# Patent

## Title
LNA antagonists targeting the androgen receptor

## Abstract

The invention relates to oligonucleotide compounds (oligomers), which target androgen receptor mRNA in a cell, leading to reduced expression of the androgen receptor. Reduction of the androgen receptor expression is beneficial for the treatment of certain disorders, such as a hyperproliferative disorders (e.g., cancer). The invention provides therapeutic compositions comprising oligomers and methods for modulating the expression of androgen receptor using said oligomers, including methods of treatment.

## Background
This application is a continuation application of U.S. application Ser. No. 12/322,033 filed on Nov. 26, 2008, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 60/990,125 filed Nov. 26, 2007, the disclosure of each of which is incorporated herein by reference in its entirety.


FIELD OF INVENTION
The invention provides compounds, compositions and methods for modulating the expression of the androgen receptor. In particular, this invention relates to oligomeric compounds (oligomers), which target the androgen receptor mRNA in a cell, leading to reduced expression of the androgen receptor. Reduction of androgen receptor expression is beneficial for a range of medical disorders, such as cancer, particularly prostate cancer or breast cancer.
BACKGROUND
The androgen receptor (“AR”) is a type of nuclear receptor which is activated by binding of either of the androgenic hormones testosterone or dihydrotestosterone. The main function of the androgen receptor is as a DNA binding transcription factor which regulates gene expression. However the androgen receptor also has additional functions independent of DNA binding. The androgen receptor is most closely related to the progesterone receptor, and progestins in higher dosages can block the androgen receptor.
Whilst in humans the AR gene is single copy and found on the X chromosome at position Xq11-12, the receptor itself exists in two iso-forms (A and B). AR-A is an 87 kDa protein which lacks the first 187 amino acids (N-terminal truncation). Isoform AR-B is the full length 110 kDa version.
The binding of an androgen to the androgen receptor induces a conformational change in the receptor, resulting in a dissociation of heat shock proteins, dimerization and transport from the cytosol to the cell nucleus where the androgen receptor dimer binds to specific DNA sequences—referred to as hormone response elements. Depending on the interaction with other nuclear proteins, the AR controls gene expression, either increasing or decreasing transcription of specific genes, such as insulin-like growth factor I (IGF-1).
Androgen receptors can also have cytoplasmic activities through interaction with signal transduction proteins in the cytoplasm. Androgen binding to cytoplasmic androgen receptors can cause rapid changes in cell function independent of gene transcription, for example ion transport, as well as indirect influence of gene transcription, for example via mediating other signal transduction pathways, thereby influencing the activity of other transcription factors.
The over-expression of androgen receptor, or expression of mutated androgen receptor genes, has been indicated in several diseases, such as cancer, including prostate cancer and breast cancer, as well as other disorders such as polyglutamate disease (Monks et al., PNAS Nov. 2, 2007, published on line) alopecia, benign prostatic hyperplasia, spinal and muscular atrophy and Kennedy disease.
WO97/11170 describes a method of treating a patient diagnosed as having benign prostatic hyperplasia or a prostate cancer comprising administering an antisense oligonucleotide which selectively hybridises to the androgen receptor mRNA. Three antisense oligonucleotide sequences of between 27-29 nucleotides are disclosed.
U.S. Pat. No. 6,733,776 and EP 0 692 972 describe a method for treating androgenic alopecia by applying liposomes comprising an antisense nucleic acid that hybridises to an androgen receptor gene. No antisense molecules having specific sequences and targeting the androgen receptor are provided.
US 2005/0164970 describes a method of treating prostate cancer using siRNA complexes targeting the androgen receptor mRNA.
WO 2005/027833 describes a method of treating prostate cancer comprising administering to a patient an oligonucleotide comprising between 12-40 morpholino sub-units.
WO 2001/083740 describes an antisense compound having an uncharged morpholino backbone of between 18 to 20 contiguous units which targets the human androgen receptor.
Morpholino antisense compounds work via binding to the nucleic acid target to block access to the mRNA by other molecules, such as molecules involved in mRNA splicing or translation initiation.
U.S. Pat. No. 7,067,256 describes a ribozyme which apparently mediates inactivation of the androgen receptor. A 19-nucleotide RNA antisense molecule targeted to a region of the androgen receptor mRNA is provided.
However, despite the application of siRNA, morpholino-containing antisense oligonucleotides and ribozymes, none of the above androgen receptor inhibitors have been successful in efficiently down-regulating the androgen-receptor in vivo and at pharmacologically acceptable dosages.
The invention provides a new class of androgen receptor antagonists which contain locked nucleic acid (“LNA”) monomers, and are targeted to particularly effective target sites on the androgen receptor mRNA.
SUMMARY OF INVENTION
The invention provides an oligomer of from 10-50 monomers, such as 10-30 monomers which comprises a first region of 10-50 monomers, such as 10-30 monomers, wherein the sequence of the first region is at least 80% (e.g., 85%, 90%, 95%, 98%, or 99%) identical to the reverse complement of a target region of a nucleic acid which encodes a mammalian androgen receptor, such as a mammalian androgen receptor gene or mRNA, such as a nucleic acid having the sequence set forth in SEQ ID NO: 1, or naturally occurring variants thereof. Thus, for example, the oligomer hybridizes to a region of a single-stranded nucleic acid molecule having the sequence shown in SEQ NO: 1.
The invention provides for a conjugate comprising the oligomer according to the invention, and at least one non-nucleotide or non-polynucleotide moiety covalently attached to the oligomer.
The invention provides for a pharmaceutical composition comprising the oligomer or the conjugate according to the invention, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
The invention provides for the oligomer or the conjugate according to the invention, for use as a medicament, such as for the treatment of a disease or a medical disorder as disclosed herein, such as a hyperproliferative disorder, such as cancer or other hyperproliferative disorder. The invention provides for the use of an oligomer or the conjugate according to the invention, for the manufacture of a medicament for the treatment of a disease or disorder as disclosed herein, such as a hyperproliferative disorder, such as cancer.
The invention provides for a method of treating a disease or disorder as disclosed herein, such as a hyperproliferative disorder, such as cancer, the method comprising administering an oligomer, a conjugate or a pharmaceutical composition according to the invention to a patient suffering from or susceptible to the disease or disorder.
The invention provides for a method for the inhibition of androgen receptor in a cell which is expressing androgen receptor, the method comprising administering an oligomer, or a conjugate according to the invention to the cell so as to effect the inhibition of androgen receptor expression in said cell.
The invention provides an oligomer of from 10-50 monomers, which comprises a first region of 10-50 contiguous monomers, wherein the base sequence is at least 80% identical to the reverse complement of a target region of a nucleic acid which encodes a mammalian androgen receptor.
The invention further provides a conjugate comprising the oligomer according to the invention, which comprises at least one non-nucleotide or non-polynucleotide moiety (“conjugated moiety”) covalently attached to the oligomer of the invention.
The invention provides for pharmaceutical compositions comprising an oligomer or conjugate of the invention, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
The invention further provides for an oligomer according to the invention, for use in medicine.
The invention further provides for the use of the oligomer of the invention for the manufacture of a medicament for the treatment of one or more of the diseases referred to herein, such as a disease selected from the group consisting of cancer, such as breast cancer or prostate cancer, alopecia, benign prostatic hyperplasia, spinal and muscular atrophy, Kennedy disease and polyglutamate disease.
The invention further provides for an oligomer according to the invention, for use for the treatment of one or more of the diseases referred to herein, such as a disease selected from the group consisting of cancer, such as breast cancer or prostate cancer, alopecia, benign prostatic hyperplasia, spinal and muscular atrophy, Kennedy disease and polyglutamate disease.
Pharmaceutical and other compositions comprising an oligomer of the invention are also provided. Further provided are methods of down-regulating the expression of AR in cells or tissues comprising contacting said cells or tissues, in vitro or in vivo, with one or more of the oligomers, conjugates or compositions of the invention.
Also disclosed are methods of treating a non-human animal or a human suspected of having, or susceptible to, a disease or condition, associated with expression, or over-expression of AR by administering to the animal or human a therapeutically or prophylactically effective amount of one or more of the oligomers, conjugates or pharmaceutical compositions of the invention. Further, methods of using oligomers for the inhibition of expression of AR, and for treatment of diseases associated with activity of AR are provided.
The invention provides for a method for treating a disease selected from the group consisting of: cancer, such as breast cancer or prostate cancer, alopecia, benign prostatic hyperplasia, spinal and muscular atrophy, Kennedy disease and polyglutamate disease, the method comprising administering an effective amount of one or more oligomers, conjugates, or pharmaceutical compositions thereof to a patient in need thereof.
The invention provides for methods of inhibiting (e.g., by down-regulating) the expression of AR in a cell or a tissue, the method comprising the step of contacting the cell or tissue with an effective amount of one or more oligomers, conjugates, or pharmaceutical compositions thereof, to effect down-regulation of expression of AR.



BRIEF DESCRIPTION OF FIGURES
FIG. 1. Oligonucleotides presented in Table 3 were evaluated for their potential to knockdown the androgen receptor mRNA at concentrations of 1, 4 and 16 nM in MCF7 cells 24 hours after transfection using Real-time PCR. All results were normalised to GAPDH and inhibition of AR mRNA is shown as percent of untreated control. Results shown are an average of three independent experiments.
FIG. 2. Oligonucleotides presented in Table 3 were evaluated for their potential to knockdown the androgen receptor mRNA at concentrations of 1, 4 and 16 nM in A549 cells 24 hours after transfection using Real-time PCR. All results were normalised to GAPDH and inhibition of AR mRNA is shown as percent of untreated control. Results shown are an average of three independent experiments.
FIG. 3. Sequence alignment of the human Androgen receptor mRNA sequence (GenBank Accession No.: NM—000044) and the mouse Androgen receptor mRNA sequence (GenBank Accession No.: NM—013476).
FIG. 4. Location of presently preferred target regions of the human AR mRNA (cDNA) targeted by oligomers according to the invention. Although 16mer target sites have been shown, in some embodiments these target regions comprise an additional 4 monomers 5′ or 3′ to the target regions shown—i.e. are target regions comprising up to 24 contiguous monomers.
FIG. 5. SEQ ID NO: 1 Homo sapiens androgen receptor (dihydrotestosterone receptor; testicular feminization; spinal and bulbar muscular atrophy; Kennedy disease) (AR), transcript variant 1, mRNA. (GenBank Accession number: NM—000044).
FIG. 6. SEQ ID NO 81: Mouse androgen receptor mRNA sequence.
FIG. 7. SEQ ID NO 82: Rhesus monkey androgen receptor mRNA sequence.
FIG. 8. SEQ ID NO 83: Homo sapiens androgen receptor protein amino acid sequence.
FIG. 9. SEQ ID NO 84: Mouse androgen receptor protein amino acid sequence.
FIG. 10. SEQ ID NO 85: Rhesus monkey androgen receptor protein amino acid sequence.
FIG. 11: AR mRNA in LNCaP, 24 h post-transfection
FIG. 12: AR mRNA in A549, 24 h post-transfection
FIG. 13: Cell proliferation assay—A549, time course post-transfection
FIG. 14: Cell proliferation assay—time course post-transfection
FIG. 15: Caspase 3/7 activity in LNCaP cells, 24, 48 or 72 hours post-transfection.
FIG. 16: Caspase 3/7 activity in A549 cells, 24, 48 or 72 hours post-transfection.
FIG. 17: Average PSA in plasma after in vivo oligomer treatment.
FIG. 18: In vivo inhibition of tumor growth



DETAILED DESCRIPTION OF INVENTION
The Oligomer
The invention employs oligomeric compounds (referred herein as oligomers), for use in modulating the function of nucleic acid molecules encoding mammalian androgen receptor, such as the androgen receptor nucleic acid shown in SEQ ID NO: 1, and naturally occurring variants of such nucleic acid molecules encoding mammalian androgen receptor. The term “oligomer” in the context of the invention, refers to a molecule formed by covalent linkage of two or more monomers (i.e. an oligonucleotide). In some embodiments, the oligomer comprises or consists of from 10-30 covalently linked monomers.
The term “monomer” includes both nucleosides and deoxynucleosides (collectively, “nucleosides”) that occur naturally in nucleic acids and that do not contain either modified sugars or modified nucleobases, i.e., compounds in which a ribose sugar or deoxyribose sugar is covalently bonded to a naturally-occurring, unmodified nucleobase (base) moiety (i.e., the purine and pyrimidine heterocycles adenine, guanine, cytosine, thymine or uracil) and “nucleoside analogues,” which are nucleosides that either do occur naturally in nucleic acids or do not occur naturally in nucleic acids, wherein either the sugar moiety is other than a ribose or a deoxyribose sugar (such as bicyclic sugars or 2′ modified sugars, such as 2′ substituted sugars), or the base moiety is modified (e.g., 5-methylcytosine), or both.
An “RNA monomer” is a nucleoside containing a ribose sugar and an unmodified nucleobase.
A “DNA monomer” is a nucleoside containing a deoxyribose sugar and an unmodified nucleobase.
A “Locked Nucleic Acid monomer,” “locked monomer,” or “LNA monomer” is a nucleoside analogue having a bicyclic sugar, as further described herein below.
The terms “corresponding nucleoside analogue” and “corresponding nucleoside” indicate that the base moiety in the nucleoside analogue and the base moiety in the nucleoside are identical. For example, when the “nucleoside” contains a 2-deoxyribose sugar linked to an adenine, the “corresponding nucleoside analogue” contains, for example, a modified sugar linked to an adenine base moiety.
The terms “oligomer,” “oligomeric compound,” and “oligonucleotide” are used interchangeably in the context of the invention, and refer to a molecule formed by covalent linkage of two or more contiguous monomers by, for example, a phosphate group (forming a phosphodiester linkage between nucleosides) or a phosphorothioate group (forming a phosphorothioate linkage between nucleosides). The oligomer consists of, or comprises, 10-50 monomers, such as 10-30 monomers.
In some embodiments, an oligomer comprises nucleosides, or nucleoside analogues, or mixtures thereof as referred to herein. An “LNA oligomer” or “LNA oligonucleotide” refers to an oligonucleotide containing one or more LNA monomers.
Nucleoside analogues that are optionally included within oligomers may function similarly to corresponding nucleosides, or may have specific improved functions. Oligomers wherein some or all of the monomers are nucleoside analogues are often preferred over native forms because of several desirable properties of such oligomers, such as the ability to penetrate a cell membrane, good resistance to extra- and/or intracellular nucleases and high affinity and specificity for the nucleic acid target. LNA monomers are particularly preferred, for example, for conferring several of the above-mentioned properties.
In various embodiments, one or more nucleoside analogues present within the oligomer are “silent” or “equivalent” in function to the corresponding natural nucleoside, i.e., have no functional effect on the way the oligomer functions to inhibit target gene expression. Such “equivalent” nucleoside analogues are nevertheless useful if, for example, they are easier or cheaper to manufacture, or are more stable under storage or manufacturing conditions, or can incorporate a tag or label. Typically, however, the analogues will have a functional effect on the way in which the oligomer functions to inhibit expression; for example, by producing increased binding affinity to the target region of the target nucleic acid and/or increased resistance to intracellular nucleases and/or increased ease of transport into the cell.
Thus, in various embodiments, oligomers according to the invention comprise nucleoside monomers and at least one nucleoside analogue monomer, such as an LNA monomer, or other nucleoside analogue monomers.
The term “at least one” comprises the integers larger than or equal to 1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and so forth. In various embodiments, such as when referring to the nucleic acid or protein targets of the compounds of the invention, the term “at least one” includes the terms “at least two” and “at least three” and “at least four.” Likewise, in some embodiments, the term “at least two” comprises the terms “at least three” and “at least four.”
In some embodiments, the oligomer comprises or consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous monomers.
In some embodiments, the oligomer comprises or consists of 10-22 contiguous monomers, such as 12-18 contiguous monomers, such as 13-17 or 12-16 contiguous monomers, such as 13, 14, 15, 16 contiguous monomers.
In certain embodiments, the oligomer comprises or consists of 10, 11, 12, 13, or 14 contiguous monomers.
In various embodiments, the oligomer according to the invention consists of no more than 22 monomers, such as no more than 20 monomers, such as no more than 18 monomers, such as 15, 16 or 17 monomers. In some embodiments, the oligomer of the invention comprises less than 20 monomers.
In various embodiments, the compounds of the invention do not comprise RNA monomers.
In various embodiments, the compounds according to the invention are linear molecules or are linear as synthesised. The oligomer, in such embodiments, is a single stranded molecule, and typically does not comprise short regions of, for example, at least 3, 4 or 5 contiguous monomers, which are complementary to another region within the same oligomer such that the oligomer forms an internal duplex. In some embodiments, the oligomer is essentially not double stranded, i.e., is not a siRNA.
In some embodiments, the oligomer of the invention consists of a contiguous stretch of monomers, the sequence of which is identified by a SEQ ID NO disclosed herein (see, e.g., Tables 1-3). In other embodiments, the oligomer comprises a first region, the region consisting of a contiguous stretch of monomers, and one or more additional regions which consist of at least one additional monomer. In some embodiments, the sequence of the first region is identified by a SEQ ID NO disclosed herein.
Gapmer Design
Typically, the oligomer of the invention is a gapmer.
A “gapmer” is an oligomer which comprises a contiguous stretch of monomers capable of recruiting an RNAse (e.g., such as RNAseH) as further described herein below, such as a region of at least 6 or 7 DNA monomers, referred to herein as region B, wherein region B is flanked both on its 5′ and 3′ ends by regions respectively referred to as regions A and C, each of regions A and C comprising or consisting of nucleoside analogues, such as affinity-enhancing nucleoside analogues, such as 1-6 nucleoside analogues.
Typically, the gapmer comprises regions, from 5′ to 3′, A-B-C, or optionally A-B-C-D or D-A-B-C, wherein: region A consists of or comprises at least one nucleoside analogue, such as at least one LNA monomer, such as 1-6 nucleoside analogues, such as LNA monomers, and region B consists of or comprises at least five contiguous monomers which are capable of recruiting RNAse (when formed in a duplex with a complementary target region of the target RNA molecule, such as the mRNA target), such as DNA monomers; region C consists of or comprises at least one nucleoside analogue, such as at least one LNA monomer, such as 1-6 nucleoside analogues, such as LNA monomers; and region D, when present, consists of or comprises 1, 2 or 3 monomers, such as DNA monomers.
In various embodiments, region A consists of 1, 2, 3, 4, 5 or 6 nucleoside analogues, such as LNA monomers, such as 2-5 nucleoside analogues, such as 2-5 LNA monomers, such as 3 or 4 nucleoside analogues, such as 3 or 4 LNA monomers; and/or region C consists of 1, 2, 3, 4, 5 or 6 nucleoside analogues, such as LNA monomers, such as 2-5 nucleoside analogues, such as 2-5 LNA monomers, such as 3 or 4 nucleoside analogues, such as 3 or 4 LNA monomers.
In certain embodiments, region B consists of or comprises 5, 6, 7, 8, 9, 10, 11 or 12 contiguous monomers which are capable of recruiting RNAse, or 6-10, or 7-9, such as 8 contiguous monomers which are capable of recruiting RNAse. In certain embodiments, region B consists of or comprises at least one DNA monomer, such as 1-12 DNA monomers, preferably 4-12 DNA monomers, more preferably 6-10 DNA monomers, such as 7-10 DNA monomers, most preferably 8, 9 or 10 DNA monomers.
In various embodiments, region A consists of 3 or 4 nucleoside analogues, such as LNA monomers, region B consists of 7, 8, 9 or 10 DNA monomers, and region C consists of 3 or 4 nucleoside analogues, such as LNA monomers. Such designs include (A-B-C) 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3, and may further include region D, which may have one or 2 monomers, such as DNA monomers.
Further gapmer designs are disclosed in WO2004/046160, which is hereby incorporated by reference.
US provisional application, 60/977,409, hereby incorporated by reference, refers to ‘shortmer’ gapmer oligomers. In some embodiments, oligomers presented here may be such shortmer gapmers.
In certain embodiments, the oligomer consists of 10, 11, 12, 13 or 14 contiguous monomers, wherein the regions of the oligomer have the pattern (5′-3′), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein: region A consists of 1, 2 or 3 nucleoside analogue monomers, such as LNA monomers; region B consists of 7, 8 or 9 contiguous monomers which are capable of recruiting RNAse when formed in a duplex with a complementary RNA molecule (such as a mRNA target); and region C consists of 1, 2 or 3 nucleoside analogue monomers, such as LNA monomers. When present, region D consists of a single DNA monomer.
In certain embodiments, region A consists of 1 LNA monomer. In certain embodiments, region A consists of 2 LNA monomers. In certain embodiments, region A consists of 3 LNA monomers. In certain embodiments, region C consists of 1 LNA monomer. In certain embodiments, region C consists of 2 LNA monomers. In certain embodiments, region C consists of 3 LNA monomers. In certain embodiments, region B consists of 7 nucleoside monomers, In certain embodiments, region B consists of 8 nucleoside monomers. In certain embodiments, region B consists of 9 nucleoside monomers. In certain embodiments, region B comprises 1-9 DNA monomers, such as 2, 3, 4, 5, 6, 7 or 8 DNA monomers. In certain embodiments, region B consists of DNA monomers. In certain embodiments, region B comprises at least one LNA monomer which is in the alpha-L configuration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA monomers in the alpha-L-configuration. In certain embodiments, region B comprises at least one alpha-L-oxy LNA monomer. In certain embodiments, all the LNA monomers in region B that are in the alpha-L-configuration are alpha-L-oxy LNA units. In certain embodiments, the number of monomers present in the A-B-C regions are selected from the group consisting of (nucleoside analogue monomers—region B—nucleoside analogue monomers): 1-8-1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4, 2-8-4, or; 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1, 4-9-1, 1-9-4, or; 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1. In certain embodiments, the number of monomers present in the A-B-C regions of the oligomer of the invention is selected from the group consisting of: 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3. In certain embodiments, each of regions A and C consists of two LNA monomers, and region B consists of 8 or 9 nucleoside monomers, preferably DNA monomers.
In various embodiments, other gapmer designs include those where regions A and/or C consists of 3, 4, 5 or 6 nucleoside analogue, such as monomers containing a 2′-O-methoxyethyl-ribose sugar (2′-MOE) or monomers containing a 2′-fluoro-deoxyribose sugar, and region B consists of 8, 9, 10, 11 or 12 nucleosides, such as DNA monomers, where regions A-B-C have 5-10-5 or 4-12-4 monomers. Further gapmer designs are disclosed in WO 2007/146511A2, hereby incorporated by reference.
Internucleoside Linkages
The monomers of the oligomers described herein are coupled together via linkage groups. Suitably, each monomer is linked to the 3′ adjacent monomer via a linkage group.
The terms “linkage group” or “internucleoside linkage” means a group capable of covalently coupling together two contiguous monomers. Specific and preferred examples include phosphate groups (forming a phosphodiester between adjacent nucleoside monomers) and phosphorothioate groups (forming a phosphorothioate linkage between adjacent nucleoside monomers).
Suitable linkage groups include those listed in PCT/DK2006/000512, for example in the first paragraph of page 34 of PCT/DK2006/000512 (hereby incorporated by reference).
It is, in various embodiments, preferred to modify the linkage group from its normal phosphodiester to one that is more resistant to nuclease attack, such as phosphorothioate or boranophosphate—these two being cleavable by RNase H, thereby permitting RNase-mediated antisense inhibition of expression of the target gene.
In some embodiments, suitable sulphur (S) containing linkage groups as provided herein are preferred. In various embodiments, phosphorothioate linkage groups are preferred, particularly for the gap region (B) of gapmers. In certain embodiments, phosphorothioate linkages are used to link together monomers in the flanking regions (A and C). In various embodiments, phosphorothioate linkages are used for linking regions A or C to region D, and for linking together monomers within region D.
In various embodiments, regions A, B and C, comprise linkage groups other than phosphorothioate, such as phosphodiester linkages, particularly, for instance when the use of nucleoside analogues protects the linkage groups within regions A and C from endo-nuclease degradation—such as when regions A and C comprise LNA monomers.
In various embodiments, adjacent monomers of the oligomer are linked to each other by means of phosphorothioate groups.
It is recognised that the inclusion of phosphodiester linkages, such as one or two linkages, into an oligomer with a phosphorothioate backbone, particularly with phosphorothioate linkage groups between or adjacent to nucleoside analogue monomers (typically in region A and/or C), can modify the bioavailability and/or bio-distribution of an oligomer—see WO2008/053314, hereby incorporated by reference.
In some embodiments, such as the embodiments referred to above, where suitable and not specifically indicated, all remaining linkage groups are either phosphodiester or phosphorothioate, or a mixture thereof.
In some embodiments all the internucleoside linkage groups are phosphorothioate.
When referring to specific gapmer oligonucleotide sequences, such us those provided herein, it will be understood that, in various embodiments, when the linkages are phosphorothioate linkages, alternative linkages, such as those disclosed herein may be used, for example phosphate (phosphodiester) linkages may be used, particularly for linkages between nucleoside analogues, such as LNA monomers. Likewise, in various embodiments, when referring to specific gapmer oligonucleotide sequences, such as those provided herein, when one or more monomers in region C comprises a 5-methylcytosine base, other monomers in that region may contain unmodified cytosine bases.
Target Nucleic Acid
The terms “nucleic acid” and “polynucleotide” are used interchangeably herein, and are defined as a molecule formed by covalent linkage of two or more monomers, as above-described. Including 2 or more monomers, “nucleic acids” may be of any length, and the term is generic to “oligomers”, which have the lengths described herein. The terms “nucleic acid” and “polynucleotide” include single-stranded, double-stranded, partially double-stranded, and circular molecules.
The term “target nucleic acid”, as used herein, refers to DNA or RNA (e.g., mRNA or pre-mRNA) encoding a mammalian androgen receptor polypeptide, such as human androgen receptor, such as the nucleic acid having the sequence shown in SEQ ID NO: 1, and naturally occurring allelic variants of such nucleic acids. In certain embodiments, the mammalian androgen receptor is a mouse androgen receptor. In some embodiments, for example when used in research or diagnostics, the “target nucleic acid” is a cDNA or a synthetic oligonucleotide derived from the above DNA or RNA nucleic acid targets. The oligomers according to the invention are typically capable of hybridising to the target nucleic acid.
Exemplary target nucleic acids include mammalian androgen receptor-encoding nucleic acids having the GenBank Accession numbers shown in the table below, along with their corresponding protein sequences:














GenBank Accession Number
GenBank



Nucleic acid (mRNA/cDNA
Accession Number



sequence)
Polypeptide (deduced)


















Human
NM_000044
NP_000035


Mouse
NM_013476
NP_038504


Rhesus monkey
NM_001032911
NP_001028083









It is recognised that the above-disclosed GenBank Accession numbers for nucleic acids refer to cDNA sequences and not to mRNA sequences per se. The sequence of a mature mRNA can be derived directly from the corresponding cDNA sequence with thymine bases (T) being replaced by uracil bases (U).
The term “naturally occurring variant thereof” refers to variants of the androgen receptor polypeptide or nucleic acid sequence which exist naturally within the defined taxonomic group, such as mammalian, such as mouse, monkey, and preferably human AR. Typically, when referring to “naturally occurring variants” of a polynucleotide the term also encompasses any allelic variant of the androgen receptor encoding genomic DNA which is found at the Chromosome X: 66.68-66.87 Mb by chromosomal translocation or duplication, and the RNA, such as mRNA derived therefrom. “Naturally occurring variants” may also include variants derived from alternative splicing of the androgen receptor mRNA. When referenced to a specific polypeptide sequence, e.g., the term also includes naturally occurring forms of the protein which may therefore be processed, e.g. by co- or post-translational modifications, such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc.
It is recognised that the human androgen receptor gene exhibits allelic variations that are associated with disease phenotypes (Mooney et al, NAR 15; 31(8) 2003). For example, a (CAG)n repeat expansion is associated with polyglutamine expansion disorder. Other characterised allelic variants include a (GGC)n trinucleotide repeat and single nucleotide polymorphisms R726L, T887A and L710H, of which the latter two single nucleotide polymorphisms have been shown to be correlated to enhanced promiscuity of the AR receptor for other steroid ligands. In one embodiment “n” ranges from 5-31. CAG repeats of less than 22 have been associated with an enhanced risk of prostate cancer in African American males.
In various embodiments, the target nucleic acid is an AR allelic variant which comprises a (CAG)n trinucleotide repeat, or (GGC)n trinucleotide repeat. In other embodiments, the target nucleic acid is an AR allelic variant which comprises one or more single nucleotide polymorphisms, including R726L, T887A and L710H.
In certain embodiments, oligomers described herein bind to a region of the target nucleic acid (the “target region”) by either Watson-Crick base pairing, Hoogsteen hydrogen bonding, or reversed Hoogsteen hydrogen bonding, between the monomers of the oligomer and monomers of the target nucleic acid. Such binding is also referred to as “hybridisation.” Unless otherwise indicated, binding is by Watson-Crick pairing of complementary bases (i.e., adenine with thymine (DNA) or uracil (RNA), and guanine with cytosine), and the oligomer binds to the target region because the sequence of the oligomer is identical to, or partially-identical to, the sequence of the reverse complement of the target region; for purposes herein, the oligomer is said to be “complementary” or “partially complementary” to the target region, and the percentage of “complementarity” of the oligomer sequence to that of the target region is the percentage “identity” to the reverse complement of the sequence of the target region.
Unless otherwise made clear by context, the “target region” herein will be the region of the target nucleic acid having the sequence that best aligns with the reverse complement of the sequence of the specified oligomer (or region thereof), using the alignment program and parameters described herein below.
In determining the degree of “complementarity” between oligomers of the invention (or regions thereof) and the target region of the nucleic acid which encodes mammalian androgen receptor, such as those disclosed herein, the degree of “complementarity” (also, “homology”) is expressed as the percentage identity between the sequence of the oligomer (or region thereof) and the reverse complement of the sequence of the target region that best aligns therewith. The percentage is calculated by counting the number of aligned bases that are identical as between the 2 sequences, dividing by the total number of contiguous monomers in the oligomer, and multiplying by 100. In such a comparison, if gaps exist, it is preferable that such gaps are merely mismatches rather than areas where the number of monomers within the gap differs between the oligomer of the invention and the target region.
Amino acid and polynucleotide alignments, percentage sequence identity, and degree of complementarity may be determined for purposes of the invention using the ClustalW algorithm using standard settings: see http://www.ebi.ac.uk/emboss/align/index.html, Method: EMBOSS::water (local): Gap Open=10.0, Gap extend=0.5, using Blosum 62 (protein), or DNAfull for nucleotide/nucleobase sequences.
As will be understood, depending on context, “mismatch” refers to a non-identity in sequence (as, for example, between the nucleobase sequence of an oligomer and the reverse complement of the target region to which it binds; as for example, between the base sequence of two aligned AR encoding nucleic acids), or to noncomplementarity in sequence (as, for example, between an oligomer and the target region to which it binds).
The androgen receptor is known to regulate the expression of several genes, such as a gene selected from the group consisting of Protein kinase C delta (PRKCD), Glutathione S— transferase theta 2 (GSTT2), transient receptor potential cation channel subfamily V member 3 (TRPV3), Pyrroline-5-carboxylate reductase 1 (PYCR1) and ornithine aminotransferase (OAT). Such genes regulated by AR are referred to herein as “androgen receptor (AR) target genes”. In various embodiments, the oligomers according to the invention are capable of inhibiting (such as, by down-regulating) the expression of one or more AR target genes in a cell which is expressing, or is capable of expressing (i.e. by alleviating AR repression of the AR target gene in a cell) an AR target gene.
The oligomers which target the androgen receptor mRNA, may hybridize to any site along the target mRNA nucleic acid, such as the 5′ untranslated leader, exons, introns and 3′ untranslated tail. However, it is preferred that the oligomers which target the androgen receptor mRNA hybridise to the mature mRNA form of the target nucleic acid.
Suitably, the oligomer of the invention or conjugate thereof is capable of down-regulating expression of the androgen receptor gene. In various embodiments, the oligomer (or conjugate) of the invention can effect the inhibition of androgen receptor, typically in a mammalian cell, such as a human cell. In certain embodiments, the oligomers of the invention, or conjugates thereof, bind to the target nucleic acid and effect inhibition of AR mRNA expression of at least 10% or 20% compared to the expression level immediately prior to dosing of the oligomer, more preferably of at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% as compared to the AR expression level immediately prior to dosing of the oligomer. In some embodiments, such inhibition is seen when using from about 0.04 nM to about 25 nM, such as from about 0.8 nM to about 20 nM of the oligomer or conjugate.
In various embodiments, the inhibition of mRNA expression is less than 100% (i.e., less than complete inhibition of expression), such as less than 98% inhibition, less than 95% inhibition, less than 90% inhibition, less than 80% inhibition, such as less than 70% inhibition. In various embodiments, modulation of gene expression can be determined by measuring protein levels, e.g. by the methods such as SDS-PAGE followed by western blotting using suitable antibodies raised against the target protein. Alternatively, modulation of expression levels can be determined by measuring levels of mRNA, e.g. by northern blotting or quantitative RT-PCR. When measuring via mRNA levels, the level of down-regulation when using an appropriate dosage, such as from about 0.04 nM to about 25 nM, such as from about 0.8 nM to about 20 nM, is, in various embodiments, typically to a level of 10-20% of the normal levels in the absence of the compound or conjugate of the invention.
The invention therefore provides a method of down-regulating or inhibiting the expression of the androgen receptor protein and/or mRNA in a cell which is expressing the androgen receptor protein and/or mRNA, the method comprising contacting the cell with an effective amount of the oligomer or conjugate according to the invention to down-regulate or inhibit the expression of the androgen receptor protein and/or mRNA in the cell. Suitably the cell is a mammalian cell, such as a human cell. The contacting may occur, in some embodiments, in vitro. The contacting may occur, in some embodiments, in vivo.
Oligomer Sequences
In some embodiments, the oligomers of the invention have sequences that are identical to a sequence selected from the group consisting of SEQ ID NOS: 2-22. Target regions in human AR mRNA (cDNA) that bind to the oligomers having sequences as set forth in SEQ ID NOs: 2-22 are shown in FIG. 4 (bold and underlined, with the corresponding oligomer SEQ ID NOs indicated above).
Further provided are target nucleic acids (e.g., DNA or mRNA encoding AR) that contain target regions that are complementary or partially-complementary to one or more of the oligomers of the invention. In certain embodiments, the oligomers bind to variants of AR target regions, such as allelic variants (such as an AR gene present at gene locus Xq11-12). In some embodiments, a variant of an AR target region has at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, at least 92%, at least 93%, at least 94%, at least 95% sequence identity to the target region in wild-type AR. Thus, in other embodiments, the oligomers of the invention have sequences that differ in 1, 2 or 3 bases when compared to a sequence selected from the group consisting of SEQ ID NOs: 2-22. Typically, an oligomer of the invention that binds to a variant of an AR target region is capable of inhibiting (e.g., by down-regulating) AR.
In other embodiments, oligomers of the invention are LNA oligomers, for example, those oligomers having the sequences shown in SEQ ID NOs: 44-80. In various embodiments, the oligomers of the invention are potent inhibitors of androgen receptor mRNA and protein expression. In various embodiments, oligomers of the invention are LNA oligomers having the sequences of SEQ ID NO: 58 or SEQ ID NO: 77.
In various embodiments, the oligomer comprises or consists of a region having a base sequence which is identical or partially identical to the sequence of the reverse complement of a target region in SEQ ID NO: 1. In various embodiments, the oligomer comprises or consists of a region having a sequence selected from the group consisting of SEQ ID NOS: 2-22 and 86-106.
In certain embodiments, the oligomer comprises or consists of a region having a base sequence which is fully complementary (perfectly complementary) to a target region of a nucleic acid which encodes a mammalian androgen receptor.
However, in some embodiments, the oligomer includes 1, 2, 3, or 4 (or more) mismatches as compared to the best-aligned target region of an AR target nucleic acid, and still sufficiently binds to the target region to effect inhibition of AR mRNA or protein expression. The destabilizing effect of mismatches on Watson-Crick hydrogen-bonded duplex may, for example, be compensated by increased length of the oligomer and/or an increased number of nucleoside analogues, such as LNA monomers, present within the oligomer.
In various embodiments, the oligomer base sequence comprises no more than 3, such as no more than 2 mismatches compared to the base sequence of the best-aligned target region of, for example, a target nucleic acid which encodes a mammalian androgen receptor.
In some embodiments, the oligomer base sequence comprises no more than a single mismatch when compared to the base sequence of the best-aligned target region of a nucleic acid which encodes a mammalian androgen receptor.
In various embodiments, the base sequence of the oligomer of the invention, or of a first region thereof, is preferably at least 80% identical to a base sequence selected from the group consisting of SEQ ID NOS: 2-22 and 86-106, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% identical, such as 100% identical.
In certain embodiments, the base sequence of the oligomer of the invention or of a first region thereof is at least 80% identical to the base sequence of the reverse complement of a target region present in SEQ ID NO: 1, such as at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, such as 100% identical.
In various embodiments, the base sequence of the oligomer of the invention, or of a first region thereof, is preferably at least 80% complementary to a target region of SEQ ID NO: 1, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% complementary, at least 97% complementary, at least 98% complementary, at least 99% complementary, such as 100% complementary (perfectly complementary).
In some embodiments the oligomer (or a first region thereof) has a base sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, or is selected from the group consisting of at least 10 contiguous monomers of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22. In other embodiments, the sequence of the oligomer of the invention or a first region thereof comprises one, two, or three base moieties that differ from those in oligomers having sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, or the sequences of at least 10 contiguous monomers thereof, when optimally aligned with the selected sequence or region thereof.
In some embodiments the oligomer (or a first region thereof) has a base sequence selected from the group consisting of SEQ ID NOs: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 and 106, or the sequences of at least 10 contiguous monomers thereof. In other embodiments, the sequence of the oligomer (or a first region thereof) comprises one, two, or three base moieties that differ from those in oligomers having sequences of SEQ ID NOs: 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106, or the sequences of at least 10 contiguous monomers thereof, when optimally aligned with the selected sequence or region thereof.
In various embodiments, the oligomers comprise a region of 12, 13, 14, 15 or 16 contiguous monomers having a base sequence identically present in a sequence selected from the group consisting of SEQ ID No 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22. In other embodiments, the oligomers include a region which comprises one, two, or three base moieties that differ from those in oligomers having sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
In some embodiments the region consists of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 contiguous monomers, such as 12-22, such as 12-18 monomers. Suitably, in some embodiments, the region is of the same length as the oligomer of the invention.
In some embodiments the oligomer comprises additional monomers at the 5′ or 3′ ends, such as, independently, 1, 2, 3, 4 or 5 additional monomers at the 5′ end and/or the 3′ end of the oligomer, which are non-complementary to the target region. In various embodiments, the oligomer of the invention comprises a region that is complementary to the target, which is flanked 5′ and/or 3′ by additional monomers. In some embodiments the additional 5′ or 3′ monomers are nucleosides, such as DNA or RNA monomers. In various embodiments, the 5′ or 3′ monomers represent region D as referred to in the context of gapmer oligomers herein.
In certain embodiments, the oligomer according to the invention consists of OT comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO:2, such as SEQ ID NO: 44, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID No: 3, such as SEQ ID NO: 45, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 4, such as SEQ ID NO: 46, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 5, such as SEQ ID NO: 47, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 6, such as SEQ ID NOs: 48, 49 or 50, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 7, such as SEQ ID NOs: 51, 52, or 53, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 8, such as SEQ ID NOs: 54, 55 or 56, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 9, such as SEQ ID NO: 57, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 10, such as SEQ ID NOs: 58, 59, or 60, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 11, such as SEQ ID NO: 61, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 12, such as SEQ ID NO: 62, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 13, such as SEQ ID NOs: 63, 64 or 65, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 14, such as SEQ ID NO: 66, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 15, such as SEQ ID NO: 67, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 16, such as SEQ ID NO: 68, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 17, such as SEQ ID NOs: 69, 70 or 71, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 18, such as SEQ ID NO: 72, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 19, such as SEQ ID NOs: 73, 74 or 75, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 20, such as SEQ ID NO: 76, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 21, such as SEQ ID NOs: 77, 78 or 79, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
In certain embodiments, the oligomer according to the invention consists of or comprises contiguous monomers having a nucleobase sequence according to SEQ ID NO: 22, such as SEQ ID NO: 80, or according to a region of at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.
Nucleosides and Nucleoside Analogues
In various embodiments, at least one of the monomers present in the oligomer is a nucleoside analogue that contains a modified base, such as a base selected from 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthine and hypoxanthine.
In various embodiments, at least one of the monomers present in the oligomer is a nucleoside analogue that contains a modified sugar.
In some embodiments, the linkage between at least 2 contiguous monomers of the oligomer is other than a phosphodiester linkage.
In certain embodiments, the oligomer includes at least one monomer that has a modified base, at least one monomer (which may be the same monomer) that has a modified sugar, and at least one inter-monomer linkage that is non-naturally occurring.
Specific examples of nucleoside analogues are described by e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and in Scheme 1 (in which some nucleoside analogues are shown as nucleotides):







The oligomer may thus comprise or consist of a simple sequence of naturally occurring nucleosides—preferably DNA monomers, but also possibly RNA monomers, or a combination of nucleosides and one or more nucleoside analogues. In some embodiments, such nucleoside analogues suitably enhance the affinity of the oligomer for the target region of the target nucleic acid.
Examples of suitable and preferred nucleoside analogues are described in PCT/DK2006/000512, or are referenced therein.
In some embodiments, the nucleoside analogue comprises a sugar moiety modified to provide a 2′-substituent group, such as 2′-O-alkyl-ribose sugars, 2′-amino-deoxyribose sugars, and 2′-fluoro-deoxyribose sugars.
In some embodiments, the nucleoside analogue comprises a sugar in which a bridged structure, creating a bicyclic sugar (LNA), which enhances binding affinity and may also provide some increased nuclease resistance. In various embodiments, the LNA monomer is selected from oxy-LNA (such as beta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such as beta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA (such us beta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA and alpha-L-ENA). In certain embodiments, the LNA monomers are beta-D-oxy-LNA. LNA monomers are further described below.
In various embodiments, incorporation of affinity-enhancing nucleoside analogues in the oligomer, such as LNA monomers or monomers containing 2′-substituted sugars, or incorporation of modified linkage groups provides increased nuclease resistance. In various embodiments, incorporation of affinity-enhancing nucleoside analogues allows the size of the oligomer to be reduced, and also reduces the size of the oligomer that binds specifically to a target region of a target sequence.
In some embodiments, the oligomer comprises at least 2 nucleoside analogues. In some embodiments, the oligomer comprises from 3-8 nucleoside analogues, e.g. 6 or 7 nucleoside analogues. In various embodiments, at least one of the nucleoside analogues is a locked nucleic acid (LNA) monomer; for example at least 3 or at least 4, or at least 5, or at least 6, or at least 7, or 8, nucleoside analogues are LNA monomers. In some embodiments, all the nucleoside analogues are LNA monomers.
It will be recognised that when referring to a preferred oligomer base sequence, in certain embodiments, the oligomers comprise a corresponding nucleoside analogue, such as a corresponding LNA monomer or other corresponding nucleoside analogue, which raise the duplex stability (Tm) of the oligomer/target region duplex (i.e. affinity enhancing nucleoside analogues).
In various embodiments, any mismatches (i.e., non-complementarities) between the base sequence of the oligomer and the base sequence of the target region, if present, are preferably located other than in the regions of the oligomer that contain affinity-enhancing nucleoside analogues (e.g., regions A or C), such as within region 13 as referred to herein, and/or within region D as referred to herein, and/or in regions consisting of DNA monomers, and/or in regions which are 5′ or 3′ to the region of the oligomer that is complementary to the target region.
In some embodiments the nucleoside analogues present within the oligomer of the invention (such as in regions A and C mentioned herein) are independently selected from, for example: monomers containing 2′-O-alkyl-ribose sugars, monomers containing 2′-amino-deoxyribose sugars, monomers containing 2′-fluoro-deoxyribose sugars, LNA monomers, monomers containing arabinose sugars (“ANA monomers”), monomers containing 2′-fluoro-arabinose sugars, monomers containing d-arabino-hexitol sugars (“HNA monomers”), intercalating monomers as defined in Christensen (2002) Nucl. Acids. Res. 30: 4918-4925, hereby incorporated by reference, and 2′-O-methoxyethyl-ribose (2′MOE) sugars. In some embodiments, there is only one of the above types of nucleoside analogues present in the oligomer of the invention, or region thereof.
In certain embodiments, the nucleoside analogues contain 2′MOE sugars, 2% fluoro-deoxyribose sugars, or LNA sugars, and as such the oligonucleotide of the invention may comprise nucleoside analogues which are independently selected from these three types. In certain oligomer embodiments containing nucleoside analogues, at least one of said nucleoside analogues contains a 2′-MOE-ribose sugar, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleoside analogues containing 2′-MOE-ribose sugars. In some embodiments, at least one nucleoside analogue contains a 2′-fluoro-deoxyribose sugar, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleoside analogues containing 2′-fluoro-DNA nucleotide sugars.
In various embodiments, the oligomer according to the invention comprises at least one Locked Nucleic Acid (LNA) monomer, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA monomers, such as 3-7 or 4 to 8 LNA monomers, or 3, 4, 5, 6 or 7 LNA monomers. In various embodiments, all the nucleoside analogues are LNA monomers. In certain embodiments, the oligomer comprises both beta-D-oxy-LNA monomers, and one or more of the following LNA monomers: thio-LNA monomers, amino-LNA monomers, oxy-LNA monomers, and/or ENA monomers in either the beta-D or alpha-L configurations, or combinations thereof. In certain embodiments, the cytosine base moieties of all LNA monomers in the oligomer are 5-methylcytosines. In certain embodiments of the invention, the oligomer comprises both LNA and DNA monomers. Typically, the combined total of LNA and DNA monomers is 10-25, preferably 10-20, even more preferably 12-16. In some embodiments of the invention, the oligomer or region thereof consists of at least one LNA monomer, and the remaining monomers are DNA monomers. In certain embodiments, the oligomer comprises only LNA monomers and nucleosides (such as RNA or DNA monomers, most preferably DNA monomers) optionally with modified linkage groups such as phosphorothioate.
In various embodiments, at least one of the nucleoside analogues present in the oligomer has a modified base selected from the group consisting of 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.
LNA
The term “LNA monomer” refers to a nucleoside analogue containing a bicyclic sugar (an “LNA sugar”). The terms “LNA oligonucleotide” and “LNA oligomer” refer to an oligomer containing one or more LNA monomers.
The LNA used in the oligonucleotide compounds of the invention preferably has the structure of the general formula I:




wherein X is selected from —O—, —S—, —N(RN*), —C(R6R6*)—;
B is selected from hydrogen, optionally substituted C1-4-alkoxy, optionally substituted C1-4-alkyl, optionally substituted C1-4-acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands;
P designates the radical position for an internucleoside linkage to a succeeding monomer, or a 5′-terminal group, such internucleoside linkage or 5′-terminal group optionally including the substituent R5 or equally applicable the substituent R5*;
P* designates an internucleoside linkage to a preceding monomer, or a 3′-terminal group;
R4* and R2* together designate a biradical consisting of 1-4 groups/atoms selected from —C(RaRb)—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —O—, —Si(Ra)2—, —N(R)—, and >C═Z,
wherein Z is selected from —O—, —S—, and —N(Ra)—, and Ra and Rb each is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C1-12-alkynyl, hydroxy, C2-12-alkoxyalkyl, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-4-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents Ra and Rb together may designate optionally substituted methylene (═CH2), and
each of the substituents R1*, R2, R3, R5, R5*, R6 and R6*, which are present is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxy, C2-12-alkoxyalkyl, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted, and where two geminal substituents together may designate oxo, thioxo, imino, or optionally substituted methylene, or together may form a spiro biradical consisting of a 1-5 carbon atom(s) alkylene chain which is optionally interrupted and/or terminated by one or more heteroatoms/groups selected from —O—, —S—, and —(NRN)— where RN is selected from hydrogen and C1-4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and RN*, when present and not involved in a biradical, is selected from hydrogen and C1-4-alkyl; and basic salts and acid addition salts thereof;
In some embodiments, R5* is selected from H, —CH3, —CH2—CH3, —CH2—O—CH3, and —CH═CH2.
In various embodiments, R4* and R2* together designate a biradical selected from —C(RaRb)—O—, —C(RaRb)—C(RcRd)—O—, —C(RaRb)—C(RcRd)—C(ReRf)—O—, —C(RaRb)—O—C(RcRd)—, —C(RaRb)—O—C(RcRd)—O—, —C(RaRb)—C(RcRd)—, —C(RaRb)—C(RcRd)—C(ReRf)—, —C(Ra)═C(Rb)—C(RcRd)—, —C(RaRb)—N(Rc—, —C(RaRb)—C(RcRd)—N(Rc)—, —C(RaRb)—N(Rc)—O—, and —C(RaRb)—S—, —C(RaRb)—C(RcRd)—S—, wherein Ra, Rb, Rc, Rd, Re, and Rf each is independently selected from hydrogen, optionally substituted C1-12-alkyl, optionally substituted C2-12-alkenyl, optionally substituted C2-12-alkynyl, hydroxy, C1-12-alkoxyalkyl, C2-12-alkenyloxy, carboxy, C1-12-alkoxycarbonyl, C1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C1-6-alkyl)amino, carbamoyl, mono- and di(C1-6-alkyl)-amino-carbonyl, amino-C1-6-alkyl-aminocarbonyl, mono- and di(C1-6-alkyl)amino-C1-6-alkyl-aminocarbonyl, C1-6-alkyl-carbonylamino, carbamido, C1-6-alkanoyloxy, sulphono, C1-6-alkylsulphonyloxy, nitro, azido, sulphanyl, C1-6-alkylthio, halogen, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, where aryl and heteroaryl may be optionally substituted and where two geminal substituents Ra and Rb together may designate optionally substituted methylene (═CH2),
In a further embodiment R4* and R2* together designate a biradical (bivalent group) selected from —CH2—O—, —CH2—S—, —CH2—NH—, —CH2—N(CH3)—, —CH2—CH2—O—, —CH2—CH(CH3)—, —CH2—CH2—S—, —CH2—CH2—NH—, —CH2—CH2—CH2—, —CH2—CH2—CH2—O—, —CH2—CH2—CH(CH3)—, —CH═CH—CH2—, —CH2—O—CH2—O—, —CH2—NH—O—, —CH2—N(CH3)—O—, —CH2—O—CH2—, —CH(CH3)—O—, —CH(CH2—O—CH3)—O—.
For all chiral centers, asymmetric groups may be found in either R or S orientation.
Preferably, the LNA monomer used in the oligomer of the invention comprises at least one LNA monomer according to any of the formulas




wherein Y is —O—, —O—CH2-, —S—, —NH—, or N(RH); Z and Z* are independently selected among an internucleotide linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety, and RH is selected from hydrogen and C1-4-alkyl.
Specifically preferred LNA monomers are shown in Scheme 2:




The term “thio-LNA” refers to an LNA monomer in which Y in the general formula above is selected from S or —CH2—S—. Thio-LNA can be in either the beta-D or alpha-L-configuration.
The term “amino-LNA” refers to an LNA monomer in which Y in the general formula above is selected from —N(H)—, N(R)—, CH2—N(H)—, and —CH2—N(R)— where R is selected from hydrogen and C1-4-alkyl. Amino-LNA can be hi either the beta-D or alpha-L-configuration.
The term “oxy-LNA” refers to an LNA monomer in which Y in the general formula above represents —O— or —CH2—O—. Oxy-LNA can be in either the beta-D or alpha-L-configuration.
The term “ENA” refers to an LNA monomer in which Y in the general formula above is —CH2—O— (where the oxygen atom of —CH2—O— is attached to the 2′-position relative to the base B).
In various embodiments, the LNA monomer is selected from a beta-D-oxy-LNA monomer, and alpha-L-oxy-LNA monomer, a beta-D-amino-LNA monomer, and beta-D-thio-LNA monomer, in particular a beta-D-oxy-LNA monomer.
In the present context, the term “C1-4alkyl” means a linear or branched saturated hydrocarbon chain wherein the chain has from one to four carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.
RNAse H Recruitment
In some embodiments, an oligomer functions via non-RNase-mediated degradation of a target mRNA, such as by steric hindrance of translation, or other mechanisms; however, in various embodiments, oligomers of the invention are capable of recruiting an endo-ribonuclease (RNase), such as RNase H.
Typically, the oligomer, comprises a region of at least 6, such as at least 7 contiguous monomers, such as at least 8 or at least 9 contiguous monomers, including 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguous monomers, which, when forming a duplex with the target region of the target RNA, is capable of recruiting RNase. The region of the oligomer which is capable of recruiting RNAse may be region B, as referred to in the context of a gapmer as described herein. In some embodiments, the region of the oligomer which is capable of recruiting RNAse, such as region B, consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 monomers.
EP 1 222 309 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability of the oligomers of the invention to recruit RNaseH. An oligomer is deemed capable of recruiting RNaseH if, when contacted with the complementary region of the RNA target, it has an initial rate, as measured in pmol/l/min, of at least 1%, such as at least 5%, such as at least 10% or less than 20% of an oligonucleotide having the same base sequence but containing only DNA monomers, with no 2′ substitutions, with phosphorothioate linkage groups between all monomers in the oligonucleotide, using the methodology provided by Examples 91-95 of EP 1 222 309, incorporated herein by reference.
In some embodiments, an oligomer is deemed essentially incapable of recruiting RNaseH if, when contacted with the complementary target region of the RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is less than 1%, such as less than 5%, such as less than 10% or less than 20% of the initial rate determined using an oligonucleotide having the same base sequence, but containing only DNA monomers, with no 2′ substitutions, with phosphorothioate linkage groups between all monomers in the oligonucleotide, using the methodology provided by Examples 91-95 of EP 1 222 309.
In other embodiments, an oligomer is deemed capable of recruiting RNaseH if, when contacted with the complementary target region of the RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is at least 20%, such as at least 40%, such as at least 60%, such as at least 80% of the initial rate determined using an oligonucleotide having the same base sequence, but containing only DNA monomers, with no 2′ substitutions, with phosphorothioate linkage groups between all monomers in the oligonucleotide, using the methodology provided by Examples 91-95 of EP 1 222 309.
Typically, the region of the oligomer which forms the duplex with the complementary target region of the target RNA and is capable of recruiting RNase contains DNA monomers and LNA monomers and forms a DNA/RNA-like duplex with the target region. The LNA monomers are preferably in the alpha-L configuration, particularly preferred being alpha-L-oxy LNA.
In various embodiments, the oligomer of the invention comprises both nucleosides and nucleoside analogues, and is in the form of a gapmer, a headmer or a mixmer.
A “headmer” is defined as an oligomer that comprises a first region and a second region that is contiguous thereto, with the 5′-most monomer of the second region linked to the 3′-most monomer of the first region. The first region comprises a contiguous stretch of non-RNase recruiting nucleoside analogues and the second region comprises a contiguous stretch (such as at least 7 contiguous monomers) of DNA monomers or nucleoside analogue monomers recognizable and cleavable by the RNase
A “tailmer” is defined as an oligomer that comprises a first region and a second region that is contiguous thereto, with the 5′-most monomer of the second region linked to the 3′-most monomer of the first region. The first region comprises a contiguous stretch (such as at least 7 contiguous monomers) of DNA monomers or nucleoside analogue monomers recognizable and cleavable by the RNase, and the second region comprises a contiguous stretch of non-RNase recruiting nucleoside analogues.
Other “chimeric” oligomers, called “mixmers”, consist of an alternating composition of (i) DNA monomers or nucleoside analogue monomers recognizable and cleavable by RNase, and (ii) non-RNase recruiting nucleoside analogue monomers.
In some embodiments, in addition to enhancing affinity of the oligomer for the target region, some nucleoside analogues also mediate RNase (e.g., RNaseH) binding and cleavage. Since •-L-LNA monomers recruit RNaseH activity to a certain extent, in some embodiments, gap regions (e.g., region B as referred to herein) of oligomers containing •-L-LNA monomers consist of fewer monomers recognizable and cleavable by the RNaseH, and more flexibility in the mixmer construction is introduced.
Conjugates
In the context of this disclosure, the term “conjugate” indicates a compound formed by the covalent attachment (“conjugation”) of an oligomer as described herein, to one or more moieties that are not themselves nucleic acids or monomers (“conjugated moieties”). Examples of such conjugated moieties include macromolecular compounds such as proteins, fatty acid chains, sugar residues, glycoproteins, polymers, or combinations thereof. Typically proteins may be antibodies for a target protein. Typical polymers may be polyethylene glycol.
Accordingly, provided herein are conjugates comprising an oligomer as herein described, and at least one conjugated moiety that is not a nucleic acid or monomer, covalently attached to said oligomer. Therefore, in certain embodiments where the oligomer of the invention consists of contiguous monomers having a specified sequence of bases, as herein disclosed, the conjugate may also comprise at least one conjugated moiety that is covalently attached to the oligomer.
In various embodiments of the invention, the oligomer is conjugated to a moiety that increases the cellular uptake of oligomeric compounds. WO2007/031091 provides suitable ligands and conjugates, which are hereby incorporated by reference.
In various embodiments, conjugation (to a conjugated moiety) may enhance the activity, cellular distribution or cellular uptake of the oligomer of the invention. Such moieties include, but are not limited to, antibodies, polypeptides, 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 phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or a polyethylene glycol chain, an adamantane acetic acid, a palmityl moiety, an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
In certain embodiments, the oligomers of the invention are conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
In certain embodiments the conjugated moiety is a sterol, such as cholesterol.
In various embodiments, the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptides of, for example 1-50, such as 2-20 such as 3-10 amino acid residues in length, and/or polyalkylene oxide such as polyethylene glycol (PEG) or polypropylene glycol—see WO 2008/034123, hereby incorporated by reference. Suitably the positively charged polymer, such as a polyalkylene oxide may be attached to the oligomer of the invention via a linker such as the releasable linker described in WO 2008/034123.
By way of example, the following moieties may be used in the conjugates of the invention:




Activated Oligomers

The term “activated oligomer,” as used herein, refers to an oligomer of the invention that is covalently linked (i.e., functionalized) to at least one functional moiety that permits covalent linkage of the oligomer to one or more conjugated moieties, i.e., moieties that are not themselves nucleic acids or monomers, to form the conjugates herein described. Typically, a functional moiety will comprise a chemical group that is capable of covalently bonding to the oligomer via, e.g., a 3′-hydroxyl group or the exocyclic NH2 group of the adenine base, a spacer that is preferably hydrophilic and a terminal group that is capable of binding to a conjugated moiety (e.g., an amino, sulfhydryl or hydroxyl group). In some embodiments, this terminal group is not protected, e.g., is an NH, group. In other embodiments, the terminal group is protected, for example, by any suitable protecting group such as those described in “Protective Groups in Organic Synthesis” by Theodora W. Greene and Peter G. M. Wuts, 3rd edition (John Wiley & Sons, 1999). Examples of suitable hydroxyl protecting groups include esters such as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, or triphenylmethyl, and tetrahydropyranyl. Examples of suitable amino protecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl, triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groups such as trichloroacetyl or trifluoroacetyl.
In some embodiments, the functional moiety is self-cleaving. In other embodiments, the functional moiety is biodegradable. See e.g., U.S. Pat. No. 7,087,229, which is incorporated by reference herein in its entirety.
In some embodiments, oligomers of the invention are functionalized at the 5′ end in order to allow covalent attachment of the conjugated moiety to the 5′ end of the oligomer. In other embodiments, oligomers of the invention can be functionalized at the 3′ end. In still other embodiments, oligomers of the invention can be functionalized along the backbone or on the heterocyclic base moiety. In yet other embodiments, oligomers of the invention can be functionalized at more than one position independently selected from the 5′ end, the 3′ end, the backbone and the base.
In some embodiments, activated oligomers of the invention are synthesized by incorporating during the synthesis one or more monomers that is covalently attached to a functional moiety. In other embodiments, activated oligomers of the invention are synthesized with monomers that have not been functionalized, and the oligomer is functionalized upon completion of synthesis.
In some embodiments, the oligomers are functionalized with a hindered ester containing an aminoalkyl linker, wherein the alkyl portion has the formula (CH2)w, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group is attached to the oligomer via an ester group (—O—C(O)—(CH2)wNH).
In other embodiments, the oligomers are functionalized with a hindered ester containing a (CH2)w-sulfhydryl (SH) linker, wherein w is an integer ranging from 1 to 10, preferably about 6, wherein the alkyl portion of the alkylamino group can be straight chain or branched chain, and wherein the functional group attached to the oligomer via an ester group (—O—C(O)—(CH2)wSH) In some embodiments, sulfhydryl-activated oligonucleotides are conjugated with polymer moieties such as polyethylene glycol or peptides (via formation of a disulfide bond).
Activated oligomers containing hindered esters as described above can be synthesized by any method known in the art, and in particular, by methods disclosed in PCT Publication No. WO 2008/034122 and the examples therein, which is incorporated herein by reference in its entirety.
Activated oligomers covalently linked to at least one functional moiety can be synthesized by any method known in the art, and in particular, by methods disclosed in U.S. Patent Publication No. 2004/0235773, which is incorporated herein by reference in its entirety, and in Zhao et al. (2007) J. Controlled Release 119:143-152; and Zhao et al. (2005) Bioconjugate Chem. 16:758-766.
In still other embodiments, the oligomers of the invention are functionalized by introducing sulfhydryl, amino or hydroxyl groups into the oligomer by means of a functionalizing reagent substantially as described in U.S. Pat. Nos. 4,962,029 and 4,914,210, i.e., a substantially linear reagent having a phosphoramidite at one end linked through a hydrophilic spacer chain to the opposing end which comprises a protected or unprotected sulfhydryl, amino or hydroxyl group. Such reagents primarily react with hydroxyl groups of the oligomer. In some embodiments, such activated oligomers have a functionalizing reagent coupled to a 5′-hydroxyl group of the oligomer. In other embodiments, the activated oligomers have a functionalizing reagent coupled to a 3′-hydroxyl group. In still other embodiments, the activated oligomers of the invention have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer. In yet further embodiments, the oligomer of the invention is functionalized with more than one of the functionalizing reagents as described in U.S. Pat. Nos. 4,962,029 and 4,914,210, incorporated herein by reference in their entirety. Methods of synthesizing such functionalizing reagents and incorporating them into monomers or oligomers are disclosed in U.S. Pat. Nos. 4,962,029 and 4,914,210.
In some embodiments, the 5′-terminus of a solid-phase bound oligomer is functionalized with a dienyl phosphoramidite derivative, followed by conjugation of the deprotected oligomer with, e.g., an amino acid or peptide via a Diels-Alder cycloaddition reaction.
In various embodiments, the incorporation of monomers containing 2′-sugar modifications, such as a 2′-carbamate substituted sugar or a 2′-(O-pentyl-N-phthalimido)-deoxyribose sugar into the oligomer facilitates covalent attachment of conjugated moieties to the sugars of the oligomer. In other embodiments, an oligomer with an amino-containing linker at the 2′-position of one or more monomers is prepared using a reagent such as, for example, 5′-dimethoxytrityl-2′-O-(e-phthalimidylaminopentyl)-2′-deoxyadenosine-3′-N,N-diisopropyl-cyanoethoxy phosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters, 1991, 34, 7171.
In still further embodiments, the oligomers of the invention have amine-containing functional moieties on the nucleobase, including on the N6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or 5 positions of cytosine. In various embodiments, such functionalization may be achieved by using a commercial reagent that is already functionalized in the oligomer synthesis.
Some functional moieties are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from the Pierce Co. (Rockford, Ill.). Other commercially available linking groups are 5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents, both available from Glen Research Corporation (Sterling, Va.). 5′-Amino-Modifier C6 is also available from ABI (Applied Biosystems Inc., Foster City, Calif.) as Aminolink-2, and 3′-Amino-Modifier is also available from Clontech Laboratories Inc. (Palo Alto, Calif.).
Compositions
In various embodiments, the oligomer of the invention is used in pharmaceutical formulations and compositions. Suitably, such compositions comprise a pharmaceutically acceptable diluent, carrier, salt or adjuvant. PCT/DK2006/000512 provides suitable and preferred pharmaceutically acceptable diluents, carriers and adjuvants—which are hereby incorporated by reference. Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in PCT/DK2006/000512—which are also hereby incorporated by reference. Details on techniques for formulation and administration also may be found in the latest edition of “REMINGTON'S PHARMACEUTICAL SCIENCES” (Maack Publishing Co, Easton Pa.).
In some embodiments, an oligomer of the invention is covalently linked to a conjugated moiety to aid in delivery of the oligomer across cell membranes. An example of a conjugated moiety that aids in delivery of the oligomer across cell membranes is a lipophilic moiety, such as cholesterol. In various embodiments, an oligomer of the invention is formulated with lipid formulations that form liposomes, such as Lipofectamine 2000 or Lipofectamine RNAiMAX, both of which are commercially available from Invitrogen. In some embodiments, the oligomers of the invention are formulated with a mixture of one or more lipid-like non-naturally occurring small molecules (“lipidoids”). Libraries of lipidoids can be synthesized by conventional synthetic chemistry methods and various amounts and combinations of lipidoids can be assayed in order to develop a vehicle for effective delivery of an oligomer of a particular size to the targeted tissue by the chosen route of administration. Suitable lipidoid libraries and compositions can be found, for example in Akinc et al. (2008) Nature Biotechnol., available at http://www.nature.corn/nbt/journal/vaop/ncurrent/abs/nbt1402.html, which is incorporated by reference herein.
As used herein, the term “pharmaceutically acceptable salts” refers to salts that retain the desired biological activity of the herein identified compounds and exhibit acceptable levels of undesired toxic effects. Non-limiting examples of such salts can be formed with organic amino acid and base addition salts formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with a cation formed from ammonia, N,N′-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and (b); e.g., a zinc tannate salt or the like.
In certain embodiments, the pharmaceutical compositions according to the invention comprise other active ingredients in addition to an oligomer or conjugate of the invention, including active agents useful for the treatment of cancer, such as prostate cancer or breast cancer, particularly agents used in conventional antiandrogen therapy.
In some embodiments, additional active agents are non-steroidal antiandrogens (NSAAs), which block the binding of androgens at the receptor site, or luteinizing hormone-releasing hormone analogues (LHRH-As) that suppress testicular production of androgens to castrate levels.
NSAAs such as CASODEX, when used with an LHRH-A as part of Combined Androgen Blockade therapy, help to inhibit the growth of prostate cancer cells. In one embodiment, the invention provides for a combined androgen blockade therapy, characterised in that the therapy comprises administering the pharmaceutical composition according to the invention, and an NSAA and/or LHRH-A agent, which in certain embodiments are administered prior to, during or subsequent to the administration of the pharmaceutical compositions of the invention.
The invention also provides a kit of parts wherein a first part comprises at least one oligomer, conjugate and/or the pharmaceutical composition according to the invention and a further part comprises a non-steroidal antiandrogen and/or a luteinizing hormone-releasing hormone analogue. It is therefore envisaged that the kit of parts may be used in a method of treatment, as referred to herein, where the method comprises administering both the first part and the further part, either simultaneously or one after the other.
Applications
The term “treatment” as used herein refers to both treatment of an existing disease (e.g., a disease or disorder as referred to herein below), or prevention of a disease, i.e., prophylaxis. It will therefore be recognised that, in certain embodiments, “treatment” includes prophylaxis.
In various embodiments, the oligomers of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.
In some embodiments, such oligomers may be used for research purposes to specifically inhibit the expression of androgen receptor protein (typically by degrading or inhibiting the AR mRNA and thereby preventing protein formation) in cells and experimental animals, thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention.
In certain embodiments, the oligomers may be used in diagnostics to detect and quantitate androgen receptor expression in cells and tissues by Northern blotting, in-situ hybridisation or similar techniques.
In various therapeutic embodiments, a non-human animal or a human suspected of having a disease or disorder which can be treated by modulating the expression of androgen receptor is treated by administering an effective amount of an oligomer in accordance with this invention. Further provided are methods of treating a mammal, such as treating a human, suspected of having or being prone to a disease or condition, associated with expression of androgen receptor by administering a therapeutically or prophylactically effective amount of one or more of the oligomers, conjugates or compositions of the invention.
In certain embodiments, the invention also provides for the use of the compounds or conjugates of the invention as described for the manufacture of a medicament for the treatment of a disorder as referred to herein, or for a method of the treatment of a disorder as referred to herein.
In various embodiments, the invention also provides for a method for treating a disorder as referred to herein, said method comprising administering a compound according to the invention as herein described, and/or a conjugate according to the invention, and/or a pharmaceutical composition according to the invention to a patient in need thereof.
Medical Indications
In certain therapeutic embodiments, the disorder to be treated is cancer, such as prostate cancer or breast cancer. In various embodiments, the treatment of such a disease or condition according to the invention may be combined with one or more other anti-cancer treatments, such as radiotherapy, chemotherapy or immunotherapy.
In certain other embodiments, the disorder to be treated is selected from alopecia, benign prostatic hyperplasia, spinal and muscular atrophy and Kennedy disease and polyglutamate disease.
In various embodiments, the disease or disorder is associated with a mutation of the AR gene or a gene whose protein product is associated with or interacts with AR. Therefore, in various embodiments, the target snRNA is a mutated form of the AR sequence, for example, it comprises one or more single point mutations or triplet repeats.
In other embodiments, the disease or disorder is associated with abnormal levels of a mutated form of androgen receptor. In various embodiments, the disease or disorder is associated with abnormal levels of a wild-type form of AR.
In various embodiments, the invention relates to methods of modulating the expression of the gene product of an androgen receptor target gene, i.e., a gene that is regulated by AR. Such AR receptor target gene products are selected form the group consisting of Protein kinase C delta (PRKCD), Glutathione S-transferase theta 2 (GSTT2), transient receptor potential cation channel subfamily V member 3 (TRPV3), Pyrroline-5-carboxylate reductase 1 (PYCR1) and ornithine aminotransferase (OAT). In some embodiments, modulation of an AR target gene results in increased expression or activity of the target gene. In other embodiments, modulation of an AR target gene results in decreased expression or activity of the target gene.
The invention further provides use of a compound of the invention in the manufacture of a medicament for the treatment of any and all conditions disclosed herein.
In various embodiments, the invention is directed to a method of treating a mammal suffering from or susceptible to a condition associated with abnormal levels of androgen receptor mRNA or protein, comprising administering to the mammal a therapeutically effective amount of an oligomer of the invention, or a conjugate thereof, that comprises one or more LNA monomers.
An interesting aspect of the invention is directed to the use of an oligomer (compound) as defined herein or a conjugate as defined herein for the preparation of a medicament for the treatment of a condition as disclosed herein above.
In various embodiments, the invention encompasses a method of preventing or treating a disease comprising administering a therapeutically effective amount of an oligomer according to the invention, or a conjugate thereof, to a human in need of such therapy.
In certain embodiments, the LNA oligomers of the invention, or conjugates thereof, are administered for a short period time rather than continuously.
In certain embodiments of the invention, the oligomer (compound) is linked to a conjugated moiety, for example, in order to increase the cellular uptake of the oligomer. In one embodiment the conjugated moiety is a sterol, such as cholesterol.
In various embodiments, the invention is directed to a method for treating abnormal levels of androgen receptor, the method comprising administering an oligomer of the invention, or a conjugate or a pharmaceutical composition thereof, to a patient in need of such treatment, and further comprising the administration of a further chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is conjugated to the oligomer, is present in the pharmaceutical composition, or is administered in a separate formulation.
The invention also relates to an oligomer, a composition or a conjugate as defined herein for use as a medicament.
The invention further relates to use of a compound, composition, or a conjugate as defined herein for the manufacture of a medicament for the treatment of abnormal levels of androgen receptor or expression of mutant forms of AR (such as allelic variants, such as those associated with one of the diseases referred to herein).
Moreover, in various embodiments, the invention relates to a method of treating a subject suffering from a disease or condition selected from cancer, such as breast cancer or prostate cancer, alopecia, benign prostatic hyperplasia, spinal and muscular atrophy, Kennedy disease and polyglutamate disease, the method comprising the step of administering a pharmaceutical composition as defined herein to the subject in need thereof.
Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in PCT/DK2006/000512—which is hereby incorporated by reference.
The invention also provides for a pharmaceutical composition comprising a compound or a conjugate as herein described or a conjugate, and a pharmaceutically acceptable diluent, carrier or adjuvant. PCT/DK2006/000512 provides suitable and preferred pharmaceutically acceptable diluents, carriers and adjuvants—which are hereby incorporated by reference.
EMBODIMENTS
The following embodiments of the invention may be used in combination with the other embodiments described herein.
1. An oligomer of between 10-50 nucleobases in length which comprises a contiguous nucleobase sequence of a total of between 10-50 nucleobases, wherein said contiguous nucleobase sequence is at least 80% homologous to a corresponding region of a nucleic acid which encodes a mammalian androgen receptor.
2. The oligomer according to embodiment 1, wherein said oligomer comprises at least one LNA unit.
3. The oligomer according to embodiment 1 or 2, wherein the contiguous nucleobase sequence comprises no more than 3, such as no more than 2 mismatches to the corresponding region of a nucleic acid which encodes a mammalian androgen receptor.
4. The oligomer according to embodiment 3, wherein said contiguous nucleobase sequence comprises no more than a single mismatch to the corresponding region of a nucleic acid which encodes a mammalian androgen receptor.
5. The oligomer according to embodiment 4, wherein said contiguous nucleobase sequence comprises no mismatches, (i.e. is complementary to) the corresponding region of a nucleic acid which encodes a mammalian androgen receptor.
6. The oligomer according to any one of embodiments 1-5, wherein the nucleobase sequence of the oligomer consists of the contiguous nucleobase sequence.
7. The oligomer according to any one of embodiments 1-6, wherein the nucleic acid which encodes a mammalian androgen receptor is the human androgen receptor nucleotide sequence such as SEQ ID No 1, or a naturally occurring allelic variant thereof.
8. The oligomer according to any one of embodiments 1-7, wherein the contiguous nucleobase sequence is complementary to a corresponding region of both the human androgen receptor nucleic acid sequence and a non-human mammalian androgen receptor nucleic acid sequence, such as the mouse androgen receptor nucleic acid sequence.
9. The oligomer according to any one of embodiments 1 to 8, wherein the contiguous nucleobase sequence comprises a contiguous subsequence of at least 7, nucleobase residues which, when formed in a duplex with the complementary androgen receptor target RNA is capable of recruiting RNaseH.
10. The oligomer according to embodiment 9, wherein the contiguous nucleobase sequence comprises of a contiguous subsequence of at least 8, at least 9 or at least 10 nucleobase residues which, when formed in a duplex with the complementary androgen receptor target RNA is capable of recruiting RNaseH.
11. The oligomer according to any one of embodiments 9 or 10 wherein said contiguous subsequence is at least 9 or at least 10 nucleobases in length, such as at least 12 nucleobases or at least 14 nucleobases in length, such as 14, 15 or 16 nucleobases residues which, when formed in a duplex with the complementary androgen receptor target RNA is capable of recruiting RNaseH.
12. The oligomer according to embodiment any one of embodiments 1-11 wherein said oligomer is conjugated with one or more non-nucleobase compounds.
13. The oligomer according to any one of embodiments 1-12, wherein said oligomer has a length of between 10-22 nucleobases.
14. The oligomer according to any one of embodiments 1-13, wherein said oligomer has a length of between 12-18 nucleobases.
15. The oligomer according to any One of embodiments 1-14, wherein said oligomer has a length of 14, 15 or 16 nucleobases.
16. The oligomer according to any one of embodiments 1-15, wherein said continuous nucleobase sequence corresponds to a contiguous nucleotide sequence present in a nucleic acid sequence selected from the group consisting of SEQ ID NO 86-106.
17. The oligomer according to any one of embodiments 1-16, wherein the oligomer or contiguous nucleobase sequence comprises, or is selected from a corresponding nucleobase sequence present in a nucleotide sequence selected from the group consisting of SEQ ID NO 2-22.
18. The oligomer according to any one of embodiments 1-17, wherein said contiguous nucleobase sequence comprises at least one affinity enhancing nucleotide analogue.
19. The oligomer according to embodiment 18, wherein said contiguous nucleobase sequence comprises a total of 2, 3, 4, 5, 6, 7, 8, 9 or 10 affinity enhancing nucleotide analogues, such as between 5 and 8 affinity enhancing nucleotide analogues.
20. The oligomer according to any one of embodiments 1-19 which comprises at least one affinity enhancing nucleotide analogue, wherein the remaining nucleobases are selected from the group consisting of DNA nucleotides and RNA nucleotides, preferably DNA nucleotides.
21. The oligomer according to any one of embodiments 1-20, wherein the oligomer comprises of a sequence of nucleobases of formula, in 5′ to 3′ direction, A-B-C, and optionally of formula A-B-C-D, wherein:

    
    
        (a) consists or comprises of at least one nucleotide analogue, such as 1, 2, 3, 4, 5 or 6 nucleotide analogues, preferably between 2-5 nucleotide analogues, preferably 2, 3 or 4 nucleotide analogues, most preferably 2, 3 or 4 consecutive nucleotide analogues and;
        (b) consists or comprises at least five consecutive nucleobases which are capable of recruiting RNAseH (when formed in a duplex with a complementary RNA molecule, such as the AR mRNA target), such as DNA nucleobases, such as 5, 6, 7, 8, 9, 10, 11 or 12 consecutive nucleobases which are capable of recruiting RNAseH, or between 6-10, or between 7-9, such as 8 consecutive nucleobases which are capable of recruiting RNAseH, and;
        (c) consists or comprises of at least one nucleotide analogue, such as 1, 2, 3, 4, 5, or 6 nucleotide analogues, preferably between 2-5 nucleotide analogues, such as 2, 3 or 4 nucleotide analogues, most preferably 2, 3 or 4 consecutive nucleotide analogues, and;
        (d) when present, consists or comprises, preferably consists, of one or more DNA nucleotide, such as between 1-3 or 1-2 DNA nucleotides.
    
    


22. The oligomer according to embodiment 21, wherein region A consists or comprises of 2, 3 or 4 consecutive nucleotide analogues.
23. The oligomer according to any one of embodiments 21-22, wherein region B consists or comprises of 7, 8, 9 or 10 consecutive DNA nucleotides or equivalent nucleobases which are capable of recruiting RNAseH when formed in a duplex with a complementary RNA, such as the androgen receptor mRNA target.
24. The oligomer according to any one of embodiments 21-23, wherein region C consists or comprises of 2, 3 or 4 consecutive nucleotide analogues.
25. The oligomer according to any one of embodiments 21-24, wherein region D consists, where present, of one or two DNA nucleotides.
26. The oligomer according to any one of embodiments 21-25, wherein:

    
    
        (a) Consists or comprises of 3 contiguous nucleotide analogues;
        (b) Consists or comprises of 7, 8, 9 or 10 contiguous DNA nucleotides or equivalent nucleobases which are capable of recruiting RNAseH when formed in a duplex with a complementary RNA, such as the androgen receptor mRNA target;
        (c) Consists or comprises of 3 contiguous nucleotide analogues;
        (d) Consists, where present, of one or two DNA nucleotides.
    
    


27. The oligomer according to embodiment 26, wherein the contiguous nucleobase sequence consists of 10, 11, 12, 13 or 14 nucleobases, and wherein;

    
    
        (a) Consists of 1, 2 or 3 contiguous nucleotide analogues;
        (b) Consists of 7, 8, or 9 consecutive DNA nucleotides or equivalent nucleobases which are capable of recruiting RNAseH when formed in a duplex with a complementary RNA, such as the androgen receptor mRNA target;
        (c) Consists of 1, 2 or 3 contiguous nucleotide analogues;
        (d) Consists, where present, of one DNA nucleotide.
    
    


28. The oligomer according to anyone of embodiments 21-27, wherein B comprises at least one LNA nucleobase which is in the alpha-L configuration, such as alpha-L-oxy LNA.
29. The oligomer according to any one of embodiments 1-28, wherein the nucleotide analogue(s) are independently or collectively selected from the group consisting of: Locked Nucleic Acid (LNA) units; 2′-O-alkyl-RNA units, 2′-OMe-RNA units, 2′-amino-DNA units, 2′-fluoro-DNA units, PNA units, HNA units, and INA units.
30. The oligomer according to embodiment 29 wherein all the nucleotide analogues(s) are LNA units.
31. The oligomer according to any one of embodiments 1-30, which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA units such as between 2 and 8 nucleotide LNA units.
32. The oligomer according to any one of the embodiments 29-31, wherein the LNAs are independently selected from oxy-LNA, thio-LNA, and amino-LNA, in either of the beta-D and alpha-L configurations or combinations thereof.
33. The oligomer according to embodiment 32, wherein the LNAs are all beta-D-oxy-LNA.
34. The oligomer according to any one of embodiments 21-33, wherein the nucleotide analogues or nucleobases of regions A and C are beta-D-oxy-LNA.
35. The oligomer according to any one of embodiments 1-34, wherein at least one of the nucleobases present in the oligomer is a modified nucleobase selected from the group consisting of 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine.
36. The oligomer according to any one of embodiments 1-35, wherein said oligomer hybridises with a corresponding mammalian androgen receptor mRNA with a Tm of at least 50° C.
37. The oligomer according to any one of embodiments 1-36, wherein said oligomer hybridises with a corresponding mammalian androgen receptor mRNA with a Tm, of no greater than 80° C.
38. The oligomer according to any one of embodiments 1-37, wherein the internucleoside linkages are independently selected from the group consisting of: phosphodiester, phosphorothioate and boranophosphate.
39. The oligomer according to embodiment 38, wherein the oligomer comprises at least one phosphorothioate internucleoside linkage.
40. The oligomer according to embodiment 39, wherein the internucleoside linkages adjacent to or between DNA or RNA units, or within region B are phosphorothioate linkages.
41. The oligomer according to embodiment 39 or 40, wherein the linkages between at least one pair of consecutive nucleotide analogues is a phosphodiester linkage.
42. The oligomer according to embodiment 39 or 40, wherein all the linkages between consecutive nucleotide analogues are phosphodiester linkages.
43. The oligomer according to embodiment 42 wherein all the internucleoside linkages are phosphorothioate linkages.
44. A conjugate comprising the oligomer according to any one of the embodiments 1-43 and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said compound.
45. A pharmaceutical composition comprising an oligomer as defined in any of embodiments 1-43 or a conjugate as defined in embodiment 44, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.
46. A pharmaceutical composition according to 45, wherein the oligomer is constituted as a pro-drug.
47. A pharmaceutical composition according to embodiment 45 or 46, which further comprises a further therapeutic agent selected from the group consisting of Non-steroidal Antiandrogens and Luteinizing hormone-releasing hormone analogues.
48. Use of an oligomer as defined in any one of the embodiments 1-43, or a conjugate as defined in embodiment 44, for the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of: Cancer such as breast cancer or prostate cancer, alopecia, benign prostatic hyperplasia, spinal and muscular atrophy, Kennedy disease and polyglutamate disease.
49. An oligomer as defined in any one of the embodiments 1-43, or a conjugate as defined in embodiment 44, for use in the treatment of a disease or disorder selected from the group consisting of: Cancer such as breast cancer or prostate cancer, alopecia, benign prostatic hyperplasia, spinal and muscular atrophy, Kennedy disease and polyglutamate disease.
50. A method for treating a disease or disorder selected from the group consisting of: Cancer such as breast cancer or prostate cancer, alopecia, benign prostatic hyperplasia, spinal and muscular atrophy, Kennedy disease and polyglutamate disease, said method comprising administering an oligomer as defined in one of the embodiments 1-43, or a conjugate as defined in embodiment 44, or a pharmaceutical composition as defined in any one of the embodiments 45-47, to a patient in need thereof.
51. A method for treating an cancer such as prostate cancer or breast cancer, said method comprising administering an oligomer as defined in one of the embodiments 1-43, or a conjugate as defined in embodiment 44, or a pharmaceutical composition as defined in any one of the embodiments 45-47, to a patient in need thereof.
52. A method of reducing or inhibiting the expression of androgen receptor in a cell or a tissue, the method comprising the step of contacting said cell or tissue with a compound as defined in one of the embodiments 1-43, or a conjugate as defined in embodiment 44, or a pharmaceutical composition as defined in any one of the embodiments 45-47, so that expression of androgen receptor is reduce or inhibited.
A method for modulating the expression of a gene which is regulated by the androgen receptor (i.e. an androgen receptor target) in a cell which is expressing said gene, said method comprising the step of contacting said cell or tissue with a compound as defined in one of the embodiments 1-43, or a conjugate as defined in embodiment 44, or a pharmaceutical composition as defined in any one of the embodiments 45-47, so that expression of androgen receptor target is modulated.
EXAMPLES
Example 1
Monomer Synthesis
The LNA monomer building blocks and derivatives were prepared following published procedures and references cited therein—see WO07/031,081 and the references cited therein.
Example 2
Oligonucleotide Synthesis
Oligonucleotides were synthesized according to the method described in WO07/031,081. Table 1 shows examples of sequences of antisense oligonucleotides of the invention. Tables 2 and 3 show examples of antisense oligonucleotides (oligomers) of the invention.
Example 3
Design of the Oligonucleotides
In accordance with the invention, a series of oligomers were designed to target different regions of human androgen receptor mRNA (GenBank Accession number NM—000044; SEQ ID NO: 1).
SEQ ID NOS: 2-22, shown in Table 1, below, are sequences of oligomers designed to target human androgen receptor mRNA. The target region of the target nucleic acid is indicated, in the table.







TABLE 1







Antisense Oligonucleotide Sequences













Target site


SEQ ID NO
Sequence (5′-3′)
Length (bases)
NM_000044





SEQ ID NO: 2
GAGAACCATCCTCACC
16
1389-1404


 


SEQ ID NO: 3
GGACCAGGTAGCCTGT
16
1428-1443


 


SEQ ID NO: 4
CCCCTGGACTCAGATG
16
1881-1896


 


SEQ ID NO: 5
GCACAAGGAGTGGGAC
16
1954-1969


 


SEQ ID NO: 6
GCTGTGAAGAGAGTGT
16
2422-2437


 


SEQ ID NO: 7
TTTGACACAAGTGGGA
16
2663-2678


 


SEQ ID NO: 8
GTGACACCCAGAAGCT
16
2813-2828


 


SEQ ID NO: 9
CATCCCTGCTTCATAA
16
2975-2990


 


SEQ ID NO: 10
ACCAAGTTTCTTCAGC
16
3008-3023


 


SEQ ID NO: 11
CTTGGCCCACTTGACC
16
3263-3278


 


SEQ ID NO: 12
TCCTGGAGTTGACATT
16
3384-3399


 


SEQ ID NO: 13
CACTGGCTGTACATCC
16
3454-3469


 


SEQ ID NO: 14
CATCCAAACTCTTGAG
16
3490-3505


 


SEQ ID NO: 15
GCTTTCATGCACAGGA
16
3529-3544


 


SEQ ID NO: 16
GAAGTTCATCAAAGAA
16
3594-3609


 


SEQ ID NO: 17
AGTTCCTTGATGTAGT
16
3616-3631


 


SEQ ID NO: 18
TTGCACAGAGATGATC
16
3809-3824


 


SEQ ID NO: 19
GATGGGCTTGACTTTC
16
3845-3860


 


SEQ ID NO: 20
CAGGCAGAAGACATCT
16
3924-3939


 


SEQ ID NO: 21
CCCAAGGCACTGCAGA
16
3960-3975


 


SEQ ID NO: 22
GCTGACATTCATAGCC
16
3114-3129


 


SEQ ID NO: 86
TGGGGAGAACCATCCTCACCCTGC
24
1385-1408


 


SEQ ID NO: 87
TCCAGGACCAGGTAGCCTGTGGGG
24
1424-1447


 


SEQ ID NO: 88
TGTTCCCCTGGACTCAGATGCTCC
24
1877-1990


 


SEQ ID NO: 89
TGGGGCACAAGGAGTGGGACGCAC
24
1950-1973


 


SEQ ID NO: 90
TTCGGCTGTGAAGAGAGTGTGCCA
24
2418-2441


 


SEQ ID NO: 91
CGCTTTTGACACAAGTGGGACTGG
24
2659-2682


 


SEQ ID NO: 92
CATAGTGACACCCAGAAGCTTCAT
24
2809-2832


 


SEQ ID NO: 93
GAGTCATCCCTGCTTCATAACATT
24
2971-2994


 


SEQ ID NO: 94
GATTACCAAGTTTCTTCAGCTTCC
24
3004-3027


 


SEQ ID NO: 95
AGGCCTTGGCCCACTTGACCACGT
24
3259-3282


 


SEQ ID NO: 96
AGCATCCTGGAGTTGACATTGGTG
24
3380-3403


 


SEQ ID NO: 97
GACACACTGGCTGTACATCCGGGA
24
3450-3473


 


SEQ ID NO: 98
GAGCCATCCAAACTCTTGAGAGAG
24
3486-3509


 


SEQ ID NO: 99
CAGTGCTTTCATGCACAGGAATTC
24
35254548


 


SEQ ID NO: 100
ATTCGAAGTTCATCAAAGAATTTT
24
3590-3613


 


SEQ ID NO: 101
ATCGAGTTCCTTGATGTAGTTCAT
24
3612-3635


 


SEQ ID NO: 102
GCACTTGCACAGAGATGATCTCTG
24
3805-3828


 


SEQ ID NO: 103
AATAGATGGGCTTGACTTTCCCAG
24
3841-3864


 


SEQ ID NO: 104
ATAACAGGCAGAAGACATCTGAAA
24
3920-3943


 


SEQ ID NO: 105
ATTCCCCAAGGCACTGCAGAGGAG
24
3956-3979


 


SEQ ID NO: 106
ATGGGCTGACATTCATAGCCTTCA
24
3110-3133









In SEQ ID NOs: 23-43, shown below in Table 2, upper case, boldface letters indicate nucleoside analogue monomers (e.g., •-D-oxy LNA monomers) and subscript “s” represents phosphorothiote linkage groups between the monomers. The absence of a subscript “s” (if any) indicates a phosphodiester linkage group. Lower case letters represent DNA monomers.







TABLE 2







Oligonucleotide designs








SEQ ID NO
Sequence (5′-3′)





SEQ ID NO: 23






 


SEQ ID NO: 24






 


SEQ ID NO: 25






 


SEQ ID NO: 26






 


SEQ ID NO: 27






 


SEQ ID NO: 28






 


SEQ ID NO: 29






 


SEQ ID NO: 30






 


SEQ ID NO: 31






 


SEQ ID NO: 32






 


SEQ ID NO: 33






 


SEQ ID NO: 34






 


SEQ ID NO: 35






 


SEQ ID NO: 36






 


SEQ ID NO: 37






 


SEQ ID NO: 38






 


SEQ ID NO: 39






 


SEQ ID NO: 40






 


SEQ ID NO: 41






 


SEQ ID NO: 42






 


SEQ ID NO: 43













Example 4
In Vitro Model: Cell Culture
The effect of antisense oligonucleotides 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 target can be expressed endogenously or by transient or stable transfection of a nucleic acid encoding said target. The expression level of target nucleic acid can be routinely determined using, for example, Northern blot analysis, Real-Time PCR, Ribonuclease protection assays. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen.
Cells were cultured in the appropriate medium as described below and maintained at 37° C. at 95-98% humidity and 5% CO2. Cells were routinely passaged 2-3 times weekly.
A549 The human lung cancer cell line A5439 was cultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+2 mM Glutamax I+gentamicin (25 μg/ml).
MCF7 The human breast cancer cell line MCF7 was cultured in EagleMEM (Sigma)+10% fetal bovine serum (PBS)+2 mM Glutamax I+1×NEAA+gentamicin (25 μg/ml).
Example 5
In Vitro Model: Treatment with Antisense Oligonucleotide
The cell lines listed in Example 4 were treated with an oligomer using the cationic liposome formulation LipofectAMINE 2000 (Gibco) as transfection vehicle. Cells were seeded in 6-well cell culture plates (NUNC) and treated when 80-90% confluent. Oligomer concentrations used ranged from 1 nM to 16 nM final concentration. Formulation of oligomer-lipid complexes were carried out essentially as described by the manufacturer using serum-free OptiMEM (Gibco) and a final lipid concentration of 5 μg/mL LipofectAMINE 2000. Cells were incubated at 37° C. for 4 hours and treatment was stopped by removal of oligomer-containing culture medium. Cells were washed and serum-containing media was added. After oligomer treatment, cells were allowed to recover for 20 hours before they were harvested for RNA analysis.
Example 6
In Vitro Model: Extraction of RNA and cDNA Synthesis
Total RNA Isolation and First Strand Synthesis
Total RNA was extracted from cells transfected as described above and using the Qiagen RNeasy kit (Qiagen cat. no. 74104) according to the manufacturer's instructions. First strand synthesis was performed using Reverse Transcriptase reagents from Ambion according to the manufacturer's instructions.
For each sample, the volume of 0.5 •g total RNA was adjusted to 10.8 •l with RNase free H2O and mixed with 2 •l random decamers (50 •M) and 4 •l dNTP mix (2.5 mM each dNTP) and heated to 70° C. for 3 min, after which the samples were rapidly cooled on ice. After cooling the samples on ice, 2 •l 10× Buffer RT, 1 •l MMLV Reverse Transcriptase (100 U/•l) and 0.25 •l RNase inhibitor (10 U/•l) were added to each sample, followed by incubation at 42° C. for 60 min, heat inactivation of the enzyme at 95° C. for 10 min and then cooling of the sample to 4° C.
Example 7
In Vitro Model: Analysis of Oligonucleotide Inhibition of Androgen Receptor Expression by Real-Time PCR
Antisense modulation of androgen receptor expression can be assayed in a variety of ways known in the art. For example, androgen receptor mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or mRNA.
Methods of RNA isolation and RNA analysis such as Northern blot analysis are routine in the art and are taught in, for example, Current Protocols in Molecular Biology, John Wiley and Sons.
Real-time quantitative (PCR) can be conveniently accomplished using the commercially available Multi-Color Real Time PCR Detection System, available from Applied Biosystems.
Real-Time Quantitative PCR Analysis of Androgen Receptor mRNA Levels
The amount of human androgen receptor mRNA in the samples was quantified using the human androgen receptor ABI Prism Pre-Developed TaqMan Assay Reagents (Applied Biosystems cat. no. Hs00171172_m1) according to the manufacturer's instructions.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA quantity was used as an endogenous control for normalizing any variance in sample preparation.
The amount of human GAPDH mRNA in the samples was quantified using the human GAPDH ABI Prism Pre-Developed TaqMan Assay Reagent (Applied Biosystems cat. no. 4310884E) according to the manufacturer's instructions.
Real-time Quantitative PCR is a technique well known in the art and is taught in for example Heid et al. Real time quantitative PCR, Genome Research (1996), 6: 986-994.
Real Time PCR
The cDNA from the first strand synthesis performed as described in Example 6 was diluted 2-20 times, and analyzed by real time quantitative PCR using Taqman 7500 FAST or 7900 FAST from Applied Biosystems. The primers and probe were mixed with 2× Taqman Fast Universal PCR master mix (2×) (Applied Biosystems Cat. #4364103) and added to 4 μl cDNA to a final volume of 10 μl. Each sample was analysed in duplicate. Standard curves were generated by assaying 2-fold dilutions of a cDNA that had been prepared on material purified from a cell line expressing the RNA of interest. Sterile H2O was used instead of cDNA for the no-template control. PCR program: 95° C. for 30 seconds, followed by 40 cycles of 95° C., 3 seconds, 60° C., 20-30 seconds. Relative quantities of target mRNA were determined from the calculated Threshold cycle using the Applied Biosystems Fast System SDS Software Version 1.3.1.21. or SDS Software Version 2.3.
Example 8
In Vitro Analysis: Antisense Inhibition of Human Androgen Receptor mRNA Expression by Oligonucleotide Compounds
Oligonucleotides presented in Table 3 were evaluated for their potential to knock down androgen receptor mRNA expression at concentrations of 1, 4 and 16 nM (see FIGS. 1 and 2).
The data in Table 3 are presented as percentage down-regulation relative to mock transfected cells at 16 nM. Lower case letters represent DNA monomers, bold, upper case letters represent β-D-oxy-LNA monomers. All cytosine bases in the LNA monomers are 5-methylcytosines. Subscript “s” represents a phosphorothioate linkage.







TABLE 3







Inhibition of human androgen receptor mRNA expression by oligonucleotides












Percent inhibition
Percent inhibition




of Androgen
of Androgen


Test substance
Sequence (5′-3′)
recpetor MCF7
receptor A549













SEQ ID NO: 44




80.1
63.8


 


SEQ ID NO: 45




89.0
88.2


 


SEQ ID NO: 46




89.4
82.8


 


SEQ ID NO: 47




83.1
77.7


 


SEQ ID NO: 48




93.8
96.7


 


SEQ ID NO: 49




n.d.
n.d.


 


SEQ ID NO: 50




n.d.
n.d.


 


SEQ ID NO: 51




96.9
95.5


 


SEQ ID NO: 52




n.d.
n.d.


 


SEQ ID NO: 53




n.d.
n.d.


 


SEQ ID NO: 54




95.4
98.3


 


SEQ ID NO: 55




n.d.
n.d.


 


SEQ ID NO: 56




n.d.
n.d.


 


SEQ ID NO: 57




89.5
88.9


 


SEQ ID NO: 58




95.6
98.9


 


SEQ ID NO: 59




n.d.
n.d.


 


SEQ ID NO: 60




n.d.
n.d.


 


SEQ ID NO: 61




86.7
93.3


 


SEQ ID NO: 62




81.3
93.0


 


SEQ ID NO: 63




90.9
98.4


 


SEQ ID NO: 64




n.d.
n.d.


 


SEQ ID NO: 65




n.d.
n.d.


 


SEQ ID NO: 66




79.8
95.3


 


SEQ ID NO: 67




83.5
97.0


 


SEQ ID NO: 68




88.2
85.6


 


SEQ ID NO: 69




92.7
94.0


 


SEQ ID NO: 70




n.d.
n.d.


 


SEQ ID NO: 71




n.d.
n.d.


 


SEQ ID NO: 72




79.2
90.4


 


SEQ ID NO: 73




91.1
97.3


 


SEQ ID NO: 74




n.d.
n.d.


 


SEQ ID NO: 75




n.d.
n.d.


 


SEQ ID NO: 76




85.9
94.3


 


SEQ ID NO: 77




93.0
98.5


 


SEQ ID NO: 78




n.d.
n.d.


 


SEQ ID NO: 79




n.d.
n.d.


 


SEQ ID NO: 80




n.d.
n.d.









As shown in Table 3, oligonucleotides having the sequences set forth in SEQ ID NOs: 48, 51, 54, 58, 63, 69, 73 and 77 at 16 nM demonstrated greater than 90% inhibition of androgen receptor mRNA expression in A549 and MCF7 cells in these experiments.
In certain embodiments, oligomers based on the tested antisense oligomer sequences and designs, but having, for example, different lengths (shorter or longer) and/or monomer content (e.g. the type and/or number of nucleoside analogues) than those shown, e.g., in Table 3, could also provide suitable inhibition of androgen receptor expression.
Example 9
In Vivo Analysis: Antisense Inhibition of Mouse Androgen Receptor mRNA Liver Expression by Oligonucleotide Compounds
Nude mice were dosed i.v. q3dx4 with 100 mg/kg oligonucleotide (group size of 5 mice). The antisense oligonucleotides (SEQ ID:48, SEQ ID:51, SEQ ID:58, SEQ ID:63, SEQ ID:77) were dissolved in phosphate buffered saline. Animals were sacrificed 24 h after last dosing and liver tissue was sampled and stored in RNA later until RNA extraction and QPCR analysis. Total RNA was extracted and AR mRNA expression in liver samples was measured by QPCR as described, in Example 7 using a mouse AR QPCR assay (cat. Mm01238475_m1, Applied Biosystems). Results were normalised to mouse GAPDH (cat. no. 4352339E, Applied Biosystems) and knock-down was quantitated relative to saline treated controls. The data in Table 4 are presented as percentage down-regulation relative to saline treated animals.







TABLE 4







In vivo knock-down of AR mRNA expression










Compound
Liver (% KD)







Saline
0



SEQ ID: 51 100 mg/kg
 65.0 +/− 12.6



SEQ ID: 58 100 mg/kg
95.2 +/− 1.0



SEQ ID: 77 100 mg/kg
91.9 +/− 3.9










As shown in Table 4, oligonucleotides of SEQ ID NOs: 58 and 77 at 100 mg/kg demonstrated greater than 90% inhibition of androgen receptor mRNA expression in mouse liver cells in these experiments.
Example 10
In Vitro Analysis: Antisense Inhibition of Human Androgen Receptor mRNA
Measurement of Proliferating Viable Cells (MTS Assay)
LNCaP prostate cancer and A549 lung cancer cells were seeded to a density of 150,000 cells per well in a 6-well plate the day prior to transfection. A549 cells were cultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+2 mM Glutamax I+gentamicin (25 μg/ml) whereas LNCaP cells were cultured in RPMI 1640 Medium (Sigma)+10% fetal bovine serum (FBS)+2 mM Glutamax I+gentamicin (25 μg/ml). On the following day, medium was removed followed by addition of 1.2 ml OptiMEM containing 5 μg/ml Lipofectamine-2000 (Invitrogen). Cells were incubated for 7 min before adding 0.3 ml oligonucleotides diluted in OptiMEM. The final oligonucleotide concentrations were 4 nM and 16 nM. After 4 hours of treatment, media was removed and cells were trypsinized and seeded to a density of 5000 cells per well in a clear 96 well plate (Scientific Orange no. 1472030100) in 100 μl media. Viable cells were measured at the times indicated by adding 10 μl the tetrazolium compound [3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES) (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega). Viable cells were measured at 490 nm in a Powerwave (Biotek Instruments). The OD490 nm measurements were plotted against time/h. (See FIG. 13 and FIG. 14). As shown in FIG. 13 and FIG. 14, oligonucleotides of SEQ ID NOs: 58 and 77 inhibit growth of both LNCaP prostate and A549 lung cancer cells.
Example 11
In Vitro Analysis: Caspase 3/7 Activity by Antisense Inhibition of Hunan Androgen Receptor mRNA
LNCaP prostate cancer cells and A549 lung cancer cells were seeded to a density of 150,000 cells per well in a 6-well plate the day prior to transfection. A549 cells were cultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+2 mM Glutamax I+gentamicin (25 μg/ml) whereas LNCaP cells were cultured in RPMI 1640 Medium (Sigma)+10% fetal bovine serum (PBS)+2 mM Glutamax I+gentamicin (25 μg/ml). The next day medium was removed followed by addition of 1.2 ml OptiMEM containing 5 μg/ml Lipofectamine2000 (Invitrogen). Cells were incubated for 7 min before adding 0.3 ml oligonucleotides diluted in OptiMEM. The final oligonucleotide concentrations were 4 nM and 16 nM. After 4 hours of treatment, media was removed and cells were trypsinized and seeded to a density of 5000 cells per well in a white 96 well plate (Nunc) in 100 μl media. Caspase 3/7 activity was measured at the times indicated by adding 100 μl Caspase-Glo 3/7 assay (Promega). Caspase 3/7 activity was measured using a luminometer. The caspase 3/7 activities were measured at three different time points 14 h, 48 h and 72 h (See FIG. 15 and FIG. 16). As shown in FIG. 15 and FIG. 16, oligonucleotides of SEQ ID NOs: 58 and 77 induce caspase 3/7 activity in both LNCaP prostate and A549 lung cancer cells.
Example 12
In Vitro Analysis: Antisense Inhibition of Human Androgen Receptor mRNA Expression by Oligonucleotide Compounds in Prostate Cancer Cell Line LNCaP and Lung Cancer Cell Line A549
Oligonucleotides were evaluated for their potential to knock down androgen receptor mRNA expression at concentrations of 0.5, 1, 2, 4, 8 and 16 nM (see FIG. 11). LNCaP prostate cancer cells and A549 lung cancer cells were seeded to a density of 150,000 cells per well in a 6-well plate the day prior to transfection. A549 cells were cultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+2 mM Glutamax I+gentamicin (25 μg/ml). LNCaP cells were cultured in RPMI 1640 Medium (Sigma)+10% fetal bovine serum (FBS)+2 mM Glutamax I+gentamicin (25 μg/ml). On the following day, medium was removed followed by addition of 1.2 ml OptiMEM containing 5 μg/ml Lipofectamine2000 (Invitrogen). Cells were incubated for 7 min before adding 0.3 ml oligonucleotides diluted in OptiMEM. The final oligonucleotide concentrations were 0.5, 1, 2, 4, 8 and 16 nM. Cells were washed and serum-containing media was added. After oligomer treatment cells were allowed to recover for 20 hours before they were harvested for RNA analysis. The procedure for RNA isolation, cDNA synthesis and qPCR were as described in Examples 5, 6 and 7. As shown in FIGS. 11 and 12 oligonucleotides of SEQ ID NOs: 58 and 77 were potent in knocking down AR mRNA expression in both the lung cancer cell line A549 and in the androgen receptor-dependent LNCaP prostate cancer cell line.
Example 13
In Vivo Analysis: Effect of Antisense Oligonucleotides on PSA Levels and Androgen-Dependent Prostate Tumor Growth in Mice
Six to seven week old male athymic nu/nu mice (Harlan Sprague Dawley) weighing an average of 27.3±2.4 g were used in the study. Ten million cells of 22RV1 (androgen-independent prostate cancer line) were suspended in PBS (Gibco#14190) and Matrigel (BD#356234) with a ratio of 1:1 were injected subcutaneously into each mouse. When tumors reached an average volume of 150-200 mm3, the mice were divided into nine experimental groups. Two hundred μl of oligomer were injected intravenously when the average tumor size reached 152.66±27.97 mm3. Oligomers were given every 3 days for a total of 5 dosings. The control vehicles were given using the same dosing regimen as the oligomers. On day 16, mice were sacrificed and blood collected in EDTA laced tubes and spun for 5 min. 50 μl of the supernatants were then subjected to PSA assay using the ELISA kit from ALPCO Diagnostics in Salem (PSAHU-L01). Results of the experiment are shown in FIG. 17.
Six to seven week old male athymic nu/nu mice (Harlan Sprague Dawley) weighing an average of 27.3±2.4 g were used in the study. Ten million cells of 22RV1 (androgen-independent prostate cancer line) were suspended in PBS (Gibco#14190) and Matrigel (BD#356234) with a ratio of 1:1 were injected subcutaneously into each mouse. When tumors reached an average volume of 150-200 mm3, the mice were divided into nine experimental groups. Two hundred μl of oligomer was injected intravenously when the average tumor size reached 152.66±27.97 mm3. Oligomers were given every 3 days for a total of 5 dosings. The control vehicles were given using the same dosing regimen as the oligomers. The tumor volumes for each mouse were determined by measuring two dimensions with calipers and calculated using the formula: tumor volume=(length×width2)/2). Results of the experiment are shown in FIG. 18.
Example 14
Preparation of Conjugates of Oligomers with Polyethylene Glycol
The oligomers having sequences shown as SEQ ID NO: 48 or SEQ ID NO: 63 are functionalized on the 5′ terminus by attaching an aminoalkyl group, such as hexan-1-amine blocked with a blocking group such as Fmoc to the 5′ phosphate groups of the oligomers using routine phosphoramidite chemistry, oxidizing the resultant compounds, deprotecting them and purifying them to achieve the functionalized oligomers, respectively, having the formulas (IA) and (IB):




wherein the bold uppercase letters represent nucleoside analogue monomers, lowercase letters represent DNA monomers, the subscript “s” represents a phosphorothioate linkage, and MeC represents 5-methylcytosine.

A solution of activated PEG, such as the one shown in formula (II):




wherein the PEG moiety has an average molecular weight of 12,000, and each of the compounds of formulas (IA) and (IB) in PBS buffer are stirred in separate vessels at room temperature for 12 hours. The reaction solutions are extracted three times with methylene chloride and the combined organic layers are dried over magnesium sulphate and filtered and the solvent is evaporated under reduced pressure. The resulting residues are dissolved in double distilled water and loaded onto an anion exchange column. Unreacted PEG linker is eluted with water and the products are eluted with NH4HCO3 solution. Fractions containing pure products are pooled and lypophilized to yield the conjugates SEQ ID NOs: 48 and 63, respectively as show in formulas (IIIA) and (IIIB):





wherein each of the oligomers of SEQ ID NOs: 48 and 63 is attached to a PEG polymer having average molecular weight of 12,000 via a releasable linker.

Chemical structures of PEG polymer conjugates that can be made with oligomers having sequences shown in SEQ NOs: 51, 58 and 77 using the process described above are respectively shown in formulas (IVA), (IVB) and (IVC):




wherein bold uppercase letters represent beta-D-oxy-LNA monomers, lowercase letters represent DNA monomers, the subscript “s” represents a phosphorothioate linkage and MeC represent 5-methylcytosine.

Activated oligomers that can be used in this process to respectively make the conjugates shown in formulas (NA), (IVB) and (IVC) have the chemical structures shown in formulas (VA), (VB) and (VC):

## Claims

1. An oligomer consisting of the formula:


5′-MeCsMeCsMeCsasasgsgscsascstsgscsAsGsA-3′  (SEQ ID NO: 77),

wherein uppercase letters denote beta-D-oxy-LNA monomers and lowercase letters denote DNA monomers, the subscript “s” denotes a phosphorothioate linkage, and MeC denotes a beta-D-oxy-LNA monomer containing a 5-methylcytosine base.

2. A conjugate comprising the oligomer of claim 1, covalently attached to at least one moiety that is not a nucleic acid or a monomer.

3. A pharmaceutical composition comprising:
the oligomer of claim 1 or a conjugate comprising said oligomer covalently attached to at least one moiety that is not a nucleic acid or a monomer; and
a pharmaceutically acceptable diluent, carrier, salt or adjuvant.

4. A method of inhibiting the expression of androgen receptor in a cell, comprising:
contacting said cell with an effective amount of the oligomer of claim 1.

5. A method of inhibiting the expression of androgen receptor in a cell, comprising:
contacting said cell with an effective amount of a conjugate according to claim 2.

6. A method of inhibiting the expression of androgen receptor in a tissue of a mammal, comprising:
contacting said tissue with an effective amount of the oligomer of claim 1.

7. A method of inhibiting the expression of androgen receptor in a tissue of a mammal comprising:
contacting said tissue with an effective amount of a conjugate according to claim 2.

8. A method of inhibiting the expression of an androgen receptor target gene in a cell or tissue of a mammal, comprising:
contacting said cell or tissue with an effective amount of the oligomer of claim 1.

9. A method of treating a cancer in a mammal comprising administering to said mammal an effective amount of the oligomer of claim 1, wherein the cancer is selected from the group consisting of breast cancer and prostate cancer.

10. An activated oligomer comprising:
the oligomer of claim 1; and
at least one functional group covalently attached thereto at one or more positions independently selected from the 5′-end, the 3′ end, the 2′-OH of a ribose sugar, and the base.