said oligomeric compound specifically hybridizes with said nucleic acid molecule encoding the Gemin Gene and inhibits the expression of the Gemin Gene. In some embodiments, the oligomeric compound is 20 nucleobases in length. In further embodiments, the oligomeric compound is an antisense oligonucleotide.
The oligomeric compound can be chimeric and include at least one modified internucleoside linkage, e.g., a phosphorothioate internucleoside linkage; and/or at least one modified sugar moiety, e.g., a 2′-O-methoxyethyl sugar moiety, and/or at least one modified nucleobase, e.g., a 5-methylcytosine. In one embodiment, the oligomeric compound is an antisense, chimeric oligonucleotide 20 nucleobases in length and includes at least one phosphorothioate internucleoside linkage and at least one 2′-O-methoxyethyl sugar moiety and at least one 5-methylcytosine.
Pharmaceutical, therapeutic and other compositions comprising the oligomeric compounds of the present invention are also provided. In one embodiment, the invention provides a composition that includes an oligomeric compound of the invention and a pharmaceutically acceptable carrier, e.g., colloidal dispersion system, or diluent.
Further provided are methods of modulating the expression of a Gemin Gene, e.g., Gemin2, in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the present invention. In some embodiments, the methods are performed in vitro.
Methods of treating an animal, particularly a human, suspected of having or at risk for a disease or condition associated with expression of a Gemin Gene are also set forth herein. Such methods include administering a therapeutically or prophylactically effective amount of one or more of the oligomeric compounds or compositions of the present invention to an animal, particularly a human.
Further provided are methods of identifying the relationship between a Gemin Gene and a disease state, phenotype, or condition by detecting or modulating said Gemin Gene comprising contacting a sample, tissue, cell, or organism with one or more oligomeric compounds, measuring the nucleic acid or protein level of said Gemin Gene and/or a related phenotypic or chemical endpoint coincident with or at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound.
Further provided are methods of screening for modulators of a Gemin Gene expression by contacting a target segment of a nucleic acid molecule encoding said Gemin Gene with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding said Gemin Gene.
Also provided is the use of the compounds or compositions of the invention in the manufacture of a medicament for the treatment of one or more conditions associated with a target of the invention. Further contemplated are methods where cells or tissues are contacted in vivo with an effective amount of one or more of the disclosed compounds or compositions. Also provided are ex vivo methods of treatment that include contacting cells or tissues with an effective amount of one or more of the compounds or compositions of the invention and then introducing said cells or tissues into an animal.


DETAILED DESCRIPTION OF THE INVENTION
Overview
Disclosed herein are oligomeric compounds, including antisense oligonucleotides and other antisense compounds for use in modulating the expression of nucleic acid molecules encoding a Gemin Gene, e.g., Gemin2, Gemin4, Gemin5, Gemin6, Gemin7, and the like. This is accomplished by providing oligomeric compounds which hybridize with one or more target nucleic acid molecules encoding a Gemin Gene. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding a Gemin Gene” have been used for convenience to encompass DNA encoding a Gemin Gene, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA.
In one embodiment, the oligomeric compounds of the invention are antisense compounds, e.g., antisense oligonucleotides. The principle behind antisense technology is that an antisense compound, which hybridizes to a target nucleic acid, interferes with gene expression activities such as transcription or translation. This sequence specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in disease.
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” or “antisense oligonucleotide” refers to an oligomeric compound that is at least partially complementary to the region of a nucleic acid molecule to which it hybridizes and 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. Antisense oligonucleotides may be chemically modified or unmodified. Nonlimiting examples of oligomeric compounds include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, and siRNAs. As such, these 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, loops or mismatches. 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.
Antisense Mechanisms
Oligomeric compounds that modulate Gemin Gene expression via antisense mechanisms are one embodiment of the invention. While not wishing to be bound by theory, antisense mechanisms fall into two general non-exclusive categories. These categories are antisense mechanisms that (1) involve target degradation and (2) involve an occupancy-based mechanism wherein the cellular machinery is stalled and may or may not involve target degradation component.
A target degradation mechanism can include an RNase H mechanism. RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression.
A target degradation mechanism can include RNA interference (RNAi). RNAi is a form of posttranscriptional gene silencing that was initially defined in the nematode, Caenorhabditis elegans, resulting from exposure to double-stranded RNA (dsRNA). In many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing (Guo and Kempheus, Cell, 1995, 81, 611-620; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The RNAi compounds are often referred to as short interfering RNAs or siRNAs. Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the siRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697). Both RNAi compounds (i.e., single- or double-stranded RNA or RNA-like compounds) and single-stranded RNase H-dependent antisense compounds bind to their RNA target by base pairing (i.e., hybridization) and induce site-specific cleavage of the target RNA by specific RNAses; i.e., both work via an antisense mechanism (Vickers et al., 2003, J. Biol. Chem., 278, 7108-7118). Double-stranded ribonucleases (dsRNases) such as those in the RNase III and ribonuclease L family of enzymes have been postulated to play a role in RNA target degradation. Double-stranded ribonucleases and oligomeric compounds that trigger them are further described in U.S. Pat. Nos. 5,898,031 and 6,107,094.
Nonlimiting examples of an occupancy based mechanism include inhibition of translation, modulation of splicing, modulation of poly(A) site selection and disruption of regulatory RNA structure. 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 such a mechanism is disclosed in U.S. Pat. No. 6,210,892 and U.S. Pre-Grant Publication 20020049173.
Certain types of antisense compounds which specifically hybridize to the 5′ cap region of their target mRNA interfere with translation of the target mRNA into protein. Such oligomers include peptide-nucleic acid (PNA) oligomers, morpholino oligomers snf oligonucleosides (such as those having an MMI or amide internucleoside linkage) and oligonucleotides having modifications at the 2′ position of the sugar. This is believed to occur via interference with ribosome assembly on the target mRNA. Methods for inhibiting the translation of a selected capped target mRNA by contacting target mRNA with an antisense compound are disclosed in U.S. Pat. No. 5,789,573.
Antisense compounds targeted to specific splice variants of an RNA can be used to modulate the populations of alternatively spliced RNA products (see U.S. Patent Application entitled “Isoform-Specific Targeting of Splice Variants” filed Aug. 29, 2003, Ser. No. 10/651,772).
Antisense compounds targeted to a specific poly(A) site of mRNA can be used to modulate the populations of alternatively polyadenylated transcripts. In addition, antisense compounds can be used to disrupt RNA regulatory structure thereby affecting, for example, the stability of the targeted RNA and its subsequent expression. Methods directed to such modulation are disclosed in U.S. Pat. No. 6,210,892 and Pre-Grant Publication 20020049173.
siRNAs
In another embodiment of the invention, oligomeric compounds of the invention comprise double-stranded antisense compounds encompassing short interfering RNAs (siRNAs). As used herein, the term “siRNA” is defined as a double-stranded compound having a first and second strand and comprises a central complementary portion between said first and second strands and terminal portions that are optionally complementary between said first and second strands or with the target mRNA. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. In one nonlimiting example, the first strand of the siRNA is antisense to the target nucleic acid, while the second strand is complementary to the first strand. Once the antisense strand is designed to target a particular nucleic acid target, the sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and either strand may contain modifications or additions to either terminus. For example, in one embodiment, both strands of the siRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini. It is possible for one end of a duplex to be blunt and the other to have overhanging nucleobases. In one embodiment, the number of overhanging nucleobases is from 1 to 6 on the 3′ end of each strand of the duplex. In another embodiment, the number of overhanging nucleobases is from 1 to 6 on the 3′ end of only one strand of the duplex. In a further embodiment, the number of overhanging nucleobases is from 1 to 6 on one or both 5′ ends of the duplexed strands. In another embodiment, the number of overhanging nucleobases is zero.
In one embodiment of the invention, double-stranded antisense compounds are canonical siRNAs. As used herein, the term “canonical siRNA” is defined as a double-stranded oligomeric compound having a first strand and a second strand each strand being 21 nucleobases in length with the strands being complementary over 19 nucleobases and having on each 3′ termini of each strand a deoxy thymidine dimer (dTdT) which in the double-stranded compound acts as a 3′ overhang.
Each strand of the siRNA duplex may be from about 8 to about 80 nucleobases, 10 to 50, 13 to 80, 13 to 50, 13 to 30, 13 to 24, 19 to 23, 20 to 80, 20 to 50, 20 to 30, or 20 to 24 nucleobases. The central complementary portion may be from about 8 to about 80 nucleobases in length, 10 to 50, 13 to 80, 13 to 50, 13 to 30, 13 to 24, 19 to 23, 20 to 80, 20 to 50, 20 to 30, or 20 to 24 nucleobases. The terminal portions can be from 1 to 6 nucleobases. The siRNAs may also have no terminal portions. The two strands of an siRNA can be linked internally leaving free 3′ or 5′ termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5′ or 3′ terminus producing an extension of single-stranded character.
In another embodiment, the double-stranded antisense compounds are blunt-ended siRNAs. As used herein the term “blunt-ended siRNA” is defined as an siRNA having no terminal overhangs. That is, at least one end of the double-stranded compound is blunt. siRNAs whether canonical or blunt act to elicit dsRNAse enzymes and trigger the recruitment or activation of the RNAi antisense mechanism. In a further embodiment, single-stranded RNAi (ssRNAi) compounds that act via the RNAi antisense mechanism are contemplated.
Further modifications can be made to the double-stranded compounds and may include conjugate groups attached to one of the termini, selected nucleobase positions, sugar positions or to one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, the compounds can take the form of a self-complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the compounds can be fully or partially double-stranded. When formed from two strands, or a single strand that takes the form of a self-complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex-forming regions of a single strand) are complementary when they base pair in Watson-Crick fashion.
Compounds
The oligomeric compounds in accordance with this invention may comprise a complementary oligomeric compound from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). In other words, a single-stranded compound of the invention comprises from 8 to about 80 nucleobases, and a double-stranded antisense compound of the invention (such as a siRNA, for example) comprises two strands, each of which is from about 8 to about 80 nucleobases. In some embodiments, oligomeric compounds of the invention are 10 to 50, 13 to 80, 13 to 50, 13 to 30, 13 to 24, 19 to 23, 20 to 80, 20 to 50, 20 to 30, or 20 to 24 nucleobases in length. In one embodiment, the oligomeric compounds are 20 nucleobases in length.
Contained within the oligomeric compounds of the invention (whether single or double stranded and on at least one strand) are antisense portions. The “antisense portion” is that part of the oligomeric compound that is designed to confer antisense activity by one of the aforementioned potential antisense mechanisms. One of ordinary skill in the art will appreciate that this comprehends antisense portions 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 have antisense portions of 10 to 50 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 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, or 50 nucleobases.
In one embodiment, the antisense compounds of the invention have antisense portions of 13 to 80 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions 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 have antisense portions of 13 to 50 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases.
In one embodiment, the antisense compounds of the invention have antisense portions of 13 to 30 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 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 have antisense portions of 13 to 24 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleobases.
In one embodiment, the antisense compounds of the invention have antisense portions of 19 to 23 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 19, 20, 21, 22 or 23 nucleobases.
In one embodiment, the antisense compounds of the invention have antisense portions of 20 to 80 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 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 have antisense portions of 20 to 50 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 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 have antisense portions of 20 to 30 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases.
In one embodiment, the antisense compounds of the invention have antisense portions of 20 to 24 nucleobases. One having ordinary skill in the art will appreciate that this embodies antisense compounds having antisense portions of 20, 21, 22, 23, or 24 nucleobases.
Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.
Exemplary compounds 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 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 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 about 80 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.
Chemical Modifications
As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base (sometimes referred to as a “nucleobase” or simply a “base”). The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.
Modified Internucleoside Linkages
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, thionoalkylphosphonates, 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 intemucleoside 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 intemucleoside 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.
Modified Sugars
Oligomeric compounds may also contain one or more substituted sugar moieties. Suitable compounds can comprise one of the following at the T 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′-dimethylamino-ethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH3)2, also described in examples hereinbelow.
Other 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.
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. 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.)
One conformation of modified nucleosides and their oligomers can be estimated by various methods such as molecular dynamics calculations, nuclear magnetic resonance spectroscopy and CD measurements. Hence, modifications predicted to induce RNA-like conformations, A-form duplex geometry in an oligomeric context, are selected for use in the modified oligonucleotides of the present invention. 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., and the examples section below.)
Oligonucleotide Mimetics
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 (—C(═O)—CH2— as shown below) 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. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. 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). 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. LNA's 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 (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). The alpha-L-LNA's were incorporated into antisense gapmers and chimeras that showed potent antisense activity.
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).
The conformations of LNAs determined by 2D NMR spectroscopy have shown that the locked orientation of the LNA nucleotides, both in single-stranded LNA and in duplexes, constrains the phosphate backbone in such a way as to introduce a higher population of the N-type conformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53). These conformations are associated with improved stacking of the nucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18, 1365-1370).
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) 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. DNA:LNA 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-methylcytosine, 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 olignucleotide 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.
Modified and Alternate Nucleobases
Oligomeric compounds may also include nucleobase (often referred to in the art as heterocyclic base or simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cyto-sines, 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 ther 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.
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 (R10═O, R11—R14═H) (Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one (R10═S, R11—R14═H), (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 (R10═O, R11—R14═F) (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. Patent Application entitled “Modified Peptide Nucleic Acids” filed May 24, 2002, Ser. No. 10/155,920; and U.S. Patent Application entitled “Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser. No. 10/013,295).
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 (R10═O, R11═—O—(CH2)2—NH2, R12-14═H) (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 oligonucleotide to its complementary target DNA or RNA with a ΔTm of up to 18° 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. No. 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 Unites States Pre-Grant Publication 20030158403 filed Nov. 28, 2001.
Conjugates
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. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999).
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 riucleotide, 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). For siRNA constructs, the 5′ end (5′ cap) is commonly but not limited to 5′-hydroxyl or 5′-phosphate.
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.
Chimeric Compounds
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 flanked by non-deoxyoligonucleotides. The central region is referred to as the “gap.” The flanking regions 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 refered 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.
Oligomer Synthesis
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.
Starting Materials and Intermediates
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-methylcytidine 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-(dimethylaminooxyethyl) 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.
Oligonucleotide Synthesis
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. Nos. 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.
Oligonucleoside Synthesis
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. Nos. 5,223,618.
Peptide Nucleic Acid Synthesis
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.
Synthesis of 2′-O-Protected Oligomers/RNA Synthesis
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.
A large number of 2′-O-protecting groups have been used for the synthesis of oligoribo-nucleotides and any can be used. Some of the protecting groups used initially for oligoribonucleotide synthesis included tetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two groups are not compatible with all 5′-O-protecting groups so modified versions were used with 5′-DMT groups such as 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reese et al. have identified a number of piperidine derivatives (like Fpmp) that are useful in the synthesis of oligoribonucleotides including 1-[(chloro-4-methyl)phenyl]-4′-methoxypiperidin-4-yl (Reese et al., Tetrahedron Lett., 1986, (27), 2291). Another approach is to replace the standard 5′-DMT (dimethoxytrityl) group with protecting groups that were removed under non-acidic conditions such as levulinyl and 9-fluorenylmethoxycarbonyl. Such groups enable the use of acid labile 2′-protecting groups for oligoribonucleotide synthesis. Another more widely used protecting group, initially used for the synthesis of oligoribonucleotides, is the t-butyldimethylsilyl group (Ogilvie et al., Tetrahedron Lett., 1974, 2861; Hakimelahi et al., Tetrahedron Lett., 1981, (22), 2543; and Jones et al., J. Chem. Soc. Perkin I., 2762). The 2′-O-protecting groups can require special reagents for their removal. For example, the t-butyldimethylsilyl group is normally removed after all other cleaving/deprotecting steps by treatment of the oligomeric compound with tetrabutylammonium fluoride (TBAF).
One group of researchers examined a number of 2′-O-protecting groups (Pitsch, S., Chimia, 2001, (55), 320-324.) The group examined fluoride labile and photolabile protecting groups that are removed using moderate conditions. One photolabile group that was examined was the [2-(nitrobenzyl)oxy]methyl (nbm) protecting group (Schwartz et al., Bioorg. Med. Chem. Lett., 1992, (2), 1019.) Other groups examined included a number structurally related formaldehyde acetal-derived, 2′-O-protecting groups. Also prepared were a number of related protecting groups for preparing 2′-O-alkylated nucleoside phosphoramidites including 2′-O-[(triisopropylsilyl)oxy]methyl (2′-O—CH2-O—Si(iPr)3, TOM). One 2′-O-protecting group that was prepared to be used orthogonally to the TOM group was 2′-O—[(R)-1-(2-nitrophenyl)ethyloxy)methyl]((R)-mnbm).
Another strategy using a fluoride labile 5′-O-protecting group (non-acid labile) and an acid labile 2′-O-protecting group has been reported (Scaringe, Stephen A., Methods, 2001, (23) 206-217). A number of possible silyl ethers were examined for 5′-O-protection and a number of acetals and orthoesters were examined for 2′-O-protection. The protection scheme that gave the best results was 5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses a modified phosphoramidite synthesis approach in that some different reagents are required that are not routinely used for RNA/DNA synthesis.
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.
The primary groups being used for commercial RNA synthesis are:

    
    
        TBDMS=5′-O-DMT-2′-O-t-butyldimethylsilyl;
        TOM=2′-O-[(triisopropylsilyl)oxy]methyl;
        DOD/ACE=(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether-2′-O-bis(2-acetoxyethoxy)methyl
        FPMP=5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl].
    
    


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.
Synthesis of Chimeric Oligomeric Compounds
(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′-dimethoxy-trityl-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-(Methoxyethyl)) Chimeric Phosphorothioate Oligonucleotides
(2′-O-(2-methoxyethyl))-(2′-deoxy)-(-2′-O-(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.
Oligomer Purification and Analysis
Methods of oligomeric compound purification and analysis are well 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.
Hybridization
“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” refers 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
“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 95%, 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).
The oligomeric compounds of the invention also include variants 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, variants 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 a Gemin Gene mRNA.
In one embodiment, homology, sequence identity or complementarity, between the oligomeric compound and target nucleic acid is from about 50% to about 60%. In another embodiment, homology, sequence identity or complementarity, is from about 60% to about 70%. In another embodiment, homology, sequence identity or complementarity, is from about 70% to about 80%. In another embodiment, homology, sequence identity or complementarity, is from about 80% to about 90%. In still other embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.
Target Nucleic Acids
“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 a Gemin Gene” encompass DNA encoding a Gemin Gene, 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 a Gemin Gene.
Target Regions, Segments, and Sites
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. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as positions within a target nucleic acid.
Start Codons
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.
Coding Regions
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.
Untranslated Regions
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.
Introns and Exons
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 is implicated in disease, or where aberrant levels of an aberrant splice product is 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).
Variants
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.
Target Names, Synonyms, Features
In accordance with the present invention are compositions and methods for modulating the expression of genes which are presented in Table 1. Table 1 lists the gene target names and their respective synonyms, as well as GenBank accession numbers used to design oligomeric compounds targeted to each gene. Table 1 also describes features contained within the gene target nucleic acid sequences of the invention. Representative features include 5′UTR, start codon, coding sequence (coding), stop codon, 3′UTR, exon, intron, exon:exon junction, intron:exon junction and exon:intron junction. “Feature start site” and “feature end site” refer to the first (5′-most) and last (3′-most) nucleotide numbers, respectively, of the described feature with respect to the designated sequence. For example, for a sequence containing a start codon comprising the first three nucleotides, “feature start site” is “1” and “feature end site” is “3”.







TABLE 1







Gene Targets, Synonyms and Features



















Feature
Feature
SEQ


Target




Start
Stop
ID


Name
Synonyms
Species
Genbank #
Feature
Site
Site
NO

















Gemin2
SIP1; SMP-
Human
AB037701.1
start codon
1
3
1



interacting



protein 1;



survival of



motor neuron



protein



interacting



protein 1


Gemin2
Same as above
Human
AB037701.1
start codon
1
3
1


Gemin2
Same as above
Human
AB037701.1
CDS
1
798
1


Gemin2
Same as above
Human
AB037701.1
exon:exon
170
171
1






junction


Gemin2
Same as above
Human
AB037701.1
exon
171
255
1


Gemin2
Same as above
Human
AB037701.1
stop codon
213
215
1


Gemin2
Same as above
Human
AB037701.1
exon:exon
255
256
1






junction


Gemin2
Same as above
Human
AB037701.1
exon
256
345
1


Gemin2
Same as above
Human
AB037701.1
exon:exon
345
346
1






junction


Gemin2
Same as above
Human
AB037701.1
exon
346
405
1


Gemin2
Same as above
Human
AB037701.1
exon:exon
405
406
1






junction


Gemin2
Same as above
Human
AB037701.1
exon
406
519
1


Gemin2
Same as above
Human
AB037701.1
exon:exon
519
520
1






junction


Gemin2
Same as above
Human
AB037701.1
exon
520
588
1


Gemin2
Same as above
Human
AB037701.1
exon:exon
588
589
1






junction


Gemin2
Same as above
Human
AB037701.1
exon
589
699
1


Gemin2
Same as above
Human
AB037701.1
exon:exon
699
700
1






junction


Gemin2
Same as above
Human
AB037701.1
exon
700
758
1


Gemin2
Same as above
Human
AB037701.1
exon:exon
758
759
1






junction


Gemin2
Same as above
Human
AB037701.1
3′UTR
768
815
1


Gemin2
Same as above
Human
AB037701.1
stop codon
796
798
1


Gemin2
Same as above
Human
AB037701.1
stop codon
796
798
1


Gemin2
Same as above
Human
AB037701.1
3′UTR
799
815
1


Gemin2
Same as above
Human
AB037702.1
start codon
1
3
2


Gemin2
Same as above
Human
AB037702.1
start codon
1
3
2


Gemin2
Same as above
Human
AB037702.1
CDS
1
753
2


Gemin2
Same as above
Human
AB037702.1
exon:exon
170
171
2






junction


Gemin2
Same as above
Human
AB037702.1
exon
171
255
2


Gemin2
Same as above
Human
AB037702.1
stop codon
213
215
2


Gemin2
Same as above
Human
AB037702.1
exon:exon
255
256
2






junction


Gemin2
Same as above
Human
AB037702.1
exon
256
345
2


Gemin2
Same as above
Human
AB037702.1
exon:exon
345
346
2






junction


Gemin2
Same as above
Human
AB037702.1
exon
346
405
2


Gemin2
Same as above
Human
AB037702.1
exon:exon
405
406
2






junction


Gemin2
Same as above
Human
AB037702.1
exon
406
519
2


Gemin2
Same as above
Human
AB037702.1
exon:exon
519
520
2






junction


Gemin2
Same as above
Human
AB037702.1
exon
520
564
2


Gemin2
Same as above
Human
AB037702.1
exon:exon
564
565
2






junction


Gemin2
Same as above
Human
AB037702.1
exon
565
633
2


Gemin2
Same as above
Human
AB037702.1
exon:exon
633
634
2






junction


Gemin2
Same as above
Human
AB037702.1
exon
634
744
2


Gemin2
Same as above
Human
AB037702.1
exon:exon
744
745
2






junction


Gemin2
Same as above
Human
AB037702.1
stop codon
751
753
2


Gemin2
Same as above
Human
AB037702.1
3′UTR
754
801
2


Gemin2
Same as above
Human
AB037702.1
stop codon
782
784
2


Gemin2
Same as above
Human
AB037703.1
start codon
1
3
3


Gemin2
Same as above
Human
AB037703.1
start codon
1
3
3


Gemin2
Same as above
Human
AB037703.1
CDS
1
135
3


Gemin2
Same as above
Human
AB037703.1
exon:exon
90
91
3






junction


Gemin2
Same as above
Human
AB037703.1
exon
91
175
3


Gemin2
Same as above
Human
AB037703.1
stop codon
133
135
3


Gemin2
Same as above
Human
AB037703.1
3′UTR
136
825
3


Gemin2
Same as above
Human
AB037703.1
exon:exon
175
176
3






junction


Gemin2
Same as above
Human
AB037703.1
exon
176
265
3


Gemin2
Same as above
Human
AB037703.1
exon:exon
265
266
3






junction


Gemin2
Same as above
Human
AB037703.1
exon
266
325
3


Gemin2
Same as above
Human
AB037703.1
exon:exon
325
326
3






junction


Gemin2
Same as above
Human
AB037703.1
exon
326
439
3


Gemin2
Same as above
Human
AB037703.1
exon:exon
439
440
3






junction


Gemin2
Same as above
Human
AB037703.1
exon
440
484
3


Gemin2
Same as above
Human
AB037703.1
exon:exon
484
485
3






junction


Gemin2
Same as above
Human
AB037703.1
exon
485
553
3


Gemin2
Same as above
Human
AB037703.1
exon:exon
553
554
3






junction


Gemin2
Same as above
Human
AB037703.1
exon
554
664
3


Gemin2
Same as above
Human
AB037703.1
exon:exon
664
665
3






junction


Gemin2
Same as above
Human
AB037703.1
exon
665
723
3


Gemin2
Same as above
Human
AB037703.1
exon:exon
723
724
3






junction


Gemin2
Same as above
Human
AB037703.1
stop codon
761
763
3


Gemin2
Same as above
Human
BC028095.1
start codon
22
24
4


Gemin2
Same as above
Human
BC028095.1
exon:exon
191
192
4






junction


Gemin2
Same as above
Human
BC028095.1
exon
192
276
4


Gemin2
Same as above
Human
BC028095.1
stop codon
234
236
4


Gemin2
Same as above
Human
BC028095.1
exon:exon
276
277
4






junction


Gemin2
Same as above
Human
BC028095.1
exon
277
366
4


Gemin2
Same as above
Human
BC028095.1
exon:exon
366
367
4






junction


Gemin2
Same as above
Human
BC028095.1
exon
367
426
4


Gemin2
Same as above
Human
BC028095.1
exon:exon
426
427
4






junction


Gemin2
Same as above
Human
BC028095.1
exon
427
544
4


Gemin2
Same as above
Human
BC028095.1
exon:exon
544
545
4






junction


Gemin2
Same as above
Human
BC028095.1
exon
545
658
4


Gemin2
Same as above
Human
BC028095.1
exon:exon
658
659
4






junction


Gemin2
Same as above
Human
BC028095.1
exon
659
703
4


Gemin2
Same as above
Human
BC028095.1
exon:exon
703
704
4






junction


Gemin2
Same as above
Human
BC028095.1
exon
704
772
4


Gemin2
Same as above
Human
BC028095.1
exon:exon
772
773
4






junction


Gemin2
Same as above
Human
BC028095.1
exon
773
883
4


Gemin2
Same as above
Human
BC028095.1
exon:exon
883
884
4






junction


Gemin2
Same as above
Human
BC028095.1
exon
884
942
4


Gemin2
Same as above
Human
BC028095.1
exon:exon
942
943
4






junction


Gemin2
Same as above
Human
BC028095.1
exon
943
1476
4


Gemin2
Same as above
Human
BC028095.1
3′UTR
952
999
4


Gemin2
Same as above
Human
BC028095.1
stop codon
980
982
4


Gemin2
Same as above
Human
BC028095.1
3′UTR
983
1341
4


Gemin2
Same as above
Human
BG779734.1
exon:exon
154
155
5






junction


Gemin2
Same as above
Human
BG779734.1
exon
155
239
5


Gemin2
Same as above
Human
BG779734.1
stop codon
197
199
5


Gemin2
Same as above
Human
BG779734.1
exon:exon
239
240
5






junction


Gemin2
Same as above
Human
BG779734.1
exon
240
329
5


Gemin2
Same as above
Human
BG779734.1
exon:exon
329
330
5






junction


Gemin2
Same as above
Human
BG779734.1
exon
330
389
5


Gemin2
Same as above
Human
BG779734.1
exon:exon
389
390
5






junction


Gemin2
Same as above
Human
BG779734.1
exon
390
503
5


Gemin2
Same as above
Human
BG779734.1
exon:exon
503
504
5






junction


Gemin2
Same as above
Human
BG779734.1
exon
504
548
5


Gemin2
Same as above
Human
BG779734.1
exon:exon
548
549
5






junction


Gemin2
Same as above
Human
BG779734.1
exon
549
659
5


Gemin2
Same as above
Human
BG779734.1
exon:exon
659
660
5






junction


Gemin2
Same as above
Human
BI602162.1
exon:exon
173
174
6






junction


Gemin2
Same as above
Human
BI602162.1
exon
174
258
6


Gemin2
Same as above
Human
BI602162.1
stop codon
216
218
6


Gemin2
Same as above
Human
BI602162.1
exon:exon
258
259
6






junction


Gemin2
Same as above
Human
BI602162.1
exon
259
348
6


Gemin2
Same as above
Human
BI602162.1
exon:exon
348
349
6






junction


Gemin2
Same as above
Human
BI602162.1
exon
349
408
6


Gemin2
Same as above
Human
BI602162.1
exon:exon
408
409
6






junction


Gemin2
Same as above
Human
BI602162.1
exon
409
453
6


Gemin2
Same as above
Human
BI602162.1
exon:exon
453
454
6






junction


Gemin2
Same as above
Human
BI602162.1
exon
454
564
6


Gemin2
Same as above
Human
BI602162.1
exon:exon
564
565
6






junction


Gemin2
Same as above
Human
BI602162.1
exon
565
623
6


Gemin2
Same as above
Human
BI602162.1
exon:exon
623
624
6






junction


Gemin2
Same as above
Human
BI602162.1
3′UTR
633
680
6


Gemin2
Same as above
Human
BI602162.1
stop codon
661
663
6


Gemin2
Same as above
Human
BI602162.1
3′UTR
664
680
6


Gemin2
Same as above
Human
NM_003616.1
5′UTR
1
83
7


Gemin2
Same as above
Human
NM_003616.1
exon
1
253
7


Gemin2
Same as above
Human
NM_003616.1
start codon
84
86
7


Gemin2
Same as above
Human
NM_003616.1
CDS
84
926
7


Gemin2
Same as above
Human
NM_003616.1
exon:exon
253
254
7






junction


Gemin2
Same as above
Human
NM_003616.1
exon
254
338
7


Gemin2
Same as above
Human
NM_003616.1
stop codon
296
298
7


Gemin2
Same as above
Human
NM_003616.1
3′UTR
299
988
7


Gemin2
Same as above
Human
NM_003616.1
exon:exon
338
339
7






junction


Gemin2
Same as above
Human
NM_003616.1
exon
339
428
7


Gemin2
Same as above
Human
NM_003616.1
exon:exon
428
429
7






junction


Gemin2
Same as above
Human
NM_003616.1
exon
429
488
7


Gemin2
Same as above
Human
NM_003616.1
exon:exon
488
489
7






junction


Gemin2
Same as above
Human
NM_003616.1
exon
489
602
7


Gemin2
Same as above
Human
NM_003616.1
exon:exon
602
603
7






junction


Gemin2
Same as above
Human
NM_003616.1
exon
603
647
7


Gemin2
Same as above
Human
NM_003616.1
exon:exon
647
648
7






junction


Gemin2
Same as above
Human
NM_003616.1
exon
648
716
7


Gemin2
Same as above
Human
NM_003616.1
exon:exon
716
717
7






junction


Gemin2
Same as above
Human
NM_003616.1
exon
717
827
7


Gemin2
Same as above
Human
NM_003616.1
exon:exon
827
828
7






junction


Gemin2
Same as above
Human
NM_003616.1
exon
828
886
7


Gemin2
Same as above
Human
NM_003616.1
exon:exon
886
887
7






junction


Gemin2
Same as above
Human
NM_003616.1
3′UTR
896
943
7


Gemin2
Same as above
Human
NM_003616.1
stop codon
924
926
7


Gemin2
Same as above
Human
NM_003616.1
3′UTR
927
1285
7


Gemin2
Same as above
Human
nucleotides 19913000 to
5′UTR
1037
1119
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
1037
1289
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
start codon
1120
1122
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
1289
1290
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
1290
1642
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
1642
1643
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
1643
1727
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
stop codon
1685
1687
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
1727
1728
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
1728
4812
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
4812
4813
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
4813
4902
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
4902
4903
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
4903
5353
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
5353
5354
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
5354
5413
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
5413
5414
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
5414
8720
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
5414
9243
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
8720
8721
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
8721
8838
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
8838
8839
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
8839
9243
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
9243
9244
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
9244
9357
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
9357
9358
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
9358
11805
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
11805
11806
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
11806
11850
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
11850
11851
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
11851
15093
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
15093
15094
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
15094
15162
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
15162
15163
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
15163
18771
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
18771
18772
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
18772
18882
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
18882
18883
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
18883
20474
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
20474
20475
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
20475
20533
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
20533
20534
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
intron
20534
23253
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
intron:exon
23253
23254
8





19938000 of NT_025892.9
junction


Gemin2
Same as above
Human
nucleotides 19913000 to
exon
23254
23787
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
3′UTR
23263
23310
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
stop codon
23291
23293
8





19938000 of NT_025892.9


Gemin2
Same as above
Human
nucleotides 19913000 to
3′UTR
23294
23652
8





19938000 of NT_025892.9


Gemin4
GIP1; gem-
Human
AF177341.2
CDS
1310
4486
9



associated



protein 4


Gemin4
Same as above
Human
AF177341.2
intron:exon
1319
1320
9






junction


Gemin4
Same as above
Human
AF177341.2
CDS
1343
4486
9


Gemin4
Same as above
Human
AF177341.2
stop codon
4484
4486
9


Gemin4
Same as above
Human
BG702457.1
exon
5
146
10


Gemin4
Same as above
Human
BG702457.1
exon:exon
146
147
10






junction


Gemin4
Same as above
Human
BI458671.1
exon:exon
44
45
11






junction


Gemin4
Same as above
Human
BI458671.1
exon
45
140
11


Gemin4
Same as above
Human
BI458671.1
exon:exon
140
141
11






junction


Gemin4
Same as above
Human
NM_015721.1
5′UTR
1
23
12


Gemin4
Same as above
Human
NM_015721.1
start codon
24
26
12


Gemin4
Same as above
Human
NM_015721.1
CDS
24
3200
12


Gemin4
Same as above
Human
NM_015721.1
exon:exon
33
34
12






junction


Gemin4
Same as above
Human
NM_015721.1
stop codon
3198
3200
12


Gemin4
Same as above
Human
NM_015721.1
3′UTR
3201
3472
12


Gemin4
Same as above
Human
the complement of
exon
1686
1827
13





nucleotides 144000 to





155000 of NT_035414.1


Gemin4
Same as above
Human
the complement of
intron:exon
1827
1828
13





nucleotides 144000 to
junction





155000 of NT_035414.1


Gemin4
Same as above
Human
the complement of
intron
1828
4050
13





nucleotides 144000 to





155000 of NT_035414.1


Gemin4
Same as above
Human
the complement of
intron:exon
4050
4051
13





nucleotides 144000 to
junction





155000 of NT_035414.1


Gemin4
Same as above
Human
the complement of
exon
4051
4146
13





nucleotides 144000 to





155000 of NT_035414.1


Gemin4
Same as above
Human
the complement of
intron:exon
4146
4147
13





nucleotides 144000 to
junction





155000 of NT_035414.1


Gemin4
Same as above
Human
the complement of
intron
4147
5927
13





nucleotides 144000 to





155000 of NT_035414.1


Gemin4
Same as above
Human
the complement of
intron:exon
5927
5928
13





nucleotides 144000 to
junction





155000 of NT_035414.1


Gemin4
Same as above
Human
the complement of
exon
5928
9616
13





nucleotides 144000 to





155000 of NT_035414.1


Gemin4
Same as above
Human
the complement of
stop codon
9097
9099
13





nucleotides 144000 to





155000 of NT_035414.1


Gemin5
DKFZP586M1
Human
AL117665.1
CDS
1
3672
14



824 protein;



gem-associated



protein 5


Gemin5
Same as above
Human
AL117665.1
exon:exon
59
60
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
60
225
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
225
226
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
226
438
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
438
439
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
439
524
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
524
525
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
525
607
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
607
608
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
608
744
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
744
745
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
745
818
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
818
819
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
819
1000
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
1000
1001
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
1001
1140
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
1140
1141
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
1141
1312
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
1312
1313
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
1313
1540
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
1540
1541
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
1541
1654
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
1654
1655
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
1655
1777
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
1777
1778
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
1778
1873
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
1873
1874
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
1874
2011
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
2011
2012
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
2012
2159
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
2159
2160
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
2160
2279
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
2279
2280
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
2280
2490
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
2490
2491
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
2491
2742
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
2742
2743
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
2743
2905
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
2905
2906
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
2906
3407
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
3407
3408
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
3408
3504
14


Gemin5
Same as above
Human
AL117665.1
exon:exon
3504
3505
14






junction


Gemin5
Same as above
Human
AL117665.1
exon
3505
4569
14


Gemin5
Same as above
Human
AL117665.1
stop codon
3670
3672
14


Gemin5
Same as above
Human
AL117665.1
3′UTR
3673
4586
14


Gemin5
Same as above
Human
BC036894.1
5′UTR
1
63
15


Gemin5
Same as above
Human
BC036894.1
exon
11
229
15


Gemin5
Same as above
Human
BC036894.1
start codon
64
66
15


Gemin5
Same as above
Human
BC036894.1
CDS
64
2301
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
229
230
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
230
390
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
390
391
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
391
572
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
572
573
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
573
724
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
724
725
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
725
844
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
844
845
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
845
977
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
977
978
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
978
1143
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
1143
1144
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
1144
1356
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
1356
1357
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
1357
1442
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
1442
1443
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
1443
1525
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
1525
1526
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
1526
1662
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
1662
1663
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
1663
1736
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
1736
1737
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
1737
1918
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
1918
1919
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
1919
2058
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
2058
2059
15






junction


Gemin5
Same as above
Human
BC036894.1
exon
2059
2230
15


Gemin5
Same as above
Human
BC036894.1
exon:exon
2230
2231
15






junction


Gemin5
Same as above
Human
the complement of
exon
839
1057
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
start codon
892
894
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
1057
1058
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
1058
1839
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
1839
1840
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
1840
2000
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
2000
2001
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
2001
3002
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
3002
3003
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
3003
3184
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
3184
3185
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
3185
6774
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
6774
6775
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
6775
6926
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
6926
6927
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
6927
7447
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
7447
7448
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
7448
7567
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
7567
7568
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
7568
10365
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
10365
10366
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
10366
10498
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
10498
10499
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
10499
11474
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
11474
11475
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
11475
11640
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
11640
11641
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
11641
12950
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
12950
12951
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
12951
13163
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
13163
13164
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
13164
14470
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
14470
14471
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
14471
14556
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
14556
14557
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
14557
17599
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
17599
17600
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
17600
17682
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
17682
17683
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
17683
18921
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
18921
18922
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
18922
19058
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
19058
19059
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
19059
21020
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
21020
21021
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
21021
21094
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
21094
21095
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
21095
21845
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
21845
21846
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
21846
22027
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
22027
22028
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
22028
25986
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
25986
25987
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
25987
26126
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
26126
26127
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
26127
27126
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
27126
27127
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
27127
27298
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
27298
27299
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
27299
31206
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
31206
31207
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
31207
31434
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
31434
31435
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
31435
33548
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
33548
33549
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
33549
33662
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
33662
33663
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
33663
34395
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
34395
34396
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
34396
34518
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
34518
34519
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
34519
35849
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
35849
35850
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
35850
35945
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
35945
35946
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
35946
36348
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
36348
36349
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
36349
36486
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
36486
36487
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
36487
37540
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
37540
37541
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
37541
37688
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
37688
37689
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron
37689
39722
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
39722
39723
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
exon
39723
39842
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the complement of
intron:exon
39842
39843
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron
39843
40382
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
40382
40383
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
exon
40383
40593
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
40593
40594
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron
40594
42690
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
42690
42691
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
exon
42691
42942
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
42942
42943
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron
42943
46480
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
46480
46481
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
exon
46481
46643
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
46643
46644
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron
46644
47287
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
47287
47288
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
exon
47288
47789
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
47789
47790
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron
47790
49614
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
49614
49615
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
exon
49615
49711
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
49711
49712
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron
49712
50659
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
intron:exon
50659
50660
16





nucleotides 2065000 to
junction





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
exon
50660
51724
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
stop codon
50825
50827
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin5
Same as above
Human
the compleme of
3′UTR
50828
51741
16





nucleotides 2065000 to





2118000 of NT_034779.1


Gemin6
FLJ23459;
Human
BG944981.1
exon
2
73
17



GEM-



associated



protein 6;



gemin 6;



hypothetical



protein



FLJ23459


Gemin6
Same as above
Human
BG944981.1
5′UTR
7
92
17


Gemin6
Same as above
Human
BG944981.1
exon:exon
73
74
17






junction


Gemin6
Same as above
Human
BG944981.1
exon
74
220
17


Gemin6
Same as above
Human
BG944981.1
start codon
93
95
17


Gemin6
Same as above
Human
BG944981.1
exon:exon
220
221
17






junction


Gemin6
Same as above
Human
BG944981.1
exon
221
404
17


Gemin6
Same as above
Human
BI600222.1
5′UTR
30
86
18


Gemin6
Same as above
Human
BI600222.1
exon:exon
67
68
18






junction


Gemin6
Same as above
Human
BI600222.1
start codon
87
89
18


Gemin6
Same as above
Human
BI600222.1
exon:exon
216
217
18






junction


Gemin6
Same as above
Human
BI600222.1
stop codon
596
598
18


Gemin6
Same as above
Human
NM_024775.1
5′UTR
1
86
19


Gemin6
Same as above
Human
NM_024775.1
exon:exon
67
68
19






junction


Gemin6
Same as above
Human
NM_024775.1
exon
68
214
19


Gemin6
Same as above
Human
NM_024775.1
start codon
87
89
19


Gemin6
Same as above
Human
NM_024775.1
CDS
87
530
19


Gemin6
Same as above
Human
NM_024775.1
exon:exon
214
215
19






junction


Gemin6
Same as above
Human
NM_024775.1
stop codon
528
530
19


Gemin6
Same as above
Human
NM_024775.1
3′UTR
531
703
19


Gemin6
Same as above
Human
NM_024775.1
stop codon
589
591
19


Gemin6
Same as above
Human
NM_024775.8
5′UTR
1
57
20


Gemin6
Same as above
Human
NM_024775.8
exon:exon
38
39
20






junction


Gemin6
Same as above
Human
NM_024775.8
exon
39
185
20


Gemin6
Same as above
Human
NM_024775.8
start codon
58
60
20


Gemin6
Same as above
Human
NM_024775.8
start codon
58
60
20


Gemin6
Same as above
Human
NM_024775.8
CDS
58
561
20


Gemin6
Same as above
Human
NM_024775.8
exon:exon
185
186
20






junction


Gemin6
Same as above
Human
NM_024775.8
stop codon
498
500
20


Gemin6
Same as above
Human
NM_024775.8
stop codon
559
561
20


Gemin6
Same as above
Human
NM_024775.8
3′UTR
562
646
20


Gemin6
Same as above
Human
nucleotides 5647000 to
exon
1043
1114
21





5653000 of NT_005367.10


Gemin6
Same as above
Human
nucleotides 5647000 to
intron:exon
1114
1115
21





5653000 of NT_005367.10
junction


Gemin6
Same as above
Human
nucleotides 5647000 to
intron
1115
1804
21





5653000 of NT_005367.10


Gemin6
Same as above
Human
nucleotides 5647000 to
intron:exon
1804
1805
21





5653000 of NT_005367.10
junction


Gemin6
Same as above
Human
nucleotides 5647000 to
exon
1805
1951
21





5653000 of NT_005367.10


Gemin6
Same as above
Human
nucleotides 5647000 to
start codon
1824
1826
21





5653000 of NT_005367.10


Gemin6
Same as above
Human
nucleotides 5647000 to
intron:exon
1951
1952
21





5653000 of NT_005367.10
junction


Gemin6
Same as above
Human
nucleotides 5647000 to
intron
1952
2090
21





5653000 of NT_005367.10


Gemin6
Same as above
Human
nucleotides 5647000 to
intron:exon
2090
2091
21





5653000 of NT_005367.10
junction


Gemin6
Same as above
Human
nucleotides 5647000 to
exon
2091
2274
21





5653000 of NT_005367.10


Gemin6
Same as above
Human
nucleotides 5647000 to
intron:exon
2274
2275
21





5653000 of NT_005367.10
junction


Gemin6
Same as above
Human
nucleotides 5647000 to
intron
2275
4356
21





5653000 of NT_005367.10


Gemin6
Same as above
Human
nucleotides 5647000 to
intron:exon
4356
4357
21





5653000 of NT_005367.10
junction


Gemin6
Same as above
Human
nucleotides 5647000 to
exon
4357
4926
21





5653000 of NT_005367.10


Gemin6
Same as above
Human
nucleotides 5647000 to
stop codon
4669
4671
21





5653000 of NT_005367.10


Gemin6
Same as above
Human
nucleotides 5647000 to
stop codon
4730
4732
21





5653000 of NT_005367.10


Gemin7
Gemin 7;
Human
AI022330.1
intron:exon
117
118
22



hypothetical


junction



protein



FLJ13956


Gemin7
Same as above
Human
BM009097.1
exon
2
86
23


Gemin7
Same as above
Human
BM009097.1
exon
2
86
23


Gemin7
Same as above
Human
BM009097.1
exon:exon
86
87
23






junction


Gemin7
Same as above
Human
BM009097.1
start codon
95
97
23


Gemin7
Same as above
Human
BM009097.1
stop codon
489
491
23


Gemin7
Same as above
Human
BQ438140.1
start codon
76
78
24


Gemin7
Same as above
Human
BQ438140.1
CDS
76
471
24


Gemin7
Same as above
Human
BQ438140.1
CDS
76
471
24


Gemin7
Same as above
Human
BQ438140.1
stop codon
469
471
24


Gemin7
Same as above
Human
NM_024707.1
5′UTR
1
221
25


Gemin7
Same as above
Human
NM_024707.1
exon
2
90
25


Gemin7
Same as above
Human
NM_024707.1
exon
2
90
25


Gemin7
Same as above
Human
NM_024707.1
exon:exon
90
91
25






junction


Gemin7
Same as above
Human
NM_024707.1
exon
91
213
25


Gemin7
Same as above
Human
NM_024707.1
exon
91
213
25


Gemin7
Same as above
Human
NM_024707.1
exon:exon
213
214
25






junction


Gemin7
Same as above
Human
NM_024707.1
exon
214
1631
25


Gemin7
Same as above
Human
NM_024707.1
exon
214
1631
25


Gemin7
Same as above
Human
NM_024707.1
start codon
222
224
25


Gemin7
Same as above
Human
NM_024707.1
CDS
222
617
25


Gemin7
Same as above
Human
NM_024707.1
stop codon
615
617
25


Gemin7
Same as above
Human
NM_024707.1
3′UTR
618
1631
25


Gemin7
Same as above
Human
nucleotides 1708000 to
exon
1467
1551
26





1723000 of NT_011109.12


Gemin7
Same as above
Human
nucleotides 1708000 to
intron:exon
1551
1552
26





1723000 of NT_011109.12
junction


Gemin7
Same as above
Human
nucleotides 1708000 to
intron
1552
2178
26





1723000 of NT_011109.12


Gemin7
Same as above
Human
nucleotides 1708000 to
exon
1561
1649
26





1723000 of NT_011109.12


Gemin7
Same as above
Human
nucleotides 1708000 to
intron:exon
1649
1650
26





1723000 of NT_011109.12
junction


Gemin7
Same as above
Human
nucleotides 1708000 to
intron
1650
2178
26





1723000 of NT_011109.12


Gemin7
Same as above
Human
nucleotides 1708000 to
intron:exon
2178
2179
26





1723000 of NT_011109.12
junction


Gemin7
Same as above
Human
nucleotides 1708000 to
exon
2179
2301
26





1723000 of NT_011109.12


Gemin7
Same as above
Human
nucleotides 1708000 to
intron:exon
2301
2302
26





1723000 of NT_011109.12
junction


Gemin7
Same as above
Human
nucleotides 1708000 to
intron
2302
12378
26





1723000 of NT_011109.12


Gemin7
Same as above
Human
nucleotides 1708000 to
intron:exon
12378
12379
26





1723000 of NT_011109.12
junction


Gemin7
Same as above
Human
nucleotides 1708000 to
exon
12379
13796
26





1723000 of NT_011109.12


Gemin7
Same as above
Human
nucleotides 1708000 to
start codon
12387
12389
26





1723000 of NT_011109.12


Gemin7
Same as above
Human
nucleotides 1708000 to
CDS
12387
12782
26





1723000 of NT_011109.12


Gemin7
Same as above
Human
nucleotides 1708000 to
stop codon
12780
12782
26





1723000 of NT_011109.12


Gemin7
Same as above
Human
nucleotides 1708000 to
3′UTR
12783
13796
26





1723000 of NT_011109.12









Small Non-Coding RNA-Regulated Regions
Small non-coding RNA molecules play important roles in regulation of gene expression, developmental timing, viral surveillance, and immunity. Not only the classic transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), but also small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), small interfering RNAs (siRNAs), tiny non-coding RNAs (tncRNAs) and microRNAs (miRNAs) are now known to act in diverse cellular processes such as chromosome maintenance, gene imprinting, pre-mRNA splicing, guiding RNA modifications, transcriptional regulation, and the control of mRNA translation (Eddy, Nat. Rev. Genet., 2001, 2, 919-929; Kawasaki and Taira, Nature, 2003, 423, 838-842). RNA-mediated processes are now also believed to direct heterochromatin formation, genome rearrangements, and DNA elimination (Cerutti, Trends Genet., 2003, 19, 39-46; Couzin, Science, 2002, 298, 2296-2297). In one embodiment, regions of target genes that are targets of small non-coding RNAs are suitable targets for oligomeric compounds of the invention.
One class of small non-coding RNAs known as microRNAs (miRNAs) participates in regulation of gene expression. Mature miRNAs originate from long endogenous primary transcripts (pri-miRNAs) that are often hundreds of nucleotides in length (Lee et al., EMBO J., 2002, 21, 4663-70). These pri-miRNAs are processed by a nucleolar enzyme in the RNase III family known as Drosha, into approximately 70 nucleotide-long pre-miRNAs (also known as stem-loop, hairpin or foldback precursors) which are subsequently processed by the Dicer RNase into mature miRNAs (Lee et al., Nature, 2003, 425, 415-419). The current model is that the primary miRNA transcript is processed by Drosha in the nucleus, and the pre-miRNA hairpin precursor is exported from the nucleus through the action of the nuclear export protein exportin-5 (Bohnsack et al., RNA, 2004, 10, 185-191; Lund et al., Science, 2004, 303, 95-98; Yi et al., Genes Dev., 2003, 17, 3011-3016). Once in the cytoplasm, the pre-miRNA is cleaved by Dicer to yield a double-stranded intermediate, but only one strand of this short-lived intermediate accumulates as the mature miRNA (Ambros et al., RNA, 2003, 9, 277-279; Bartel and Bartel, Plant Physiol., 2003, 132, 709-717; Shi, Trends Genet., 2003, 19, 9-12). miRNAs are believed to primarily direct translation repression but have recently been shown to trigger cleavage events.
More than 200 miRNA genes have been detected in the human genome. Naturally occurring miRNAs are characterized by imperfect complementarity to their target sequences. Artificially modified miRNAs with sequences completely complementary to their target RNAs have been designed and found to function as siRNAs that inhibit gene expression by reducing RNA transcript levels. Accordingly, regions of Gemin Genes targeted by naturally occurring or artificially modified miRNAs are contemplated as suitable target sites for the oligomeric compounds of the present invention.
Modulation of Target Expression
As used herein, “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 and “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 a Gemin Gene. 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.
The effect of oligomeric compounds of the present invention on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. The effect of oligomeric compounds of the present invention on target nucleic acid expression can be routinely determined using, for example, PCR or Northern blot analysis. Useful cell lines include, e.g., 1321rX3-7; 3T3-L1, differentiated; 3T3-L1, undifferentiated; 70Z3; 7D4; 7F2 (osteoblast); A10; A20; A375; A431; A549; AML-12; ARIP; differentiated Adipocytes; B104; B16-F10; B50; BALC; BB88; BC3H1; BCL; BEAS2B; BHK-21 (fibroblast, kidney); BLO-11 (skeletal muscle); BT-474; BW5147.3 (ATCC TIB-47); BaF3; Mouse primary bone marrow-derived osteoclasts; C2C12; C3A; C3H/10T1/2; C58; C6; CHO (Ovary); CMT-93; COS-7; CT26.WT; Caco-2; ConA; D1 TNC1; D1B; DA-3; DDT1-MF2; DU 145 (prostate); Peripheral Blood Monocyte derived Human Primary Dendritic Cells; E14; EL4; EMT-6; F11; FAT 7 (epithelial, nasal squamous cell carcinoma); Human Primary Dermal Fibroblasts; Mouse Embryonic Primary Fibroblasts; G-361; GH1; GH3; H-4-II-E; H2.35; H8; H9 (Human T Lymphocyte); H9c2(2-1); HASMC (Human Aortic Smooth Muscle Cells); HC252 (differentiated rat neuronal progenitor cell line); HC252 (undifferentiated rat neuronal progenitor cell line); HCT116; HEK A-Z (rt-SLC tx); HEK-293; HEK-293 (Rat VR1 Transfected); HEPA1-6; HFN 36.3; HK-2 (human HPV-16 transformed proximal tubule kidney); HL-60; HMEC (Normal Human Mammary Epithelial Cells); HMVEC-L (Lung Endothelial); HMVEC-d Ad (Human Adult Dermal Endothelial); HMVEC-d Neo (Human Neonatal Dermal Endothelial); HPAEC (Human Pulmonary Artery Endothelial Cells); HT-1080; HeLa; Hec-1A; HepB3; HepG2; Human Primary Hepatocytes; Mouse Primary Hepatocytes; Rabbit Primary Hepatocytes; Rat Primary Hepatocytes; HuT 78 (Human cutaneous T lymphocyte); HuVEC; Huh7; Human H-ras transformed rat intestinal epithelial cells; IC21; IEC-6; IW32; JAR; JEG-3; JUG-3; Jurkat; K-562; K204; Mouse Primary Keratinocytes; L2 (lung); L6; LA4; LBRM-33; LC-540; LL/6; LL2; LLC1; LNcAP; M-3 (Mouse melanoma; skin; melanocyte); MCF7 (breast adenocarcinoma, w/t p53); MDA; MDA MB 468; MDA MB231; MEF; MH-S; MLE12; MLg2908; MMT 060562; MRC-5; Human Primary Macrophages; Mouse Peritoneal Macrophages; Rat Peritoneal Macrophages; Human Primary Melanocytes; Mia Paca; Human Primary Monocytes; N1S1 (liver); NBT-II; NCCIT; NCI-H292; NCTC 3749; ND7/23; NG108-15 (mouse); NG108-15 (rat); NHDC (Dendritic Cells); NHDF; NHEK (Human Primary Keratinocytes); NHEK-Ad (Human Primary Adult Keratinocytes); NHEK-Neo (Human Primary Neonatal Keratinocytes); NIH/3T3 (mouse fibroblast); NIT-1; NOR-10 (Mouse muscle); NR-8383; NRK; NTERA-2 c1.D1; Rat Primary Neurons; Mouse primary Osteoblasts; Rat primary Osteoblasts; P-19; P388D1 (IL-1) adherent; P388D1 suspension; PANC-1; PC-12; PC-3 (prostate); White Preadipocytes; R2C; R6; RAW264.7; RB++; RBL-2H3; RFL-6; RK3E; RMC; ROS 17/2.8; Raji; Rat Tissue—Cerebellum; Rat Tissue—Cerebrum; Rat Tissue—Hippocampus; Rat-2; Human Primary Renal Proximal Tubule Epithelial Cell; Rin-5F; Rin-M; SK-MEL-28; SKBR; SMT/2A LNM; SV40 MES 13; SW480; SW97; Shionogi; Human Primary Smooth Muscle Bronchial Cell; Mouse Primary Splenocytes; Human Synoviocytes; Mouse Synoviocytes; Rat Synoviocytes; T cell hybridoma 2B4; T-24; T-49 D; T3-3A1; T47D (breast adenocarcinoma, mutant p53); T47D+p53 (breast adenocarcinoma, mutant p53, transfected with wild-type p53); TCMK-1 (kidney); TF1.8; THLE-3; THP-1; TM-3; TM4; TRAMP-C1; U-20S; U-87 MG; U373; U937; UMR-106 (osteosarcoma); VERO C1008; WEHI 231; WISH; Y-1; Y13-238 (spleen); Y13-259 (spleen); YB2/0 (spleen); Yac-1; b.END; mIMCD-3; and sw872. The culture of such cells is routine to those skilled in the art. Other cell types well known to one of skilled in the art can be routinely used. Many cell lines and instructions for growing them are obtainable from the American Type Culture Collection (ATCC) (Manassas, Va.).
Assaying Modulation of Expression
Modulation of Gemin Gene expression can be assayed in a variety of ways known in the art. Gemin Gene 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 a Gemin Gene can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to a protein encoded by a Gemin Gene 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.
Suitable Target Regions
Once one or more target regions, segments or sites have been identified, oligomeric compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
The oligomeric compounds of the present invention can be targeted to features of a target nucleobase sequence, such as those described in Table 1. All regions of a nucleobase sequence to which an oligomeric compound can be targeted, wherein the regions are greater than or equal to 8 and less than or equal to 80 nucleobases, are described as follows:
Let R(n, n+m−1) be a region from a target nucleobase sequence, where “n” is the 5′-most nucleobase position of the region, where “n+m−1” is the 3′-most nucleobase position of the region and where “m” is the length of the region. A set “S(m)”, of regions of length “m” is defined as the regions where n ranges from 1 to L−m+1, where L is the length of the target nucleobase sequence and L>m. A set, “A”, of all regions can be constructed as a union of the sets of regions for each length from where m is greater than or equal to 8 and is less than or equal to 80.
This set of regions can be represented using the following mathematical notation:



  
    A
    =
    
      
        
          
            ⋃
            m
          
          ⁢
          
            
              S
              ⁡
              
                (
                m
                )
              
            
            ⁢
            
              
            
            ⁢
            where
            ⁢
            
              
            
            ⁢
            m
          
        
        ∈
        N
      
      ❘
      
        8
        ≤
        m
        ≤
        80
      
    
  




  and




  
    
      S
      ⁡
      
        (
        m
        )
      
    
    =
    
      {
      
        
          R
          
            n
            ,
            
              n
              +
              m
              -
              1
            
          
        
        ❘
        
          n
          ∈
          
            {
            
              1
              ,
              2
              ,
              3
              ,
              ...
              ⁢
              
                
              
              ,
              
                L
                -
                m
                +
                1
              
            
            }
          
        
      
      }
    
  



    
    
        where the mathematical operator | indicates “such that”,
        where the mathematical operator ε indicates “a member of a set” (e.g. y ε Z indicates that element y is a member of set Z),
        where x is a variable,
        where N indicates all natural numbers, defined as positive integers,
        and where the mathematical operator Å indicates “the union of sets”.
    
    


For example, the set of regions for m equal to 8, 20 and 80 can be constructed in the following manner. The set of regions, each 8 nucleobases in length, S(m=8), in a target nucleobase sequence 100 nucleobases in length (L=100), beginning at position 1 (n=1) of the target nucleobase sequence, can be created using the following expression:

S(8)={R1,8|nε{1,2,3, . . . , 93}}

and describes the set of regions comprising nucleobases 1-8, 2-9, 3-10, 4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19, 13-20, 14-21, 15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28, 22-29, 23-30, 24-31, 25-32, 26-33, 27-34, 28-35, 29-36, 30-37, 31-38, 32-39, 33-40, 34-41, 35-42, 36-43, 37-44, 38-45, 39-46, 40-47, 41-48, 42-49, 43-50, 44-51, 45-52, 46-53, 47-54, 48-55, 49-56, 50-57, 51-58, 52-59, 53-60, 54-61, 55-62, 56-63, 57-64, 58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71, 65-72, 66-73, 67-74, 68-75, 69-76, 70-77, 71-78, 72-79, 73-80, 74-81, 75-82, 76-83, 77-84, 78-85, 79-86, 80-87, 81-88, 82-89, 83-90, 84-91, 85-92, 86-93, 87-94, 88-95, 89-96, 90-97, 91-98, 92-99, 93-100.

An additional set for regions 20 nucleobases in length, in a target sequence 100 nucleobases in length, beginning at position 1 of the target nucleobase sequence, can be described using the following expression:

S(20)={R1,20|n ε{1,2,3, . . . , 81}}

and describes the set of regions comprising nucleobases 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72, 54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82, 64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92, 74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100.

An additional set for regions 80 nucleobases in length, in a target sequence 100 nucleobases in length, beginning at position 1 of the target nucleobase sequence, can be described using the following expression:

S(80)={R1,80|n ε{1,2,3, . . . , 21}}

and describes the set of regions comprising nucleobases 1-80, 2-81, 3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91, 13-92, 14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.

Thus, in this example, A would include regions 1-8, 2-9, 3-10 . . . 93-100, 1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82 . . . 21-100.
The union of these aforementioned example sets and other sets for lengths from 10 to 19 and 21 to 79 can be described using the mathematical expression




  A
  =
  
    
      ⋃
      m
    
    ⁢
    
      S
      ⁡
      
        (
        m
        )
      
    
  




    
    
        where Å represents the union of the sets obtained by combining all members of all sets.
    
    


The mathematical expressions described herein defines all possible target regions in a target nucleobase sequence of any length L, where the region is of length m, and where m is greater than or equal to 8 and less than or equal to 80 nucleobases and, and where m is less than L, and where n is less than L−m+1.
Validated Target Segments
The locations on the target nucleic acid to which the suitable 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 nucleobases). Similarly suitable 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 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 about 80 nucleobases.
Screening for Modulator Compounds
In another embodiment, the validated target segments identified herein can be employed in a screen for additional compounds that modulate the expression of a Gemin Gene. “Modulators” are those compounds that modulate the expression of a Gemin Gene 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 a Gemin Gene with one or more candidate modulators, and selecting for one or more candidate modulators which perturb the expression of a nucleic acid molecule encoding a Gemin Gene. Once it is shown that the candidate modulator or modulators are capable of modulating the expression of a nucleic acid molecule encoding a Gemin Gene, the modulator can then be employed in further investigative studies of the function of a Gemin Gene, 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.
Phenotypic Assays
Once modulator compounds of a Gemin Gene have been identified by the methods disclosed herein, the compounds can be further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of a Gemin Gene in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).
Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.
Analysis of the genotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the Gemin Gene modulators. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.
The following phenotypic assays are useful in the study of the compounds and compositions of the present invention.
Cell Proliferation and Survival
Unregulated cell proliferation is a characteristic of cancer cells, thus most current chemotherapy agents target dividing cells, for example, by blocking the synthesis of new DNA required for cell division. However, cells in healthy tissues are also affected by agents that modulate cell proliferation.
In some cases, a cell cycle inhibitor will cause apoptosis in cancer cells, but allow normal cells to undergo growth arrest and therefore remain unaffected (Blagosklonny, Bioessays, 1999, 21, 704-709; Chen et al., Cancer Res., 1997, 57, 2013-2019; Evan and Littlewood, Science, 1998, 281, 1317-1322; Lees and Weinberg, Proc. Natl. Acad. Sci. USA, 1999, 96, 4221-4223). An example of sensitization to anti-cancer agents is observed in cells that have reduced or absent expression of the tumor suppressor genes p53 (Bunz et al., Science, 1998, 282, 1497-1501; Bunz et al., J. Clin. Invest., 1999, 104, 263-269; Stewart et al., Cancer Res., 1999, 59, 3831-3837; Wahl et al., Nat. Med., 1996, 2, 72-79). However, cancer cells often escape apoptosis (Lowe and Lin, Carcinogenesis, 2000, 21, 485-495; Reed, Cancer J. Sci. Am., 1998, 4 Suppl 1, S8-14). Further disruption of cell cycle checkpoints in cancer cells can increase sensitivity to chemotherapy while allowing normal cells to take refuge in G1 and remain unaffected. Cell cycle assays can be employed to identify genes, such as p53, whose inhibition will sensitize cells to anti-cancer agents.
Caspase Activity
Programmed cell death, or apoptosis, is an important aspect of various biological processes, including normal cell turnover, as well as immune system and embryonic development. Apoptosis involves the activation of caspases, a family of intracellular proteases through which a cascade of events leads to the cleavage of a select set of proteins. The caspase family can be divided into two groups: the initiator caspases, such as caspase-8 and -9, and the executioner caspases, such as caspase-3, -6 and -7, which are activated by the initiator caspases. The caspase family contains at least 14 members, with differing substrate preferences (Thornberry and Lazebnik, Science, 1998, 281, 1312-1316). For example, a caspase assay can be used to identify genes whose inhibition selectively cause apoptosis in breast carcinoma cell lines, without affecting normal cells, and to identify genes whose inhibition results in cell death in p53-deficient T47D cells, and not in MCF7 cells which express p53 (Ross et al., Nat. Genet., 2000, 24, 227-235; Scherf et al., Nat. Genet., 2000, 24, 236-244).
Angiogenesis
Angiogenesis is the growth of new blood vessels (veins and arteries) by endothelial cells. This process is important in the development of a number of human diseases, and is believed to be particularly important in regulating the growth of solid tumors. Without new vessel formation it is believed that tumors will not grow beyond a few millimeters in size. In addition to their use as anti-cancer agents, inhibitors of angiogenesis have potential for the treatment of diabetic retinopathy, cardiovascular disease, rheumatoid arthritis and psoriasis (Carmeliet and Jain, Nature, 2000, 407, 249-257; Freedman and Isner, J. Mol. Cell. Cardiol., 2001, 33, 379-393; Jackson et al., Faseb J., 1997, 11, 457-465; Saaristo et al., Oncogene, 2000, 19, 6122-6129; Weber and De Bandt, Joint Bone Spine, 2000, 67, 366-383; Yoshida et al., Histol. Histopathol., 1999, 14, 1287-1294).
During the process of angiogenesis, endothelial cells perform several distinct functions, including the degradation of the extracellular matrix (ECM), migration, proliferation and the formation of tube-like structures (Liekens et al., Biochem. Pharmacol., 2001, 61, 253-270). Endothelial cells must regulate the expression of many genes in order to perform the functions necessary for angiogenesis. This gene regulation has been the subject of intense scrutiny, and many genes have been identified as being important for the angiogenic phenotype. For example, the expression levels of the following genes, previously identified as being highly expressed in angiogenic endothelial cells, can be measured as indicators: Integrin beta3, endoglin/CD105, TEM5 and MMP-14/MT-MMP1.
Integrin beta3 is part of a family of heterodimeric transmembrane receptors that consist of alpha and beta subunits (Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822). Each subunit recognizes a unique set of ECM ligands, thereby allowing cells to transmit angiogenic signals from the extracellular matrix. Integrin beta3 is prominently expressed on proliferating vascular endothelial cells, and it plays roles in allowing new blood vessels to form at tumor sites as well as allowing the epithelial cells of breast tumors to spread (Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822; Drake et al., J. Cell Sci., 1995, 108 (Pt 7), 2655-2661). Blockage of integrin beta3 with monoclonal antibodies or low molecular weight antagonists inhibits blood vessel formation in a variety of in-vivo models, including tumor angiogenesis and neovascularization during oxygen-induced retinopathy (Brooks et al., Science, 1994, 264, 569-571; Brooks et al., J. Clin. Invest., 1995, 96, 1815-1822; Hammes et al., Nat. Med., 1996, 2, 529-533).
Endoglin is a transforming growth factor receptor-associated protein highly expressed on endothelial cells, and present on some leukemia cells and minor subsets of bone marrow cells (Burrows et al., Clin. Cancer Res., 1995, 1, 1623-1634; Haruta and Seon, Proc. Natl. Acad. Sci. USA, 1986, 83, 7898-7902). Its expression is upregulated in endothelial cells of angiogenic tissues and is therefore used as a prognostic indicator in various tumors (Burrows et al., Clin. Cancer Res., 1995, 1, 1623-1634). Endoglin functions as an ancillary receptor influencing binding of the transforming growth factor beta (TGF-beta) family of ligands to signaling receptors, thus mediating cell survival (Massague and Chen, Genes Dev., 2000, 14, 627-644).
Tumor endothelial marker 5 (TEM5) is a putative 7-pass transmembrane protein (GPCR) (Carson-Walter et al., Cancer Res., 2001, 61, 6649-6655). The mRNA transcript, designated KIAA1531, encodes one of many tumor endothelium markers (TEMs) that display elevated expression (greater than 10-fold) during tumor angiogenesis (St Croix et al., Science, 2000, 289, 1197-1202). TEM5 is coordinately expressed with other TEMs on tumor endothelium in humans and mice.
Matrix metalloproteinase 14 (MMP-14), a membrane-type MMP covalently linked to the cell membrane, is involved in matrix detachment and migration. MMP-14 is thought to promote tumor angiogenesis; antibodies directed against the catalytic domain of MMP-14 block endothelial-cell migration, invasion and capillary tube formation in vitro (Galvez et al., J. Biol. Chem., 2001, 276, 37491-37500). MMP-14 can degrade the fibrin matrix that surrounds newly formed vessels potentially allowing the endothelial cells to invade further into the tumor tissue (Hotary et al., J. Exp. Med., 2002, 195, 295-308). MMP-14 null mice have impaired angiogenesis during development, further demonstrating the role of MMP-14 in angiogenesis (Vu and Werb, Genes Dev., 2000, 14, 2123-2133; Zhou et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 4052-4057).
Angiogenesis is stimulated by numerous factors that promote interaction of endothelial cells with each other and with extracellular matrix molecules, resulting in the formation of capillary tubes. This morphogenic process is necessary for the delivery of oxygen to nearby tissues and plays an essential role in embryonic development, wound healing, and tumor growth (Carmeliet and Jain, Nature, 2000, 407, 249-257). Moreover, this process can be reproduced in a tissue culture assay that evaluated the formation of tube-like structures by endothelial cells. There are several different variations of the assay that use different matrices, such as collagen I (Kanayasu et al., Lipids, 1991, 26, 271-276), Matrigel (Yamagishi et al., J. Biol. Chem., 1997, 272, 8723-8730) and fibrin (Bach et al., Exp. Cell Res., 1998, 238, 324-334), as growth substrates for the cells. For example, HUVECs can be plated on a matrix derived from the Engelbreth-Holm-Swarm mouse tumor, which is very similar to Matrigel (Kleinman et al., Biochemistry, 1986, 25, 312-318; Madri and Pratt, J. Histochem. Cytochem., 1986, 34, 85-91). Untreated HUVECs form tube-like structures when grown on this substrate. Loss of tube formation in vitro has been correlated with the inhibition of angiogenesis in vivo (Carmeliet and Jain, Nature, 2000, 407, 249-257; Zhang et al., Cancer Res., 2002, 62, 2034-2042), which supports the use of in vitro tube formation as an endpoint for angiogenesis.
Adipocyte Differentiation and Insulin Signaling Assays
Insulin is an essential signaling molecule throughout the body, but its major target organs are the liver, skeletal muscle and adipose tissue. Insulin is the primary modulator of glucose homeostasis and helps maintain a balance of peripheral glucose utilization and hepatic glucose production. The reduced ability of normal circulating concentrations of insulin to maintain glucose homeostasis manifests in insulin resistance which is often associated with diabetes, central obesity, hypertension, polycystic ovarian syndrome, dyslipidemia and atherosclerosis (Saltiel, Cell, 2001, 104, 517-529; Saltiel and Kahn, Nature, 2001, 414, 799-806).
Insulin promotes the differentiation of preadipocytes into adipocytes. The condition of obesity, which results in increases in fat cell number, occurs even in insulin-resistant states in which glucose transport is impaired due to the anti-lipolytic effect of insulin. Inhibition of triglyceride breakdown requires much lower insulin concentrations than stimulation of glucose transport, resulting in maintenance or expansion of adipose stores (Kitamura et al., Mol. Cell. Biol., 1999, 19, 6286-6296; Kitamura et al., Mol. Cell. Biol., 1998, 18, 3708-3717).
One of the hallmarks of cellular differentiation is the upregulation of gene expression. During adipocyte differentiation, the gene expression patterns in adipocytes change considerably. Some genes known to be upregulated during adipocyte differentiation include hormone-sensitive lipase (HSL), adipocyte lipid binding protein (aP2), glucose transporter 4 (Glut4), and peroxisome proliferator-activated receptor gamma (PPAR-gamma). Insulin signaling is improved by compounds that bind and inactivate PPAR-gamma, a key regulator of adipocyte differentiation (Olefsky, J. Clin. Invest., 2000, 106, 467-472). Insulin induces the translocation of GLUT4 to the adipocyte cell surface, where it transports glucose into the cell, an activity necessary for triglyceride synthesis. In all forms of obesity and diabetes, a major factor contributing to the impaired insulin-stimulated glucose transport in adipocytes is the downregulation of GLUT4. Insulin also induces hormone sensitive lipase (HSL), which is the predominant lipase in adipocytes that functions to promote fatty acid synthesis and lipogenesis (Fredrikson et al., J. Biol. Chem., 1981, 256, 6311-6320). Adipocyte fatty acid binding protein (aP2) belongs to a multi-gene family of fatty acid and retinoid transport proteins. aP2 is postulated to serve as a lipid shuttle, solubilizing hydrophobic fatty acids and delivering them to the appropriate metabolic system for utilization (Fu et al., J. Lipid Res., 2000, 41, 2017-2023; Pelton et al., Biochem. Biophys. Res. Commun., 1999, 261, 456-458). Together, these genes play important roles in the uptake of glucose and the metabolism and utilization of fats.
Leptin secretion and an increase in triglyceride content are also well-established markers of adipocyte differentiation. While it serves as a marker for differentiated adipocytes, leptin also regulates glucose homeostasis through mechanisms (autocrine, paracrine, endocrine and neural) independent of the adipocyte's role in energy storage and release. As adipocytes differentiate, insulin increases triglyceride accumulation by both promoting triglyceride synthesis and inhibiting triglyceride breakdown (Spiegelman and Flier, Cell, 2001, 104, 531-543). As triglyceride accumulation correlates tightly with cell size and cell number, it is an excellent indicator of differentiated adipocytes.
Insulin mediates its effects by suppressing the RNA expression levels of enzymes important for gluconeogenesis and glycogenolysis, and also by controlling the activities of some metabolic enzymes by post-translational mechanisms (Hall and Granner, J. Basic Clin. Physiol. Pharmacol., 1999, 10, 119-133; Moller, Nature, 2001, 414, 821-827; Saltiel and Kahn, Nature, 2001, 414, 799-806). However, the mechanisms by which insulin regulates these genes are not fully understood. Genes in liver cells cells that are involved in regulating glucose metabolism are identified by monitoring changes in the expression of selective insulin-responsive genes in a cell culture model. Primary human hepatocytes are difficult to obtain and work with in culture. Therefore, the insulin signaling assay as used in the Examples can be performed in the hepatocellular carcinoma cell line HepG2. Insulin responsive genes that can be evaluated in this assay are phosphoenolpyruvate carboxykinase (PEPCK), insulin-like growth factor binding protein 1 (IGFBP-1) and follistatin.
IGFBP-1 is one of a family of six secreted proteins that bind insulin-like growth factor (IGF) with high affinity and thereby modulate IGFs action in vivo (Baxter, Am. J. Physiol. Endocrinol. Metab., 2000, 278, E967-976; Lee et al., Proc. Soc. Exp. Biol. Med., 1997, 216, 319-357). IGFBP-1 is characterized by dynamic variability of levels in circulation due to the regulation of its hepatic secretion (Lee et al., Proc. Soc. Exp. Biol. Med., 1997, 216, 319-357). The multi-hormonal regulation of PEPCK and IGFBP-1 are similar. Glucocorticoids and cyclic AMP (cAMP) stimulate transcription of the IGFBP-1 gene expression whereas insulin acts in a dominant manner to suppress both basal and cAMP or glucocorticoid-stimulated IGFBP-1 gene transcription (O'Brien and Granner, Physiol. Rev., 1996, 76, 1109-1161). PEPCK catalyzes the rate-limiting step in gluconeogenesis, and thereby contributes to hepatic glucose output (Hall and Granner, J. Basic Clin. Physiol. Pharmacol., 1999, 10, 119-133; Moller, Nature, 2001, 414, 821-827; Saltiel and Kahn, Nature, 2001, 414, 799-806). In hepatoma cells, studies have shown that the expression of PEPCK is stimulated by glucocorticoids, glucagon (via cAMP), and retinoic acid. Insulin acts in a dominant manner to suppress these stimulations as well as basal transcription (O'Brien and Granner, Physiol. Rev., 1996, 76, 1109-1161). In HepG2 cells, prolonged serum starvation induces the expression of PEPCK and subsequent insulin stimulation significantly reduces the PEPCK mRNA level.
Follistatin is significantly stimulated by insulin in HepG2 cells. Interestingly, follistatin levels have been shown to be higher in women with polycystic ovary syndrome (PCOS) (Norman et al., Hum. Reprod., 2001, 16, 668-672). PCOS is a metabolic as well as a reproductive disorder, and an important cause of type 2 diabetes mellitus in women. It is often associated with profound insulin resistance and hyperinsulinemia as well as with a defect in insulin secretion (Dunaif, Endocr. Rev., 1997, 18, 774-800; Nestler et al., Fertil. Steril., 2002, 77, 209-215).
Inflammation Assays
Inflammation assays are designed to identify genes that regulate the activation and effector phases of the adaptive immune response. During the activation phase, T lymphocytes (also known as T-cells) receiving signals from the appropriate antigens undergo clonal expansion, secrete cytokines, and upregulate their receptors for soluble growth factors, cytokines and co-stimulatory molecules (Cantrell, Annu. Rev. Immunol., 1996, 14, 259-274). These changes drive T-cell differentiation and effector function. In the effector phase, response to cytokines by non-immune effector cells controls the production of inflammatory mediators that can do extensive damage to host tissues. The cells of the adaptive immune systems, their products, as well as their interactions with various enzyme cascades involved in inflammation (e.g., the complement, clotting, fibrinolytic and kinin cascades) all represent potential points for intervention in inflammatory disease. The inflammation assay measures hallmarks of the activation phase of the immune response.
Dendritic cells can be used to identify regulators of dendritic cell-mediated T-cell costimulation. The level of interleukin-2 (IL-2) production by T-cells, a critical consequence of T-cell activation (DeSilva et al., J. Immunol., 1991, 147, 3261-3267; Salomon and Bluestone, Annu. Rev. Immunol., 2001, 19, 225-252), is used as an endpoint for T-cell activation. T lymphocytes are important immunoregulatory cells that mediate pathological inflammatory responses. Optimal activation of T lymphocytes requires both primary antigen recognition events as well as secondary or costimulatory signals from antigen presenting cells (APC). Dendritic cells are the most efficient APCs known and are principally responsible for antigen presentation to T-cells, expression of high levels of costimulatory molecules during infection and disease, and the induction and maintenance of immunological memory (Banchereau and Steinman, Nature, 1998, 392, 245-252). While a number of costimulatory ligand-receptor pairs have been shown to influence T-cell activation, a principal signal is delivered by engagement of CD28 on T-cells by CD80 (B7-1) and CD86 (B7-2) on APCs (Boussiotis et al., Curr. Opin. Immunol., 1994, 6, 797-807; Lenschow et al., Annu. Rev. Immunol., 1996, 14, 233-258). While not adhering to a specific mechanism, inhibition of T-cell co-stimulation by APCs holds promise for novel and more specific strategies of immune suppression. In addition, blocking costimulatory signals may lead to the development of long-term immunological anergy (unresponsiveness or tolerance) that would offer utility for promoting transplantation or dampening autoimmunity. T-cell anergy is the direct consequence of failure of T-cells to produce the growth factor IL-2 (DeSilva et al., J. Immunol., 1991, 147, 3261-3267; Salomon and Bluestone, Annu. Rev. Immunol., 2001, 19, 225-252).
The cytokine signaling assay identifies genes that regulate the responses of non-immune effector cells (initially endothelial cells) to cytokines such as interferon-gamma (IFN-gamma). The effects of the oligomeric compounds of the present invention on the regulation of the production of intercellular adhesion molecule-1 (ICAM-1), interferon regulatory factor 1 (IRF1) and small inducible cytokine subfamily B (Cys-X-Cys), member 11 (SCYB 11), which regulate other parameters of the inflammatory response, can be monitored in response to cytokine treatment.
Kits, Research Reagents, and Diagnostics
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 exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
For use in kits and diagnostics, the 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).
Therapeutics
The specificity and sensitivity of antisense technology is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense drugs have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds are useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
For therapeutics, an animal, such as a human, suspected of having or at risk of having a disease or disorder which can be treated by modulating the expression of a Gemin Gene is treated by administering compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to said animal, a therapeutically effective amount of an antisense compound that inhibits expression of a Gemin Gene. In one embodiment, the antisense compounds of the present invention effectively inhibit the levels or function of a Gemin Gene RNA. Because reduction in Gemin Gene mRNA levels can lead to alteration in Gemin Gene protein products of expression as well, such resultant alterations can also be measured. In one embodiment, the antisense compounds of the invention inhibit the expression of a Gemin Gene 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%.
For example, the reduction of the expression of a Gemin Gene 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 a Gemin Gene protein and/or the Gemin Gene-encoded protein itself For example, tissues or fluids procured from patients can be evaluated for expression levels of the target mRNA or protein. Additionally, the mRNA or protein expression levels of other genes known or suspected to be associated with the specific disease state, condition or phenotype can be assessed. 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 disease or condition in the aforementioned tissues and fluids, collected from a patient or subject receiving treatment, 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.
Pharmaceuticals and Methods of Treatment
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 a Gemin Gene. Consequently, the compounds are useful in the treatment of disorders and diseases related to Gemin Gene expression. Use of the compounds, compositions and methods of the invention may also be useful prophylactically. The compounds of the invention can also be used in the manufacture of a medicament for the treatment of diseases and disorders related to Gemin Gene expression.
Methods whereby bodily fluids, cells 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, cells or tissues can be contacted with one or more of the compounds of the invention resulting in modulation of Gemin Gene expression in the bodily fluids, cells 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.
Further contemplated herein is a method for the treatment of a subject suspected of having or at risk of having a disease or disorder comprising administering to a subject an effective amount of an isolated single stranded RNA or double stranded RNA oligonucleotide directed to a Gemin Gene. The ssRNA or dsRNA oligonucleotide may be modified or unmodified. That is, the present invention provides for the use of an isolated double stranded RNA oligonucleotide targeted to a Gemin Gene, or a pharmaceutical composition thereof, for the treatment of a disease or disorder.
In one embodiment, provided are uses of a compound of an isolated double stranded RNA oligonucleotide in the manufacture of a medicament for inhibiting Gemin Gene expression or overexpression. Thus, provided herein is the use of an isolated double stranded RNA oligonucleotide targeted to a Gemin Gene in the manufacture of a medicament for the treatment of a disease or disorder by means of the method described above.
Salts, Prodrugs and Bioequivalents
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.
Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 22 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfoc acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.
For oligonucleotides, examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine. Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans. In another embodiment, sodium salts of dsRNA compounds are also provided.
Formulations
The oligomeric compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756.
The present invention also includes pharmaceutical compositions and formulations which include the oligomeric compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including but not limited to ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer (intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Sites of administration are known to those skilled in the art. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.
Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the oligomeric compound which may be present as a solution in the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860.
Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860.
Formulations of the present invention include dendrimers, which are hyperbranched polymers. Dendrimers are built around a central core to which the branch points are attached. Thus the core is encapsulated in a web of branch points. Dendrimers form a monodisperse material of uniform molecular weight providing more predictable pK and biodistribution properties. Hyperbranched dendrimers offer a unique opportunity of polyfunctionalization with desired carrier or targeting molecules for targeted drug delivery. Short dendrimers can also be used to attach multiple carriers to a single oligomeric compound thus increasing chances of uptake by the cluster effect. Dendrimers can be attached to oligomeric compounds of the invention to target solid tumors which have a leaky vasculature while avoiding glomelular filtration by virtue of their large size. Due to dendrimers' cage like structure they protect their core and thus an oligomeric compound can be protected from degradation by encapsulating it within a dendrimer. Dendrimers can be programmed to self-destruct under certain pH, oxidative and enzymatic conditions and thus can be used as osmotic bombs attached to oligomeric compounds to disrupt lysosomal membranes and release attached oligomeric compounds. Dendrimers have also been used to target molecules to specific organs and intracellular compartments; therefore, such uses can be applied to oligomeric compounds of the invention. Dendrimers can also serve as biocompatible carriers for oligomeric compounds of the invention to increase their plasma circulation time.
The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860.
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of oligomeric compounds, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic compounds across cell membranes, penetration enhancers also enhance the permeability of lipophilic compounds. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860.
In general, a composition's bioavailability is said to be “enhanced” when its relative bioavailability is greater than the bioavailability of a composition substantially consisting of pure oligomeric compound, i.e. oligomeric compound in the absence of a penetration enhancer.
Organ bioavailability refers to the concentration of compound in an organ. Organ bioavailability may be measured in test subjects by a number of means, such as by whole-body radiography. Organ bioavailability may be modified, e.g. enhanced, by one or more modifications to the oligomeric compound, by use of one or more carrier compounds or excipients, etc. as discussed in more detail herein. In general, an increase in bioavailability will result in an increase in organ bioavailability.
Suitable embodiments provided herein are compositions comprising one or more pharmaceutically acceptable penetration enhancers, and methods of using such compositions, which result in the improved bioavailability of oligonucleotides administered via non-parenteral modes of administration. Heretofore, certain penetration enhancers have been used to improve the bioavailability of certain drugs. See Muranishi, Crit. Rev. Ther. Drug Carrier Systems, 1990, 7, 1 and Lee et al., Crit. Rev. Ther. Drug Carrier Systems, 1991, 8, 91. It has been found that the uptake and delivery of oligonucleotides can be greatly improved even when administered by non-parenteral means through the use of a number of different classes of penetration enhancers.
In some embodiments, compositions for non-parenteral administration include one or more modifications from naturally-occurring oligonucleotides (i.e. full-phosphodiester deoxyribosyl or full-phosphodiester ribosyl oligonucleotides). Such modifications may increase binding affinity, nuclease stability, cell or tissue permeability, tissue distribution, or other biological or pharmacokinetic property. In some embodiments, compositions for administration to a subject, and in particular oral compositions for administration to an animal or human subject, will comprise modified oligomeric compounds having one or more modifications for enhancing affinity, stability, tissue distribution, or other biological property.
Oral compositions for administration of non-parenteral oligomeric compounds can be formulated in various dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The term “alimentary delivery” encompasses e.g. oral, rectal, endoscopic and sublingual/buccal administration. A common requirement for these modes of administration is absorption over some portion or all of the alimentary tract and a need for efficient mucosal penetration of the nucleic acid(s) so administered. Consequently, such oral oligomeric compound compositions can be referred to as “mucosal penetration enhancers.”
Delivery of a drug via the oral mucosa, as in the case of buccal and sublingual administration, has several desirable features, including, in many instances, a more rapid rise in plasma concentration of the drug than via oral delivery (Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711).
Oligomeric compounds, such as oligonucleotides, may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8, 2002.
Endoscopy may be used for drug delivery directly to an interior portion of the alimentary tract. For example, endoscopic retrograde cystopancreatography (ERCP) takes advantage of extended gastroscopy and permits selective access to the biliary tract and the pancreatic duct (Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl.), 1591). Pharmaceutical compositions, including liposomal formulations, can be delivered directly into portions of the alimentary canal, such as, e.g., the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the gastric submucosa (Akamo et al., Japanese J. Cancer Res., 1994, 85, 652) via endoscopic means. Gastric lavage devices (Inoue et al., Artif. Organs, 1997, 21, 28) and percutaneous endoscopic feeding devices (Pennington et al., Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for direct alimentary delivery of pharmaceutical compositions.
In some embodiments, oligomeric compound formulations may be administered through the anus into the rectum or lower intestine. Rectal suppositories, retention enemas or rectal catheters can be used for this purpose and may be preferred when patient compliance might otherwise be difficult to achieve (e.g., in pediatric and geriatric applications, or when the patient is vomiting or unconscious). Rectal administration can result in more prompt and higher blood levels than the oral route (Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711). Because about 50% of the drug that is absorbed from the rectum will bypass the liver, administration by this route significantly reduces the potential for first-pass metabolism (Benet et al., Chapter 1 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).
In one embodiment, oral oligomeric compound compositions comprise at least one member of the group consisting of surfactants, fatty acids, bile salts, chelating agents, and non-chelating surfactants. Further embodiments comprise oral oligomeric compound comprising at least one fatty acid, e.g. capric or lauric acid, or combinations or salts thereof. One combination is the sodium salt of lauric acid, capric acid and UDCA. Other embodiments comprise methods of enhancing the oral bioavailability of an oligomeric compound, the method comprising co-administering the oligomeric compound and at least one penetration enhancer.
Other excipients that may be added to oral oligomeric compound compositions include surfactants (or “surface-active agents”), which are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligomeric compounds through the alimentary mucosa and other epithelial membranes is enhanced. In addition to bile salts and fatty acids, surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Phamacol., 1988, 40, 252).
Fatty acids and their derivatives which act as penetration enhancers and can be used in compositions of the compounds of the present invention include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines and mono- and di-glycerides thereof and/or physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:2, 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:1, 1-33; El-Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654). Examples of some fatty acids are sodium caprate (C10) and sodium laurate (C12), used singly or in combination at concentrations of 0.5 to 5%.
In one embodiment, oligomeric compound compositions for oral delivery comprise at least two discrete phases, which phases may comprise particles, capsules, gel-capsules, microspheres, etc. Each phase may contain one or more oligomeric compounds, penetration enhancers, surfactants, bioadhesives, effervescent agents, or other adjuvant, excipient or diluent. In one embodiment, one phase comprises at least one oligomeric compound and at least one penetration enhancer. In one embodiment, a first phase comprises at least one oligomeric compound and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer. In one embodiment, a first phase comprises at least one oligomeric compound and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer and substantially no oligomeric compound. In one embodiment, at least one phase is compounded with at least one degradation retardant, such as a coating or a matrix, which delays release of the contents of that phase. In one embodiment, a first phase comprises at least one oligomeric compound and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer and a release-retardant. In particular embodiments, an oral oligomeric compound comprises a first phase comprising particles containing an oligomeric compound and a penetration enhancer, and a second phase comprising particles coated with a release-retarding agent and containing penetration enhancer.
A variety of bile salts also function as penetration enhancers to facilitate the uptake and bioavailability of drugs. The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term “bile salt” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Bile salts contemplated herein include, for example, cholic acid (or its sodium salt, sodium cholate, or other pharmaceutically acceptable salts), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583). UDCA and CDCA have been used effectively as penetration enhancers for oligonucleotides, and even more effectively when combined.
In one embodiment, penetration enhancers useful in some embodiments are mixtures of penetration enhancing compounds. One such penetration enhancer is a mixture of UDCA (and/or CDCA) with capric and/or lauric acids or salts thereof e.g. sodium. Such mixtures are useful for enhancing the delivery of biologically active substances across mucosal membranes, in particular intestinal mucosa. Other penetration enhancer mixtures comprise about 5-95% of bile acid or salt(s) UDCA and/or CDCA with 5-95% capric and/or lauric acid. Particular penetration enhancers are mixtures of the sodium salts of UDCA, capric acid and lauric acid in a ratio of about 1:2:2 respectively. Anther such penetration enhancer is a mixture of capric and lauric acid (or salts thereof) in a 0.01:1 to 1:0.01 ratio (mole basis). In particular embodiments capric acid and lauric acid are present in molar ratios of e.g. about 0.1:1 to about 1:0.1, in particular about 0.5:1 to about 1:0.5.
Other excipients include chelating agents, i.e. compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligomeric compound through the alimentary and other mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315). Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. Control Rel., 1990, 14, 43).
As used herein, non-chelating non-surfactant penetration enhancers may be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucelotides through the alimentary and other mucosal membranes (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1). This class of penetration enhancers includes, but is not limited to, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621).
Some oral oligonucleotide compositions also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which may be inert (i.e., does not possess biological activity per se) or may be necessary for transport, recognition or pathway activation or mediation, or is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177).
A “pharmaceutical carrier” or “excipient” may be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, EXPLOTAB); and wetting agents (e.g., sodium lauryl sulphate, etc.).
Oral oligomeric compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.
Formulations for topical administration include those in which the oligomeric compounds of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
For topical or other administration, oligomeric compounds of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999.
Combinations
In accordance with the present invention certain pharmaceutical compositions contain one or more antisense compounds and one or more other agents which function by a non-antisense mechanism. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one can achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
Chemotherapeutic Agents
For example, in the treatment of cancer, one or more of the agents can be chemotherapeutic agents. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin, pemetrexed and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention.
NONLIMITING DISCLOSURE AND INCORPORATION BY REFERENCE
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
The effect of oligomeric compounds on target nucleic acid expression was tested in one or more of the following cell types.
3T3-L1 cells: The mouse embryonic adipocyte-like cell line 3T3-L1 was obtained from the American Type Culture Collection (Manassas, Va.). 3T3-L1 cells were routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 80% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 4000 cells/well for use in oligomeric compound transfection experiments.
A10 cells: The rat aortic smooth muscle cell line A10 was obtained from the American Type Culture Collection (Manassas, Va.). A10 cells were routinely cultured in DMEM, high glucose (American Type Culture Collection, Manassas, Va.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 80% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 2500 cells/well for use in oligomeric compound transfection experiments.
A549 cells: The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (Manassas, Va.). A549 cells were 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 were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 5000 cells/well for use in oligomeric compound transfection experiments.
AML-12 cells: AML12 (alpha mouse liver 12) cell line was established from hepatocytes from a mouse (CD 1 strain, line MT42) transgenic for human TGF alpha. Cells are cultured in a 1:1 mixture of DMEM and Ham's F12 medium (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum, 0.005 mg/ml insulin, 0.005 mg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 5000 cells/well for use in oligomeric compound transfection experiments.
b.END cells: The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany). b.END cells were routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 3000 cells/well for use in oligomeric compound transfection experiments.
B16-F10 cells: The mouse melanoma cell line B16-F10 was obtained from the American Type Culture Collection (Manassas, Va.). B16-F10 cells were routinely cultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.), Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 7000 cells/well for use in oligomeric compound transfection experiments.
CHO cells: The Chinese hamster ovary cell line CHO was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). CHO cells were routinely cultured in Ham's F12K media (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum and 2 mM L-glutamine, which was adjusted to contain 1.5 g/L sodium bicarbonate (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 6000 cells/well for use in oligomeric compound transfection experiments.
HEPA 1-6 cells: The mouse hepatoma cell line HEPA 1-6 is a derivative of the BW7756 mouse hepatoma that arose in a C57/L mouse and is supplied by the American Type Culture Collection (Manassas, Va.). The cells are propagated in DMEM, high glucose, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were routinely passaged by trypsinization and dilution when they reached approximately 80% confluence. Cells were seeded into 96-well plates at a density of approximately 4000 cells/well for use in oligomeric compound transfection experiments.
Primary mouse hepatocytes: Primary mouse hepatocytes were prepared from CD-1 mice purchased from Charles River Labs. Primary mouse hepatocytes were routinely cultured in Hepatocyte Attachment Media supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% antibiotic-antimitotic (Invitrogen Life Technologies, Carlsbad, Calif.) and 10 nM bovine insulin (Sigma-Aldrich, St. Louis, Mo.). Cells were seeded into 96-well plates (Falcon-Primaria #3872) coated with 0.1 mg/ml collagen at a density of approximately 10,000 cells/well for use in oligomeric compound transfection experiments.
HepG2 cells: The human hepatoblastoma cell line HepG2 was obtained from the American Type Culture Collection (Manassas, Va.). HepG2 cells were routinely cultured in Eagle's MEM supplemented with 10% fetal bovine serum, 1 mM non-essential amino acids, and 1 mM sodium pyruvate (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Multiwell culture plates are prepared for cell culture by coating with a 1:100 dilution of type 1 rat tail collagen (BD Biosciences, Bedford, Mass.) in phosphate-buffered saline. The collagen-containing plates were incubated at 37° C. for approximately 1 hour, after which the collagen was removed and the wells were washed twice with phosphate-buffered saline. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 8,000 cells/well for use in oligomeric compound transfection experiments.
HUVEC cells: The human umbilical vein endothilial cell line HuVEC was obtained from the American Type Culture Collection (Manassas, Va.). HuVEC cells were routinely cultured in EBM (Cambrex Bio Science, Walkersville, Md.) supplemented with SingleQuots supplements (Cambrex Bio Science, Walkersville, Md.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence were maintained for up to 15 passages. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 10000 cells/well for use in oligomeric compound transfection experiments.
MLg2908 cells: The mouse lung cell line MLg2908 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). MLg2908 cells were routinely cultured in MEM (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 3000 cells/well for use in oligomeric compound transfection experiments.
P388D1 cells: The murine macrophage cell line P388D1 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). P388D1 cells were routinely cultured in RPMI 1640 media supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% sodium pyruvate (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 65-75% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 15,000 cells/well for use in oligomeric compound transfection experiments.
T-24 cells: The transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached approximately 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of approximately 4000-6000 cells/well for use in oligomeric compound transfection experiments.
Treatment with antisense compounds: When cells reached approximately 65-75% confluency, they were treated with oligonucleotide. Oligonucleotide was mixed with LIPOFECTINTM (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve a final concentration of 3 μg/mL LIPOFECTINTM per 100 nM oligonucleotide in 1 mL OPTI-MEMTM-1 or Eagle's MEM (Invitrogen Life Technologies, Carlsbad, Calif.). For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEMTM-1, Eagle's MEM or serum-free culture medium and then treated with 130 μL of the oligonucleotide/OPTI-MEMTM-1 or Eagle's MEM/LIPOFECTINTM cocktail. Cells were treated and data were obtained in duplicate or triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.
Control oligonucleotides are used to determine the optimal oligomeric compound concentration for a particular cell line. Furthermore, when oligomeric compounds of the invention are tested in oligomeric compound screening experiments or phenotypic assays, control oligonucleotides are tested in parallel with compounds of the invention. In some embodiments, the control oligonucleotides are used as negative control oligonucleotides, i.e., as a means for measuring the absence of an effect on gene expression or phenotype. In alternative embodiments, control oligonucleotides are used as positive control oligonucleotides, i.e., as oligonucleotides known to affect gene expression or phenotype.
Control oligonucleotides are shown in Table 2. “Target Name” indicates the gene to which the oligonucleotide is targeted. “Species of Target” indicates species in which the oligonucleotide is perfectly complementary to the target mRNA. “Motif” is indicative of chemically distinct regions comprising the oligonucleotide. Certain compounds in Table 2 are composed of 2′-O-(2-methoxyethyl) nucleotides, also known as 2′-MOE nucleotides, and designated as “Uniform MOE”. Certain compounds in Table 2 are chimeric oligonucleotides, composed of a central “gap” region consisting of 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) by “wings”. The wings are composed of 2′-O-(2-methoxyethyl) nucleotides, also known as 2′-MOE nucleotides. The “motif” of each gapmer oligonucleotide is illustrated in Table 2 and indicates the number of nucleotides in each gap region and wing, for example, “5-10-5” indicates a gapmer having a 10-nucleotide gap region flanked by 5-nucleotide wings. Similarly, the motif “5-9-6” indicates a 9-nucleotide gap region flanked by 5-nucleotide wing on the 5′ side and a 6-nucleotide wing on the 3′ side. ISIS 15839 is a “hemimer” composed of two regions of distinct chemistry, wherein the first 12-nucleotides are 2′-deoxynucleotides and the last 8 nucleotides are 2′-MOE nucleotides. ISIS 15344 is a “hemimer” composed of two regions of distinct chemistry, wherein the first 9 nucleotides are 2′-deoxynucleotides and the last 11 are 2′-MOE nucleotides. ISIS 13513 is a chimeric oligonucleotide composed of multiple regions of distinct chemistry, denoted with a motif of “6-8-5-1” and comprised of a 6-nucleotide wing flanking an 8-nucleotide gap region followed by 5 2′-MOE nucleotides and terminating with a 2′-deoxynucleotide at the 3′ end. The internucleoside (backbone) linkages are phosphorothioate throughout the oligonucleotides in Table 2. Unmodified cytosines are indicated by “uC” in the nucleotide sequence; all other cytosines are 5-methylcytosines.







TABLE 2







Control oligonucleotides for cell line testing, oligomeric 


compound screening and phenotypic assays

















SEQ




Species of


ID


ISIS #
Target Name
Target
Sequence (5′ to 3′)
Motif
NO





117386
C/EBP alpha
Human
CCCTACTCAGTAGGCATTGG
5-10-5
 27


 


 15839
CD54
Cynomolgus
GuCuCuCAAGuCTGGuCATuCuCGTuCA
Hemimer
 28




monkey;







Human;







Rhesus monkey





 


113131
CD86
Human
CGTGTGTCTGTGCTAGTCCC
5-10-5
 29


 


289865
forkhead box O1A
Human
GGCAACGTGAACAGGTCCAA
5-10-5
 30



(rhabdomyosarcoma)






 


186515
insulin-like
Human
AGGTAGCTTTGATTATGTAA
5-10-5
 31



growth factor







binding protein 1






 


 25237
integrin beta 3
Human
GCCCATTGCTGGACATGC
4-10-4
 32


 


196103
integrin beta 3
Human
AGCCCATTGCTGGACATGCA
5-10-5
 33


 


134062
Interleukin 8
Human
GCTTGTGTGCTCTGCTGTCT
5-10-5
 34


 


148715
Jagged 2
Human;
TTGTCCCAGTCCCAGGCCTC
5-10-5
 35




Mouse; Rat





 


 15346
Jun N-Terminal
Human
CTCTCTGTAGGuCuCuCGCTTGG
5-9-6
 36



Kinase-1






 


 18076
Jun N-Terminal
Human
CTTTCuCGTTGGAuCuCCCTGGG
5-9-6
 37



Kinase-1






 


 18078
Jun N-Terminal
Human
GTGCGuCGuCGAGuCuCuCGAAATC
5-9-6
 38



Kinase-2






 


101759
Jun N-Terminal
Mouse; Rat
GCTCAGTGGACATGGATGAG
5-10-5
 39



Kinase-2






 


183881
kinesin-like 1
Human
ATCCAAGTGCTACTGTAGTA
5-10-5
 40


 


342672
Mir-143
Human;
ATACCGCGATCAGTGCATCTTT
Uniform
 41




Mouse; Rat

MOE



 


342673
Mir-143
Human;
AGACTAGCGGTATCTTTATCCC
Uniform
 42




Mouse; Rat

MOE



 


 29848
none
none
NNNNNNNNNNNNNNNNNNNN
5-10-5
 43


 


129695
none
none
TTCTACCTCGCGCGATTTAC
5-10-5
 44


 


129700
none
none
TAGTGCGGACCTACCCACGA
5-10-5
 45


 


226844
Notch (Drosophila)
Human;
GCCCTCCATGCTGGCACAGG
5-10-5
 46



homolog 1
Mouse





 


105990
Peroxisome
Human
AGCAAAAGATCAATCCGTTA
5-10-5
 47



proliferator-







activated receptor







gamma






 


 13513
Protein kinase C-
Human;
GGAuCuCuCCGAAAGACuCAuCuCAG
6-8-5-1
 48



delta
Mouse





 


116847
PTEN
Human;
CTGCTAGCCTCTGGATTTGA
5-10-5
 49




Mouse;







Rabbit; Rat





 


 15344
Raf kinase B
Human
CTGCCTGGATGGGTGTTTTT
Hemimer
 50


 


 13650
Raf kinase C
Human
TCCCGCuCTGTGAuCATGCATT
6-9-6
 51


 


336806
Raf kinase C
Human
TACAGAAGGCTGGGCCTTGA
5-10-5
 52


 


 15770
Raf kinase C
Mouse;
ATGCATTuCTGuCuCuCuCuCAAGGA
5-10-5
 53




Murine







sarcoma







virus; Rat





 


 30748
Ship-2
Human;
CCAACCTCAAATGTCCCA
4-10-4
 54




Mouse; Rat





 


153704
STAT 1
Human; Rat
AGGCATGGTCTTTGTCAATA
5-10-5
 55


 


 23722
Survivin
Human
TGTGCTATTCTGTGAATT
4-10-4
 56


 


 13920
Ras-Ha
Human
TuCuCGTCATCGCTuCuCTuCAGGG
3-9-8
475









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. Positive controls are shown in Table 2. For human and non-human primate cells, the positive control oligonucleotide is selected from either ISIS 13650 (SEQ ID NO: 51), ISIS 336806 (SEQ ID NO: 52) or ISIS 18078 (SEQ ID NO: 38). For mouse or rat cells the positive control oligonucleotide is ISIS 15770 (SEQ ID NO: 53) or ISIS 15346 (SEQ ID NO: 36). The concentration of positive control oligonucleotide that results in 80% inhibition of the target mRNA, for example, human Raf kinase C for ISIS 13650, 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 the target mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.
Example 2
Real-Time Quantitative PCR Analysis of Target Gene mRNA Levels
Quantitation of Gemin Gene mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISMTM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions.
Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured were evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. After isolation the RNA is subjected to sequential reverse transcriptase (RT) reaction and real-time PCR, both of which are performed in the same well. RT and PCR reagents were obtained from Invitrogen Life Technologies (Carlsbad, Calif.). RT, real-time PCR was carried out in the same by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl2, 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5× ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
Gene target quantities obtained by RT, real-time PCR were normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression was quantified by RT, real-time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA was quantified using RiboGreenTM RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).
170 μL of RiboGreenTM working reagent (RiboGreenTM reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was pipetted into a 96-well plate containing 30 μL purified cellular RNA. The plate was read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.
Presented in Table 3 are primers and probes used to measure GAPDH expression in the cell types described herein. The GAPDH PCR probes have JOE covalently linked to the 5′ end and TAMRA covalently linked to the 3′ end, where JOE is the fluorescent reporter dye and TAMRA is the quencher dye. In some cell types, primers and probe designed to a GAPDH sequence from a different species are used to measure GAPDH expression. For example, a human GAPDH primer and probe set is used to measure GAPDH expression in monkey-derived cells and cell lines.







TABLE 3







GAPDH primers and probes for use in real-time PCR















SEQ


Target

Sequence

ID


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





GAPDH
Human
Forward Primer
CAACGGATTTGGTCGTATTGG
57


 


GAPDH
Human
Reverse Primer
GGCAACAATATCCACTTTACCAGAGT
58


 


GAPDH
Human
Probe
CGCCTGGTCACCAGGGCTGCT
59


 


GAPDH
Human
Forward Primer
GAAGGTGAAGGTCGGAGTC
60


 


GAPDH
Human
Reverse Primer
GAAGATGGTGATGGGATTTC
61


 


GAPDH
Human
Probe
CAAGCTTCCCGTTCTCAGCC
62


 


GAPDH
Human
Forward Primer
GAAGGTGAAGGTCGGAGTC
60


 


GAPDH
Human
Reverse Primer
GAAGATGGTGATGGGATTTC
61


 


GAPDH
Human
Probe
TGGAATCATATTGGAACATG
63


 


GAPDH
Mouse
Forward Primer
GGCAAATTCAACGGCACAGT
64


 


GAPDH
Mouse
Reverse Primer
GGGTCTCGCTCCTGGAAGAT
65


 


GAPDH
Mouse
Probe
AAGGCCGAGAATGGGAAGCTTGTCATC
66


 


GAPDH
Rat
Forward Primer
TGTTCTAGAGACAGCCGCATCTT
67


 


GAPDH
Rat
Reverse Primer
CACCGACCTTCACCATCTTGT
68


 


GAPDH
Rat
Probe
TTGTGCAGTGCCAGCCTCGTCTCA
69









Probes and primers for use in real-time PCR were designed to hybridize to target-specific sequences. The primers and probes and the target nucleic acid sequences to which they hybridize are presented in Table 4. The target-specific PCR probes have FAM covalently linked to the 5′ end and TAMRA covalently linked to the 3′ end, where FAM is the fluorescent dye and TAMRA is the quencher dye.







TABLE 4







Gene target-specific primers and probes for use in real-time PCR














Target


SEQ


Target

SEQ ID
Sequence

ID


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





Gemin2
Human
 7
Forward Primer
CAAATTGACCCAAAGAAGTTGAAA
70


 


Gemin2
Human
 7
Reverse Primer
GTTGCTGTTGCCATTGAAGTGT
71


 


Gemin2
Human
 7
Probe
ACCCGCCCCTGAAGGTTATTCCCC
72


 


Gemin4
Human
12
Forward Primer
GAAGTGCAGGGTCCCAATTC
73


 


Gemin4
Human
12
Reverse Primer
TTCATCCAGACGGTTTCTTTGA
74


 


Gemin4
Human
12
Probe
TCTGCCACTTTCATGGTGTCAT
75


 


Gemin5
Human
14
Forward Primer
GCAAAAGCTCCTCCTCTTACCA
76


 


Gemin5
Human
14
Reverse Primer
GTCACCCTCTCCACGAAAGG
77


 


Gemin5
Human
14
Probe
ACTTGGAACACGGGCACCGAAG
78


 


Gemin6
Human
20
Forward Primer
GAGAAGAACCACATCCCCATCA
79


 


Gemin6
Human
20
Reverse Primer
GACCCCAGCCACACAGAGA
80


 


Gemin6
Human
20
Probe
TGAACAGGGAGACGCTCCAAGGA
81


 


Gemin7
Human
25
Forward Primer
CCTTCAAGCCATAAAGATATTGTGTTC
82


 


Gemin7
Human
25
Reverse Primer
TTGGGAGGCCTGGGATACA
83


 


Gemin7
Human
25
Probe
CTTTTCTGCTTGAGGCTAAGGCA
84









Example 3
Treatment of Cultured Cells with Oligomeric Compounds
Oligomeric compounds targeted to Gemin Genes presented in Table 1 were tested for their effects on gene target expression in cultured cells. Table 5 shows the experimental conditions, including cell type, transfection method, dose of oligonucleotide and control SEQ ID NO used to evaluate the inhibition of gene expression by the oligomeric compounds of the invention. The control oligonucleotide was chosen from the group presented in Table 2, and in these experiments was used as a negative control. Each cell type was treated with the indicated dose of oligonucleotide as described by other examples herein.







TABLE 5







Treatment conditions of cultured cells with oligomeric compounds















Control


Target
Cell
Transfection
Dose of
SEQ ID


Name
Type
Method
Oligonucleotide (nM)
NO





Gemin2
A549
Lipofectin
150
38


Gemin4
T-24
Lipofectin
150
38


Gemin5
A549
Lipofectin
150
38


Gemin6
T-24
Lipofectin
150
38


Gemin7
T-24
Lipofectin
150
38









Example 4
Antisense Inhibition of Gene Targets by Oligomeric Compounds
A series of oligomeric compounds was designed to target different regions of the Gemin Genes, using sequences cited in Table 1. The oligomeric compounds and the data describing the degree to which they inhibit gene expression are shown in Table 6.
All oligomeric compounds in Table 6 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of 10 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) by five-nucleotide “wings”. The wings are composed of 2′-0-(2-methoxyethyl) nucleotides, also known as 2′-MOE nucleotides. The internucleoside (backbone) linkages are phosphorothioate throughout the oligonucleotide. All cytidine residues are 5-methylcytidines.
The oligomeric compounds were analyzed for their effect on gene target mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from experiments in which cultured cells, as indicated for each target in Table 5, were treated with the disclosed oligomeric compounds. A reduction in expression is expressed as percent inhibition in Table 6. If the target expression level of oligomeric compound-treated cell was higher than control, percent inhibition is expressed as zero inhibition. If present, “N.D.” indicates “not determined”. The target regions to which these oligomeric compounds are inhibitory are herein referred to as “validated target segments.”
As shown in Table 6, SEQ ID NOs 87, 89, 90, 91, 92, 94, 103, 104, 105, 106, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123, 125, 127, 128, 129, 130, 131, 132, 135, 136, 137, 138, 139, 140, 141, 142, 145, 146, 147, 148, 149, 150, 151, 152, 153, 156, 157, 158, 161, 163, 166, 177, 178, 180, 188, 193, 195, 210, 225, 228, 229, 232, 238, 241, 242, 243, 244, 245, 246, 249, 250, 251, 252, 253, 254, 255, 256, 257, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 291, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 311, 313, 315, 316, 317, 319, 320, 326, 328, 329, 330, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 346, 347, 348, 349, 350, 351, 352, 353, 354, 356, 357, 358, 359, 360, 361, 362, 363, 364, 366, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 381, 382, 383, 384, 385, 387, 388, 393, 394, 395, 396, 397, 398, 399, 401, 402, 403, 410, 411, 412, 414, 415, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 450, 451, 452, 457, 458, 462, 467, 468, 469, 470, 471, 472, 473, 474, demonstrated at least 50% inhibition of a Gemin Gene in this assay. In some embodiments of the invention, oligomeric compounds of the invention comprise at least one of these sequences. In other embodiments, oligomeric compounds of the invention consist of at least one of these sequences.
The target sites to which these sequences are complementary are examples of “validated target sites” and are therefore sites for designing other oligomeric compounds of the present invention.







TABLE 6







Modulation of Gemin Gene mRNA levels by  


chimeric oligonucleotides having 2′-MOE 


wings and deoxy gap













Target



SEQ



SEQ ID
Target

%
ID


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















297287
1
510
TTACTGTTGCTTGTACATAA
28
85


 


297288
2
735
GTCAAAATACCACTAAGAGC
2
86


 


297289
3
81
TGCTTCGATCCGGCAATAGC
59
87


 


297292
4
1317
CAATAAAATTTTATATGTAT
5
88


 


297293
4
1356
TAGGAGTCAAACAAAATTTT
51
89


 


297294
4
1381
GCTAAGGCCATCCATTGTCT
54
90


 


297295
4
1394
TTTTAATTCTGATGCTAAGG
53
91


 


297296
4
1412
GCCATTTAATCCAGATTATT
52
92


 


297297
4
1418
CACATTGCCATTTAATCCAG
35
93


 


297298
4
1432
TTGCTGACTATGAACACATT
57
94


 


297299
4
1439
AATTTTATTGCTGACTATGA
43
95


 


297290
5
539
GCCATCTTCCCTGATTCATT
10
96


 


297291
6
399
GAAAACCAATCATTGTCACA
8
97


 


297230
7
44
CGCAGGCGCACTAATAGACA
17
98


 


297231
7
51
GTCACAGCGCAGGCGCACTA
38
99


 


297232
7
57
TTCTAGGTCACAGCGCAGGC
37
100


 


297233
7
71
GGCGCATGCGCCCATTCTAG
13
101


 


297234
7
95
TTTTCAAACCAGCCAGTTCC
48
102


 


297235
7
174
GTCAAGTCGCAAGGCTCTAC
81
103


 


297236
7
270
GCTACCACAACATCTGGACA
77
104


 


297237
7
287
TCTTTGGGTCAATTTGAGCT
92
105


 


297238
7
294
TTCAACTTCTTTGGGTCAAT
88
106


 


297239
7
308
CACTTTGCTTCCTTTTCAAC
45
107


 


297240
7
329
ATCCTGAAAGAGAAATATTC
19
108


 


297241
7
374
GTTGCCATTGAAGTGTTGGG
81
109


 


297242
7
378
TGCTGTTGCCATTGAAGTGT
90
110


 


297243
7
389
GTGCCACTTGTTGCTGTTGC
86
111


 


297244
7
406
TCGAACAGTTGAAAACTGTG
61
112


 


297245
7
419
TGTTCACATTCTGTCGAACA
71
113


 


297246
7
577
ATAATCTATTCCAGGACTTT
68
114


 


297247
7
630
GCCTGATTCATTCTGCTAAC
78
115


 


297248
7
633
GTTGCCTGATTCATTCTGCT
83
116


 


297249
7
649
CAAGACACTAGTTACTGTTG
77
117


 


297250
7
652
TTCCAAGACACTAGTTACTG
74
118


 


297251
7
657
AGATATTCCAAGACACTAGT
74
119


 


297252
7
671
CAAACCAATTACTCAGATAT
33
120


 


297253
7
682
GTCTCTTTCTCCAAACCAAT
52
121


 


297254
7
687
GTAAAGTCTCTTTCTCCAAA
53
122


 


297255
7
692
CTGGAGTAAAGTCTCTTTCT
82
123


 


297256
7
697
CAATTCTGGAGTAAAGTCTC
47
124


 


297257
7
702
CTTCCCAATTCTGGAGTAAA
65
125


 


297258
7
706
CCATCTTCCCAATTCTGGAG
43
126


 


297259
7
735
TTTTCAAGACAAGCCAATAA
55
127


 


297260
7
738
GGCTTTTCAAGACAAGCCAA
66
128


 


297261
7
742
CAAAGGCTTTTCAAGACAAG
51
129


 


297262
7
746
GTAACAAAGGCTTTTCAAGA
57
130


 


297263
7
762
AGTGAATGAGCCTCAGGTAA
85
131


 


297264
7
873
AAATACCTGCTAACCAAGCA
57
132


 


297265
7
876
TCAAAATACCTGCTAACCAA
24
133


 


297266
7
900
GGCTCATCAGCTAAATCACG
0
134


 


297267
7
903
GATGGCTCATCAGCTAAATC
65
135


 


297268
7
908
ATCAAGATGGCTCATCAGCT
79
136


 


297269
7
916
TCAGCTACATCAAGATGGCT
71
137


 


297270
7
941
AGAAATATCTTCTATCCCTG
68
138


 


297271
7
970
GTTTTCCTCAGAGTTAGGCT
83
139


 


297272
7
1004
AAGATGTGTTGAAATCTGTA
77
140


 


297273
7
1017
TCACATAGTGTTGAAGATGT
79
141


 


297274
7
1023
AACCCTTCACATAGTGTTGA
80
142


 


297275
7
1031
AAGATGTGAACCCTTCACAT
0
143


 


297276
7
1037
CAGGTTAAGATGTGAACCCT
18
144


 


297277
7
1055
GTATCAATCTGAATTGCACA
81
145


 


297278
7
1105
GTGGGATTTTCCATTGATAT
69
146


 


297279
7
1110
ACTGAGTGGGATTTTCCATT
83
147


 


297280
7
1114
AAAAACTGAGTGGGATTTTC
82
148


 


297281
7
1121
GTTCATCAAAAACTGAGTGG
74
149


 


297282
7
1131
TGTTCAAACTGTTCATCAAA
75
150


 


297283
7
1144
GATTACAGAAAACTGTTCAA
64
151


 


297284
7
1150
CTGCTTGATTACAGAAAACT
79
152


 


297285
7
1164
AATTTCTATGCAAGCTGCTT
89
153


 


297286
7
1182
TGTAAAATTTCATCATACAA
48
154


 


297300
8
1981
TAAGTAACCCATTTAAAGAC
36
155


 


297301
8
4803
ATCCTGAAAGCTAGAGATCA
58
156


 


297302
8
5344
TGTTCACATTCTAAAGGAAG
60
157


 


297303
8
7356
GGAATAAGGTTATCTACCTT
71
158


 


297304
8
14948
CAAAGAAAGCTAATTTGTTT
42
159


 


297305
8
18873
TGCAACTTACCACTAAGAGC
36
160


 


297306
8
19918
TAATCACTGTACAGTCAAGA
64
161


 


297307
8
20524
TTAACTATACCTGCTAACCA
28
162


 


297452
9
6
TGGGCACAGAGCAATCACAC
75
163


 


297450
9
129
CAATCACACGGCCACAGGAT
45
164


 


297451
9
427
CAATCACACAGCCACAGGAT
41
165


 


297453
9
892
CAAGACGGTGAATGAGATCC
53
166


 


297446
10
7
CTTAGGCCTGCTCACAACCT
0
167


 


297447
10
12
CCGCGCTTAGGCCTGCTCAC
0
168


 


297448
10
41
CACGATGGGAGACGCAGGAG
13
169


 


297449
10
80
CTCCCCTCCGAGAACTCGAA
44
170


 


297454
11
63
TCCTTCCAGTCAAAAACAAG
31
171


 


297455
11
90
TTTCAGGGAAACGGTCCACC
49
172


 


297386
12
15
CCTAGGTCCATGGCGGCGAC
0
173


 


297387
12
22
CAAGGGTCCTAGGTCCATGG
0
174


 


297388
12
24
TTCAAGGGTCCTAGGTCCAT
0
175


 


297389
12
53
CATGCAGAATAGTCATTTCT
40
176


 


297390
12
108
GTTAATTCTGCCAGTGCCTT
53
177


 


297391
12
203
TCTTCCAGGCAAAGGGCTGG
59
178


 


297392
12
210
GCTTTCTTCTTCCAGGCAAA
27
179


 


297393
12
282
TGCCACCGTGTCTCTGTGTC
62
180


 


297394
12
471
TCTTCGGCAGAAGTGTCAAC
43
181


 


297395
12
694
GATCCGACTCTGGATCTGTG
35
182


 


297396
12
767
CCTCTGTCAGCGCAAACACA
25
183


 


297397
12
837
GAGTTCCACACAGAGATCAC
28
184


 


297398
12
843
GTGTCCGAGTTCCACACAGA
19
185


 


297399
12
849
TTCTGGGTGTCCGAGTTCCA
42
186


 


297400
12
980
CCCGCAGCAGGTGGTGCAGG
0
187


 


297401
12
1076
TCTGGCTGAAGGAAGTCAGA
52
188


 


297402
12
1164
AGGAAGTCCCTGACACACTC
0
189


 


297403
12
1173
GTTTTCCTCAGGAAGTCCCT
48
190


 


297404
12
1199
AGGCCCTGTTCTTCAGCACC
42
191


 


297405
12
1215
GCTGTGATATCCTCCAAGGC
42
192


 


297406
12
1269
CACACTTCCATATGGCGGTC
62
193


 


297407
12
1302
AAGGCCCACTTCTTCTCAGA
18
194


 


297408
12
1337
TGTTACTCCCCAGGCAGGCT
55
195


 


297409
12
1374
AGCCTCAACACCAAGTCTGG
31
196


 


297410
12
1469
CTGCGTAACATTCCAGGATC
45
197


 


297411
12
1559
CATAAGCCAGCAACTTTTCA
32
198


 


297412
12
1579
GTCTTCCTGAAAACCCTCCA
38
199


 


297413
12
1584
TTGAGGTCTTCCTGAAAACC
43
200


 


297414
12
1655
GGGCCACGGAGGCCACAGCT
18
201


 


297415
12
1686
ACCGTGACTTCCGGGTGCAC
0
202


 


297416
12
1724
TGCCGAGATTGACCACAGCC
48
203


 


297417
12
1762
AGGGAAGGCAGTGAGAATCT
33
204


 


297418
12
1781
CTTCCACAAACCTAAGGGCA
46
205


 


297419
12
1857
GGTGTAGAGAACTTCATCCA
31
206


 


297420
12
1887
AGGAGCTCTAAAAATTGCTT
23
207


 


297421
12
1938
AGAGCAGCCACTGGAATCCC
25
208


 


297422
12
1982
AGAAAGGCAGGACAAATTCC
20
209


 


297423
12
2018
TCAGACTGAGGTCTACCTCT
51
210


 


297424
12
2244
AAGGTCTCAGCATTGGCTGA
25
211


 


297425
12
2282
GGAGCCAGGACAGGGACTTG
49
212


 


297426
12
2289
TTGCGGTGGAGCCAGGACAG
49
213


 


297427
12
2323
CCTCAGGCCCACAGTCCAGT
45
214


 


297428
12
2471
AGCAGCACTCCATCCAGGCC
0
215


 


297429
12
2541
ACCTCCTCAGGATTGCCCAC
43
216


 


297430
12
2550
AACAGTCTGACCTCCTCAGG
26
217


 


297431
12
2616
CGCTGCCACTCCTGAGGGCT
38
218


 


297432
12
2633
TGGTCAGCTGGTGAAGGCGC
38
219


 


297433
12
2719
AAAGGGCTTCAGGTTGAGCA
40
220


 


297434
12
2778
TGGCTGCAGAGAAACTGGAA
44
221


 


297435
12
2793
CAATTACGACAGCTATGGCT
43
222


 


297436
12
3037
GCAGGTGAGGGTTTCCAAGG
0
223


 


297437
12
3077
AGGAGCTGACAGAAGGGTTG
12
224


 


297438
12
3086
TCTGGAGCAAGGAGCTGACA
53
225


 


297439
12
3189
ACGCCAAGTCAGAAGCTGCT
0
226


 


297440
12
3228
GATCTTCTGCAGACCCGCCA
34
227


 


297441
12
3263
CAGCTTTTTCGCTCCCGCAC
61
228


 


297442
12
3291
ACCCCTACAGACCTGCCATG
53
229


 


297443
12
3304
CTGCTCGGGTCTGACCCCTA
48
230


 


297444
12
3326
TCACATAACTGTAAAGTCCA
45
231


 


297445
12
3348
CCATGACTTTTTGTGGACAG
71
232


 


297456
13
1818
CGGCACCCACCTAGGTCCAT
25
233


 


297457
13
2360
GTGATACCTCAGGAGCGCAG
0
234


 


297458
13
2691
ATAAGGACACTGAGGCGCAG
38
235


 


297459
13
4041
GGCCCTAGCTCTGTGGGAGA
19
236


 


297460
13
4137
ATAAACTTGCCTTGGGAACA
46
237


 


297461
13
4300
AATCACACAGCCACAGGGTA
54
238


 


297462
13
5440
GGCGAGACCCTCCAGCAAAG
15
239


 


297463
13
5918
TTCAAGGGTCCTGCACACAG
43
240


 


297551
14
43
CCTCCAAAACAGCTAGATAC
54
241


 


297552
14
64
AGATCCCATTGCAACAGTTC
87
242


 


297553
14
112
TCTGATGAGGCACTGAAGAG
83
243


 


297554
14
203
TCTATCCATTGATGTAGAAA
64
244


 


297555
14
275
GTATGCAAACCCACCAAGGG
76
245


 


297556
14
531
GATATGTGCTAGAAATCTGT
67
246


 


297557
14
612
GGGAAGGTCTGTCTCCTTCT
39
247


 


297558
14
650
TAAGACAATCCCTTCTCCTC
30
248


 


297559
14
677
TCCACTAAGCTTCCAGGGAT
84
249


 


297560
14
939
TGGAGCCAGAGGCCATCAGA
85
250


 


297561
14
960
GCACGTAAATGACTGCATTG
77
251


 


297562
14
991
GGGCTGCTCTCTATGACAGT
52
252


 


297563
14
1076
ATGTGGGCTCCACGCCACAC
62
253


 


297564
14
1230
CCCCTGAATAGATGCAGTCT
81
254


 


297565
14
1291
TGAGGAGGCCGGGAATGATC
66
255


 


297566
14
1377
TTCTCAAGGTGGGCTTTTTC
66
256


 


297567
14
1843
ATCATTCTATACAGGGTAGC
63
257


 


297568
14
1864
TGACCTTTTCCTTCAATATC
33
258


 


297569
14
1893
GAAATAACTCAGGGTGGCCA
56
259


 


297570
14
1935
GGAGAACACCTTTGAGATCC
70
260


 


297571
14
1982
CATAGCCACAAGGTTGTCTG
64
261


 


297572
14
2002
TGGTAGCCAGCTGCTGGTGC
75
262


 


297573
14
2073
AAGCAGCCTTGACATACTGA
88
263


 


297574
14
2117
CAGCTCCACCGCTTCATACA
70
264


 


297575
14
2369
ATCCTCTCCTACGATGGCAG
83
265


 


297576
14
2395
CTGAGAGCCAGGGAAGCAGA
83
266


 


297577
14
2431
ACCCAGTTGTTGGCCAGAAG
79
267


 


297578
14
2477
CTGACCCTGTAGACTTTCAT
71
268


 


297579
14
2681
GTTCTGCAGCTTCTGAAAGG
68
269


 


297580
14
2748
AGTCATGGCAAATGTGAAGC
78
270


 


297581
14
2782
GAGGCCATCTGTTGGCTCAG
58
271


 


297582
14
2865
ACACTTCCTGCATGATGGTG
79
272


 


297583
14
2896
TGGTCACAGCCATCAGGGAG
25
273


 


297584
14
3088
GCAGTGTCTAACTGCTGGCT
74
274


 


297585
14
3125
TGGCTGAGAAGTTTCAGGGT
66
275


 


297586
14
3390
TACACTGGCTCTGAGAACTG
69
276


 


297587
14
3460
ATTCTCTGATTTGCCTCGGT
79
277


 


297588
14
3662
TGTGAAAATTCACATACAGA
57
278


 


297589
14
3739
GAGTGAATGTCTGGTGAGGC
78
279


 


297590
14
3794
AAACTTAATCCTTGTTTGTA
54
280


 


297591
14
3822
GTTGTCTATGGGTAGGCAGG
80
281


 


297592
14
3982
TTTGGGTGACGACAGAAGTT
78
282


 


297593
14
4024
GAAGTATTTACAAAGGTAGC
74
283


 


297594
14
4057
TCTCAAACAAGCATGACACA
51
284


 


297595
14
4092
AAAGGTATCAATAGTCCTAA
77
285


 


297596
14
4182
GGCCTATTTCATATTCAAAC
87
286


 


297597
14
4360
TTCCTGAGCTATTTGACTGC
42
287


 


297598
14
4375
TGAAACAGTAATTTATTCCT
37
288


 


297599
14
4492
AATAATCCAAAGGTCATCTA
45
289


 


297600
14
4527
TTAAATAGTATTTAGGGTCC
45
290


 


297601
15
20
GAAGCTGCCACAGCCGACCG
57
291


 


297602
15
35
CCGTCAGAGACAAGAGAAGC
49
292


 


297603
15
55
TCCTGCCCCATAACTACAAG
31
293


 


297604
15
163
CGGACAAGGAAGACGGAGGT
63
294


 


297605
15
233
GTGTCCCACCAACTCTCCTA
64
295


 


297606
15
249
CAGAGACCCTTTCGGTGTGT
86
296


 


297607
15
359
ATGTTCTGTCACAACTGTTT
76
297


 


297608
15
421
ACTATTAAGTCCTTTACTCG
54
298


 


297609
15
477
GGCTGTCATTTCTGTTAAAC
62
299


 


297610
15
498
TGGGTTCTATAAAGAGGTGC
76
300


 


297611
15
580
ATTATCACCACTATGCCATC
63
301


 


297612
15
632
ATCATCATGGCCTCGAAGCC
82
302


 


297613
15
658
GGACACCAGGCTATGGAGTG
65
303


 


297614
15
769
AAGTAGCAACCTTTTGTTAC
65
304


 


297615
15
783
TGCTTCCAGTGGCTAAGTAG
86
305


 


297616
15
803
GATTCGAATGGTTTGATCTT
60
306


 


297617
15
827
CCCTCGGCCTCTAGAACAGC
77
307


 


297618
15
890
TTTAACAGTTGGGTCTATAC
67
308


 


297619
15
931
GGTTGATTGCTGGGCCAATG
62
309


 


297620
15
1516
CCTTCTCCTCCAAGTGACAT
39
310


 


297621
15
2049
CATCCCACACCTGGGCTGTA
54
311


 


297622
16
19049
AAGAACTCACTTTGATTGAA
0
312


 


297623
16
19178
CAGCCTTATAAGAACATTTA
62
313


 


297624
16
22018
CCAAAGTTACCTATGACAGT
4
314


 


297625
16
30470
GGTTGCCATGCCTTTCTGGT
60
315


 


297626
16
34509
ATTTTTGTACCTCTGGAGTG
58
316


 


297627
16
44650
AGCTGCTGTAAACATTTGTG
52
317


 


297628
16
49702
TAACTCTTACCTTAATGCTC
5
318


 


297694
17
28
GGTACTCAGCAACCATGCTT
83
319


 


297695
17
33
CTCTGGGTACTCAGCAACCA
80
320


 


297696
17
207
AGAACCTTGGCAGAGACTGG
44
321


 


297697
18
713
TTTACCGACACATCCAAGAA
0
322


 


297698
18
770
GCCCTAAGTTCATTAATTTC
3
323


 


297699
18
807
CTGCGCCTAATCTTAAGCCA
7
324


 


297700
18
916
ACCTGAAGATTGCCCCATGG
7
325


 


297630
20
8
CTCCTCGCAACTCTGGGTAC
70
326


 


297631
20
29
CTGGCTAAATCAGTTAAAAA
16
327


 


297632
20
36
TTGCCACCTGGCTAAATCAG
63
328


 


297633
20
40
ATGATTGCCACCTGGCTAAA
63
329


 


297634
20
50
CCATTCACTCATGATTGCCA
87
330


 


297635
20
55
TTCATCCATTCACTCATGAT
44
331


 


297636
20
84
TGTAATCTTGCCATTCTAAG
58
332


 


297637
20
90
TGTAAATGTAATCTTGCCAT
73
333


 


297638
20
93
CTTTGTAAATGTAATCTTGC
65
334


 


297639
20
103
ACTCGGACCTCTTTGTAAAT
51
335


 


297640
20
112
CTGGCTGTCACTCGGACCTC
78
336


 


297641
20
116
CTCACTGGCTGTCACTCGGA
88
337


 


297642
20
119
CTTCTCACTGGCTGTCACTC
58
338


 


297643
20
125
CTCATTCTTCTCACTGGCTG
81
339


 


297644
20
148
GTAGTTAAAACCCATCCTTT
56
340


 


297645
20
152
GTCTGTAGTTAAAACCCATC
57
341


 


297646
20
156
CTGGGTCTGTAGTTAAAACC
66
342


 


297647
20
159
AGACTGGGTCTGTAGTTAAA
57
343


 


297648
20
166
TTGGCAGAGACTGGGTCTGT
82
344


 


297649
20
176
AAGGACAATATTGGCAGAGA
24
345


 


297650
20
190
TCAAGGAAGTTCACAAGGAC
59
346


 


297651
20
197
GCCATCTTCAAGGAAGTTCA
78
347


 


297652
20
199
CTGCCATCTTCAAGGAAGTT
58
348


 


297653
20
211
GTCACAGACATGCTGCCATC
77
349


 


297654
20
214
CCGGTCACAGACATGCTGCC
88
350


 


297655
20
232
ACAGCATGTCCCATAATTCC
53
351


 


297656
20
240
CAGTCTGCACAGCATGTCCC
80
352


 


297657
20
244
TCAACAGTCTGCACAGCATG
68
353


 


297658
20
256
TCATTCATAGTTTCAACAGT
53
354


 


297659
20
262
TCCCCTTCATTCATAGTTTC
36
355


 


297660
20
267
TATGGTCCCCTTCATTCATA
63
356


 


297661
20
269
TCTATGGTCCCCTTCATTCA
62
357


 


297662
20
297
TGAACAAATGCATCAGCTTC
59
358


 


297663
20
303
CAGACGTGAACAAATGCATC
76
359


 


297664
20
315
CTTTGCAGTCTCCAGACGTG
84
360


 


297665
20
327
CTGGGCTGTATGCTTTGCAG
79
361


 


297666
20
333
GATCCTCTGGGCTGTATGCT
68
362


 


297667
20
336
CCAGATCCTCTGGGCTGTAT
74
363


 


297668
20
346
TTTCTCTCTTCCAGATCCTC
54
364


 


297669
20
369
CAAGCCATTTCTTTAGGCTG
32
365


 


297670
20
372
TCTCAAGCCATTTCTTTAGG
60
366


 


297671
20
374
CTTCTCAAGCCATTTCTTTA
48
367


 


297672
20
377
GTTCTTCTCAAGCCATTTCT
68
368


 


297673
20
379
TGGTTCTTCTCAAGCCATTT
71
369


 


297674
20
381
TGTGGTTCTTCTCAAGCCAT
80
370


 


297675
20
385
GGGATGTGGTTCTTCTCAAG
65
371


 


297676
20
404
GTCTCCCTGTTCAGTGATGG
86
372


 


297677
20
424
ACACAGAGAGTCCTTGGAGC
65
373


 


297678
20
429
CAGCCACACAGAGAGTCCTT
66
374


 


297679
20
433
ACCCCAGCCACACAGAGAGT
69
375


 


297680
20
447
GGTCTATAGTCAGGACCCCA
62
376


 


297681
20
454
TATGGTGGGTCTATAGTCAG
54
377


 


297682
20
457
TCATATGGTGGGTCTATAGT
59
378


 


297683
20
468
AATTTTCTGGATCATATGGT
21
379


 


297684
20
472
CTGCAATTTTCTGGATCATA
83
380


 


297685
20
481
TTAGAGCTGCTGCAATTTTC
84
381


 


297686
20
485
CTCATTAGAGCTGCTGCAAT
81
382


 


297687
20
499
CGCGACAGAATAATCTCATT
89
383


 


297688
20
511
AGATCCTGAACACGCGACAG
63
384


 


297689
20
550
CTGGCCTCTCATTGGGAAGC
58
385


 


297690
20
590
GATAAAATATAGTCTTTTTC
27
386


 


297691
20
595
TGAGGGATAAAATATAGTCT
76
387


 


297692
20
603
ACATTTTATGAGGGATAAAA
70
388


 


297693
20
610
ATTTAAAACATTTTATGAGG
32
389


 


297701
21
1302
TCTCCGCTGGATCCCGCCAT
36
390


 


297702
21
1452
ACATTCCAAATCGCTTTCAC
21
391


 


297703
21
1795
CTGGCTAAATCTGAAACAAC
18
392


 


297704
21
1942
GCATACTCACTTGGCAGAGA
81
393


 


297705
21
2844
TCTTTTAAGAGATATAGTGA
58
394


 


297706
21
4327
AAAGTAAGTTTAGAGGCATC
55
395


 


297707
21
4347
AAGGACAATACTTTGGAGGA
65
396


 


297769
22
144
AGAGCCCAACACTTAGGTCA
55
397


 


297770
22
152
TAGATTCAAGAGCCCAACAC
61
398


 


297771
22
269
TCCCTGGTGACCTGGCCCCA
80
399


 


297772
22
325
TTAAGAAAGATCACACAGGG
34
400


 


297773
22
349
GGTGTGACCTGATAAGAAAC
59
401


 


297774
22
383
GTGCCCCATGTAGAAAGGCA
73
402


 


297775
22
410
AGAGGGAGCAAACTCAGGTC
66
403


 


297776
22
416
GGAGGAAGAGGGAGCAAACT
39
404


 


297766
23
58
CGGCTACTGGTGTCCCCACT
23
405


 


297767
23
66
GAGGTCCGCGGCTACTGGTG
30
406


 


297768
23
77
ATTGTCTTGGCGAGGTCCGC
26
407


 


297785
24
56
TGTCTTGGCTCTTTCCGTCA
29
408


 


297708
25
81
GCCGCCAGACCTCTTTCCGT
19
409


 


297709
25
94
TGTCAACAGAGAAGCCGCCA
82
410


 


297710
25
98
GAGTTGTCAACAGAGAAGCC
68
411


 


297711
25
106
AACCAGCTGAGTTGTCAACA
77
412


 


297712
25
113
GGTGTGGAACCAGCTGAGTT
48
413


 


297713
25
167
GAGAGGAGATCAGCTCAGTG
62
414


 


297714
25
176
GGTGCTCCAGAGAGGAGATC
72
415


 


297715
25
204
ATTGTCTTGGCTCCCTCCTG
36
416


 


297716
25
213
GGAGTTTGCATTGTCTTGGC
68
417


 


297717
25
224
GAATGTTCACTGGAGTTTGC
83
418


 


297718
25
231
GGCACGGGAATGTTCACTGG
76
419


 


297719
25
328
CTGGATTTCAGGAACCTCTG
74
420


 


297720
25
339
ATGGGACACTCCTGGATTTC
65
421


 


297721
25
347
CTTGAGCTATGGGACACTCC
86
422


 


297722
25
374
CCCGCTGCTCCTGGGATTCC
75
423


 


297723
25
507
AAGTTGGCCACATCCAGGTC
82
424


 


297724
25
513
ACGTAGAAGTTGGCCACATC
83
425


 


297725
25
527
TCTGCAGCTGTGACACGTAG
89
426


 


297726
25
537
CCTATGGGAGTCTGCAGCTG
83
427


 


297727
25
540
ACACCTATGGGAGTCTGCAG
90
428


 


297728
25
544
TTGCACACCTATGGGAGTCT
86
429


 


297729
25
550
CTCTGCTTGCACACCTATGG
87
430


 


297730
25
572
TGTCACTACATCGGAGCAGC
0
431


 


297731
25
594
GGCTTGAAGGTATATGAAAT
76
432


 


297732
25
607
ACAATATCTTTATGGCTTGA
85
433


 


297733
25
680
GCCCAGAGTTCCTGGCTCGG
70
434


 


297734
25
701
GGGAGCTCATAACTCCATGG
72
435


 


297735
25
725
TTAAAGCTTGGCTCAAAATT
51
436


 


297736
25
741
AGGAGTCCAGACTTGCTTAA
74
437


 


297737
25
751
AGGAGGTCTCAGGAGTCCAG
84
438


 


297738
25
792
CTTAGAATTCCTAGAGTTGC
78
439


 


297739
25
808
TTCCTTCCAATGGGATCTTA
84
440


 


297740
25
822
TGTGAGGTAGAGCATTCCTT
93
441


 


297741
25
831
TCAGAGTTCTGTGAGGTAGA
86
442


 


297742
25
858
GGCAGCAGGCCCATATTTCT
76
443


 


297743
25
885
ACCTCGATGCCCCGGTCTTC
76
444


 


297744
25
907
AGGTTGTATCCTTTATCACC
83
445


 


297745
25
941
ACTTTGCAGCCTCTTTAATA
37
446


 


297746
25
1020
AAACAGGAGTCGAATTTTCC
30
447


 


297747
25
1059
AAGATGACTCCATCCCCGTC
28
448


 


297748
25
1070
ACCAGCCCCTAAAGATGACT
38
449


 


297749
25
1083
GGATAACCACCCTACCAGCC
61
450


 


297750
25
1114
ACTTGGCCCAGCTCCACTGG
50
451


 


297751
25
1161
GACTTGCGGCCGTGCAGCCC
66
452


 


297752
25
1171
CTTTCCAGGAGACTTGCGGC
43
453


 


297753
25
1188
ATGCCGGTCACTCCAGCCTT
44
454


 


297754
25
1303
TCCACAGCAACCACAGGTGG
19
455


 


297755
25
1331
GGGACAGCGAATCTGCTGTC
20
456


 


297756
25
1405
CACCCTGATCGCGAATCCTC
56
457


 


297757
25
1414
GTGAAGTTGCACCCTGATCG
59
458


 


297758
25
1442
TGCAAGGCTGGAGAGAAGAG
48
459


 


297759
25
1477
AGGCTTAGTGAAGCGCGTGA
40
460


 


297760
25
1504
ATGCGCTGGAAGGTTACTAG
38
461


 


297761
25
1515
CTTTTTAAACAATGCGCTGG
54
462


 


297762
25
1524
AACTGCTTTCTTTTTAAACA
14
463


 


297763
25
1574
GAAGGCTTCGCTTACATCCT
37
464


 


297764
25
1592
ACACACCTTTACTTAATGGA
0
465


 


297765
25
1601
CGAAACGACACACACCTTTA
10
466


 


297777
26
1542
TTGTACTCACCGAGGTCCGC
51
467


 


297778
26
1640
GCGGTCCTACCTCTTTCCGT
51
468


 


297779
26
1647
TGCAGGCGCGGTCCTACCTC
68
469


 


297780
26
2169
GCCGCCAGACCTAATTAATT
54
470


 


297781
26
4301
AGACACTAGGAACGCAATGA
80
471


 


297782
26
8747
ATGCTATGATGTTGAATGTG
63
472


 


297783
26
8992
CTATGCTGTTATCAGGATTC
78
473


 


297784
26
12369
ATTGTCTTGGCTGTTGAGTG
61
474

## Claims

1. A method of modulating expression of a Gemin gene in a cell comprising contacting the cell with an oligomeric compound of from 13 to 30 nucleobases in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 1110 to 1183 of SEQ ID NO. 7, and wherein said nucleobase sequence is at least 90% complementary to SEQ ID NO:7 as measured over the entirety of the oligomeric compound, and thereby modulating expression of the Gemin gene.

2. The method of claim 1, wherein said oligomeric compound is 20 nucleobases in length.

3. The method of claim 1, wherein said oligomeric compound is chimeric.

4. The method of claim 1, wherein said oligomeric compound comprises at least one modified internucleoside linkage.

5. The method of claim 4, wherein the modified internucleoside linkage comprises a phosphorothioate internucleoside linkage.

6. The method of claim 1, wherein said oligomeric compound comprises at least one modified sugar moiety.

7. The method of claim 6, wherein said modified sugar moiety comprises a 2′-O-methoxyethyl sugar moiety.

8. The method of claim 1, wherein said oligomeric compound comprises at least one modified nucleobase.

9. The method of claim 8, wherein said modified nucleobase comprises a 5-methylcytosine.

10. The method of claim 1, wherein said oligomeric compound is an antisense, chimeric oligonucleotide 20 nucleobases in length and comprises at least one phosphorothioate internucleoside linkage and at least one 2′-O-methoxyethyl sugar moiety and at least one 5-methylcytosine.

11. The method of claim 1, wherein the expression of the Gemin gene is reduced by at least 50%.

12. The method of claim 1, wherein the cell is in an animal.

13. The method of claim 12, wherein the animal is a human.

14. The method of claim 1, wherein said nucleobase sequence is at least 95% complementary to SEQ ID NO:7 as measured over the entirety of the oligomeric compound.

15. The method of claim 1, wherein said nucleobase sequence is 100% complementary to SEQ ID NO:7 as measured over the entirety of the oligomeric compound.

16. The method of claim 10, wherein said nucleobase sequence is 100% complementary to SEQ ID NO:7 as measured over the entirety of the oligomeric compound.