WO2012068000A2 - Methods of treating cancers with her3 and pik3ca antisense oligonucleotides - Google Patents

Abstract

One aspect of the invention provides methods for treating hyperproliferative disorders such as cancers in a mammal using a combination of antisense oligomers directed against HER3 and PIK3CA. Cancers treatable according to the invention include those resistant to treatment with one or more protein tyrosine kinase inhibitors. Also provided are pharmaceutical compositions including the antisense oligomers.

Description

METHODS OF TREATING CANCERS WITH HER3 AND PIK3CA

ANTISENSE OLIGONUCLEOTIDES

1. CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims priority to U.S. Provisional Patent Application Serial No. 61/413,784 filed November 15, 2010, which is hereby incorporated by reference in its entirety.

2. BACKGROUND

[2] HER3 is a member of the ErbB family of receptor tyrosine kinases, which includes four different receptors: ErbB-1 (EGFR, HERl), ErbB-2 (neu, HER2), ErbB-3 (HER3) and ErbB-4 (HER4) (Yarden et al, Nat. Rev. Mol. Cell. Biol, 2001, 2(2): 127-137). The receptor proteins of this family are composed of an extracellular ligand-binding domain, a single hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase-containing domain. There are at least 12 growth factors in the EGF family that bind to one or more of the ErbB receptors and effect receptor homo- or hetero-dimerization. Dimerization triggers internalization and recycling of the ligand-bound receptor (or its degradation), as well as downstream intracellular signaling pathways that regulate, inter alia, cell survival, apoptosis and proliferative activity. HER3 (ErbB3) is understood by those skilled in the art to lack tyrosine kinase activity.

[3] EGFR, HER2 and recently HER3 have been associated with tumor formation. Recent studies have shown that EGFR is over expressed in a number of malignant human tissues when compared to their normal tissue counterparts. A high incidence of over-expression, amplification, deletion and structural rearrangement of the gene coding for EGFR has been found in tumors of the breast, lung, ovaries and kidney. For example, EGFR is overexpressed in 80% of head and neck cancers, activated by amplification and/or mutation in about 50% of glioblastomas, and activated by mutation in 10-15% of non-small cell lung carcinomas (NSCLCs) in the west and in 30-50% of NSCLCs in Asia (Frederick, L, Wang, XY, Eley, G, James, CD (2000) Cancer Res 60: 1383-1387; Riely et al. (2006) Clin. Cancer Res. 12(24):7232-7241). Amplification of the EGFR gene in glioblastoma multiforme tumors is one of the most consistent genetic alterations known. EGFR overexpression has also been noted in many non-small cell lung carcinomas. HER2 is amplified or overexpressed in approximately 25-30% of breast cancers (Slamon et al. (1989) Science 244:707-712). Elevated levels of HER3 mRNA have been detected in human mammary carcinomas.

[4] U.S. Patent No. 6,277,640 to Bennett et al. discloses antisense compounds, compositions and methods for inhibiting the expression of HER3.

[5] Phosphatidylinositol 3 -kinase (PI3K) is a ubiquitous lipid kinase involved in receptor signal transduction by tyrosine kinase receptors. PI3K comprises a large and complex family that includes 3 classes with multiple subunits and isoforms. The class I PDKs are composed of a Src homology-2 domain containing an 85 kDa regulatory subunit (p85) and a 100-kDa catalytic subunit (pi 10), which catalyses the phosphorylation of phosphoinositol 4-phosphate and phosphoinisitol 4,5 -phosphate at their D3 positions. The PI3K regulatory subunits include p85alpha and its truncated splice variants p50alpha and p55alpha, as well as p85beta and p55gamma; the catalytic subunits include pl lOalpha, pl lObeta, and pl lOdelta. The human catalytic subunit pl lOalpha is encoded by the PIK3CA gene, located on the human chromosome 3 [Chr 3: 180.35 - 180.44 M bp] specifically [chr3: 180,349,005-180,435, 191 bp](NCBI reference sequence annotation) (3q26.3), which is frequently mutated in a variety of human cancers; PIK3CA has been shown to be mutated in 32 % of colorectal cancers, 27% of glioblastomas, 25% of gastric cancers, 36% of hepatocellular carcinomas, 18-40% of breast cancers, 4-12 % of ovarian cancers and 4 % of lung cancers (Samuels et al., 2006). Most of these mutations map to three mutational hot-spots within the PIK3CA coding sequence, which are E542K, E545K and H1047R (Kang et al, 2005).

[6] PI3K has been indicated in a wide range of cancers, such as colorectal carcinoma, where it is has been shown that the activation of PI3K/Akt is associated with colorectal carcinoma and can convert differentiated human gastric or colonic carcinoma cells to a less differentiated and more malignant phenotype (Rychahou et al 2006). The effects of PI3K on tumor growth and progression are thought to be mediated by Akt, a downstream effector of PI3K. In humans there are three members of the Akt gene family, Akt 1, Akt 2 and Akt3. Akt is over expressed in a number of cancers, including colon, pancreatic, ovarian and some steroid hormone-insensitive breast cancers.

[7] Inhibitors of proteins that are involved in the PI3K/Akt signalling, which have been suggested as potential therapeutic agents, include both siRNAs and antisense oligonucleotides (US2006/030536A), however to date most research in this area appears to have focused on the use of siRNAs. [8] W02005/091849 describes antisense down-regulation of PI3K, however no specific antisense oligonucleotides are disclosed.

[9] Zhang et al., 2004 (Cancer Biology and Therapy 3 : 12 1283-1289) discloses siRNAs targeting pi lOalpha and suggests its use in gene therapy in ovarian cancer.

[10] Rychahou et al 2006 (Annals of Surgery 243833 - 844) discloses siRNA complexes targeting p85alpha and pl lOalpha which were found to decrease in vitro colon cancer cell survival and to increase apoptosis in human colon cancer cells, and decreased liver metastasis in in vivo experiments.

[11] Meng et al, 2006 (Cellular Signaling 18 2262-2271) discloses siRNAs targeting pl lOalpha for inhibiting PI3K activity in ovarian cancer cells. The authors determined that inhibition of AKT 1 is sufficient to affect cell migration, invasion and proliferation.

[12] Hsieh et al, 2004 (NAR 32 893-901) reports on the use of 148 siRNA duplexes targeting 30 genes within the PI3K pathway.

[13] US 2005/0272682 discloses siRNA complexes targeting a phosphoinositide 3 -kinase (PI3K) signal transduction pathway.

[14] US 2009/0192110 discloses RNA antagonist compounds for the modulation of PIK3CA expression.

[15] Several protein tyrosine kinase ("PTK") inhibitors have been approved as selective therapies for certain cancers in which protein tyrosine kinase expression is dysregulated. Gleevec® (imatinib), which was initially approved in 2001, has been approved for the treatment of certain types leukemia in adults and children, aggressive systemic mastocytosis, hypereosinophilic syndrome, metastatic dermatofibrosarcoma protuberans, and certain types of metastatic malignant gastrointestinal stromal tumors. The small molecule PTK inhibitor Iressa® (gefitinib) has been approved for the treatment of locally advanced or metastatic non- small lung cancer after failure of platinum and docetaxel therapies. Tarceva™ (erlotinib) has been approved as a monotherapy for the treatment of locally advanced or metastatic non- small cell lung cancer or in combination with gemcitabine for the treatment of locally advanced, unresectable or metastatic pancreatic cancer. However, the efficacy of such therapies is limited because a resistance to the inhibitors develops over time. Arora et al. (2005) J. Pharmacol, and Exp. Therapies 315(3):971-971 -979. Recently, it has been shown that inhibition of HER2 and EGFR tyrosine kinase activity using protein tyrosine kinase inhibitors show limited effect on HER2-driven breast cancers due to a compensatory increase in HER3 expression and subsequent signaling through the PI3K/Akt pathway (Sergina et al, Nature, 2007, 445:437-441).

[16] There is a need for agents capable of effectively treating cancers that are resistant to or have become less responsive to treatment with protein tyrosine kinase inhibitors and/or that have become resistant to or less responsive to treatment with HER2 inhibitors.

3. SUMMARY OF THE INVENTION

[17] In one embodiment, the invention provides methods of treating cancer in a mammal, comprising administering to the mammal a therapeutically effective amount of a HER3 antisense oligomer or conjugate thereof and a PIK3CA antisense oligomer or conjugate thereof, wherein each oligomer consists of 10 to 50 contiguous monomers wherein adjacent monomers are covalently linked by a phosphate group or a phosphorothioate group, wherein each oligomer comprises a first region of at least 10 contiguous monomers; wherein at least one monomer of the first region is a nucleoside analogue; wherein the sequence of the first region is at least 80% identical to the reverse complement of the best-aligned target region of a mammalian HER3 gene or a mammalian HER3 mRNA and a mammalian PIK3CA gene or a mammalian PIK3CA mRNA, respectively; and wherein the cancer is resistant to treatment with a protein tyrosine kinase inhibitor and/or HER2 inhibitor and/or HER2 pathway inhibitor, or is susceptible to developing such resistance. Said resistance may be at least partially reversed as a result of reducing expression of HER3 and PIK3CA using the oligomers. A related variation includes administering both the HER3 antisense oligomer, the PIK3CA oligomer and the protein tyrosine kinase inhibitor and/or HER2 inhibitor and/or HER2 pathway inhibitor such that the respective inhibitory effects of the oligomers and said inhibitor are temporally overlapping. In this manner, the invention provides treatments that at least partially prevent the development of resistance to such an inhibitor by a cancer (if not already developed) or at least partially reverse resistance to such an inhibitor by a cancer (if already developed).

[18] The HER3 antisense oligomer may, for example, have the sequence of SEQ ID NO: 180. The cancer may, for example, be a cancer resistant to treatment with gefitinib.

[19] In some embodiments, the invention provides a method of treating cancer in a mammal, comprising administering to the mammal an effective amount of a HER3 antisense oligomer consisting of the sequence 5'-T_sA_sG_sc_sc_st_sg_st_sc_sa_sc_st_st_s ^MeC_sT_s ^MeC -3' (SEQ ID NO: 180) or a conjugate thereof and a PIK3CA antisense oligomer selected from the group consisting of 5'A_sG_s ^MeC_sc_sa_st_st_s c_sa_st_st_sc_sc_sA_s ^MeC_s ^MeC-3' (SEQ ID NO: 254) and 5'- T_sT_sA_st_st_sg_st_sg_sc_sa_st_sc_st_s ^MeC_sA_sG -3' (SEQ ID NO: 257) or a conjugate thereof, 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, and wherein the cancer is resistant to treatment with a protein tyrosine kinase inhibitor such as but not limited to gefitinib or lapitinib, and/or resistant to treatment with a HER2 pathway inhibitor, such as but not limited to trastuzumab or pertuzumab, or is susceptible to .developing one or both of such resistances.

[20] In certain embodiments, the invention provides a method of inhibiting the proliferation of a mammalian cancer cell comprising contacting the cell with an effective amount of an oligomer targeting HER3 or a conjugate thereof and an oligomer targeting PIK3CA or a conjugate thereof, wherein each oligomer consists of 10 to 50 contiguous monomers wherein adjacent monomers are covalently linked by a phosphate group or a phosphorothioate group, wherein each oligomer comprises a first region of at least 10 contiguous monomers; wherein at least one monomer of the first region is a nucleoside analog; wherein the sequence of the first region is at least 80% identical to the reverse complement of the best-aligned target region of a mammalian HER3 gene or a mammalian HER3 mRNA or a mammalian PIK3CA gene or a mammalian PIK3CA mRNA, respectively; and wherein proliferation of the mammalian cancer cell is not inhibited by a protein tyrosine kinase inhibitor such as but not limited to gefitinib or lapitinib.

[21] Still another embodiment of the invention provides methods for treating cancers in a mammal by administering antisense oligomers that down-modulate (reduce) the expression of HER3 and PIK3CA while, concurrently or in conjunction therewith, the mammal is treated with at least one protein tyrosine kinase inhibitor (PTKI) such as but not limited to gefitinb or any of those described herein. Said oligomers and PTKI may or may not be co-administered; what is important is that oligomers and PTKI are active together in therapeutically effective amounts in the mammal patient at the same time and/or the respective inhibitory effects of each are temporally overlapping. The cancers may be those that have been become resistant to or less responsive to treatment with PTKI, or they may be cancers which have never developed resistance to one or more PTKIs. The cancer may, for example, be a cancer at least initially responsive to treatment with one or more PTKIs, such as breast cancer, or may be any of the cancers described herein. Where the cancer is not substantially resistant to treatment with a PTKI, one embodiment provides for at least partially preventing resistance (or further resistance) to a PTKI by reducing the expression of HER3 in any of the manners described.

[22] A related embodiment provides the use of at least one antisense oligomer or a conjugate thereof that down-modulates (reduces) the expression of HER3 and at least one antisense oligomer or conjugate thereof that down-modulates (reduces) the expression of PIK3CA as described herein for the preparation of a medicament in treating a hyperproliferative disorder, such as a cancer, in a mammal, such as a cancer of a human patient, for example, breast cancer. The medicament may, for example, be for use concurrently with or in conjunction with a PTKI, such as but not limited to gefitinib. Another embodiment provides the combination use of at least one antisense oligomer or a conjugate thereof that down-modulates (reduces) the expression of HER3 and at least one antisense oligomer or conjugate thereof that down-modulates (reduces) the expression of PIK3CA as described herein for the treatment of a cancer in a mammal, such as a human patient, such as a PTKI-resistant cancer in a mammal such as a human, for example, a PTKI-resistant human breast cancer patient. The combination use may further include use of a protein tyrosine kinase inhibitor such as those described herein, for example one to which the cancer is resistant or capable of developing resistance. A further embodiment of the invention provides an improved method for treating a cancer in a mammal, such as a human patient, with at least one PTKI such as but not limited to gefitinib, in which the improvement comprises concurrently inhibiting the expression of HER3 and PIK3CA in the mammal (e.g., in the cancer cells in the mammal), for example, by administering to the mammal at least one antisense oligomer that down-modulates the expression of HER3 and at least one antisense oligomer or conjugate thereof that down-modulates (reduces) the expression of PIK3CA, such as those described herein. The at least one PTKI may, for example, be any of those described herein. The cancer may, for example, be a cancer at least initially responsive to a PTKI, such as breast cancer, or may be any of the cancers described herein.

[23] In some embodiments, the proliferation of the mammalian cancer cell is inhibited by at least 50% when compared to the proliferation of an untreated cell of the same type.

[24] Still another embodiment of the invention provides methods for treating cancers in a mammal by administering antisense oligomers that down-modulate the expression of HER3 and PIK3CA (or conjugates thereof) while, concurrently or in conjunction therewith, the mammal is treated with at least one inhibitor of HER2 or of the HER2 pathway. Said oligomers and inhibitor of HER2 may or may not be co-administered; what is important is that oligomers and inhibitor of HER2 or HER2 pathway are active together in therapeutically effective amounts in the mammal patient at the same time and/or the respective inhibitory effects of each are temporally overlapping. The cancers may be those that have been become resistant to or less responsive to treatment with HER2 inhibitors, such as HER2-binding antibodies or binding fragments thereof, for example, trastuzumab or pertuzumab, or HER2 pathway inhibitors such as lapatinib, or they may be cancers which have never developed resistance to HER2 inhibitors. The cancer may, for example, be a cancer at least initially responsive to inhibition of HER2 or the HER2 pathway, such as breast cancer, or may be any of the cancers described herein. Where the cancer is not substantially resistant to treatment with a HER2 inhibitor or HER2 pathway inhibitor, one embodiment provides for at least partially preventing resistance (or further resistance) to a HER2 inhibitor or HER2 pathway inhibitor by reducing the expression of HER3 in any of the manners described.

[25] A related embodiment provides the use of at least one antisense oligomer or conjugate thereof that down-modulates (reduces) the expression of HER3 and at least one antisense oligomer or conjugate thereof that down-modulates (reduces) the expression of PIK3CA, as described herein, for the preparation of a medicament for use concurrently with or in conjunction with at least one inhibitor of HER2 or HER2 pathway in treating a cancer in a mammal, such as a human patient. Another embodiment provides the use of at least one oligomer or conjugate thereof that reduces the expression of HER3 and at least one antisense oligomer or conjugate thereof that down-modulates (reduces) the expression of PIK3CA in the preparation of a medicament for the treatment of a cancer that has become resistant to or less responsive to treatment with an inhibitor of HER2 or the HER2 pathway, such as but not limited to trastuzumab or pertuzumab, or HER2 pathway inhibitors such as lapatinib, in a mammal such as a human, for example, a human with breast cancer that has become resistant to or less responsive to treatment with a HER2 inhibitor or inhibitor of the HER2 pathway.

[26] A further embodiment of the invention provides an improved method for treating a cancer in a mammal, such as a human patient, with an inhibitor of HER2 or the HER2 pathway, in which the improvement comprises concurrently inhibiting the expression of HER3 and PIK3CA in the mammal (e.g., in the cancer cells in the mammal), for example, by administering to the mammal at least one antisense oligomer or conjugate thereof that down- modulates the expression of HER3 and at least one antisense oligomer or conjugate thereof that down-modulates (reduces) the expression of PIK3CA, such as those described herein. The inhibitor of HER2 or the HER2 pathway may, for example, be any of those described herein. The cancer may, for example, be a cancer at least initially responsive to inhibition of HER2 or the HER2 pathway, such as breast cancer, or may be any of the cancers described herein.

[27] For any of the aforementioned embodiments and variations thereof, the one or more antisense oligomers that reduce the expression of HER3 or PIK3CA may, for example, be gapmers having terminal LNA monomers at each of the 5' and 3 ' ends, such as 1, 2, 3 or 4 contiguous LNA monomers at each end, which bound a central portion of DNA monomers. At least some, for example all, of the inter-monomer linkages maybe phosphorothioate linkages.

[28] In one embodiment, the present invention provides a composition comprising at least one antisense oligomer or a conjugate thereof that reduces the expression of HER3 and at least one antisense oligomer or a conjugate thereof that reduces the expression of PIK3CA for use in the treatment of a hyperproliferative disease, such as cancer, in a mammal.

[29] Additional features, advantages, and embodiments of the invention may be set forth or apparent from consideration of the following detailed description, drawings, and claims. Moreover, it is to be understood that both the foregoing summary of the invention and the following detailed description are exemplary and intended to provide further explanation without limiting the scope of the invention as claimed.

4. BRIEF DESCRIPTION OF THE FIGURES

[30] FIG. 1. The HER3 target sequences that are targeted by the oligomers having the sequence of SEQ ID NOS: 1, 16, 17, 18, 19, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 74, 75, 76, 91, 92, 107, 122, 137, 138, 139, and 140, respectively, are shown in bold and underlined, indicating their position in the HER3 transcript (GenBank Accession number NM_001982 - SEQ ID NO: 197).

[31] FIG. 2. HER3 niRNA expression in 15PC3, 24 hours after transfection, SEQ ID NOS: 169-179

[32] FIG. 3. EGFR niRNA expression in 15PC3, 24 hours after transfection, SEQ ID NOS: 169-179

[33] FIG. 4. HER-2 mRNA expression in 15PC3, 24 hours after transfection, SEQ ID NOS: 169-179 [34] FIG. 5: HER3 mRNA expression in 15PC3, 24 hours after transfection, SEQ ID NOS: 180-194

[35] FIG. 6: Data show apoptosis induction measured as activated Caspase 3/7 at different time points in HUH7 cells transfected with oligonucleotides at 5 and 25 nM concentrations. Results are plotted relative to cells mock treated with a scrambled control oligonucleotide having SEQ ID NO: 235.

[36] FIG. 7: Data show viable cells measured as OD490 using MTS assay at different time points in HUH-7 cells transfected with oligonucleotides at 5 and 25 nM concentrations. SEQ ID NO: 235 is a scrambled control oligonucleotide.

[37] FIG. 8A: Data show percent change in tumor volume in 15PC3 xenograft tumors transplanted onto female nude mice treated with SEQ ID NO: 180 i.v. at 25 and 50 mg/kg q3dxl0. Saline treated mice were used as control.

[38] FIG. 8B: Data show HER3 mRNA expression in 15PC3 xenograft tumors transplanted onto female nude mice treated with SEQ ID NO: 180 i.v. at 25 and 50 mg/kg q3dxl0. Results are normalized to GAPDH and presented as % of saline treated controls.

[39] FIG. 9: Data show HER3 mRNA expression in mouse liver after treatment i.v. with 1 or 5 mg/kg oligonucleotides on three consecutive days having sequences shown in SEQ ID NO: 180 or SEQ ID NO: 234. Results are normalized to GAPDH and presented as % of saline treated controls.

[40] FIG. 10: Data show the generation of HCC827 human lung adenocarcinoma cells that are resistant to gefitinib at a concentration as high as 10μΜ.

[41] FIG. 1 1 : Data show that levels of phosphorylated EFGR are much lower in gefitinib- resistant HCC827 cells than in parent HCC827 gefitinib-sensitive cells.

[42] FIG. 12: Data show that levels of unphosphorylated and phosphorylated EGFR are significantly reduced in HCC827 gefitinib-resistant clones, either in the presence ("+") or absence ("-") of gefitinib, as compared to the levels of unphosphorylated and phosphorylated EGFR in untreated ("-") parent cells. In contrast, the levels of ErbB3 or MET, which are also involved in the EGFR signaling pathway, are not significantly decreased in the resistant clones compared to the parent cells.

[43] FIG. 13 : Data show that treatment with Ι μΜ of the oligonucleotide having SEQ ID NO: 180 over a 10-day period has a greater effect on inhibition of the growth of gefitinib- resistant HCC827 cells (greater than 80% reduction in growth as compared to untreated control) than on the growth of HCC827 cells that are sensitive to gefitinib.

[44] FIG. 14: Data show that HER3 expression-reducing LNA antisense oligomer, but not trastuzumab, is able to prevent feedback upregulation of HER3 and P-HER3 expression by lapatinib in three human cancer cell lines.

[45] FIG. 15: Data show that synergistic promotion of apoptosis in three human cancer cell lines is greater for a combination of lapatinib and a HER3 expression-reducing LNA antisense oligomer than for a combination of lapatinib and trastuzumab.

[46] FIG. 16: Data show that antisense HER3 inhibitor SEQ ID NO: 180 inhibits tumor growth in an in vivo mouse xenograft model of human non-small cell lung cancer.

[47] FIG. 17 shows HCC827 cells are sensitive to gefitinib, while R1-R5 cells was not affected by gefitinib up to 10 uM, the highest concentration tested.

[48] FIG. 18 shows that Rl, R3, R4, and R5 gefitinib-resistant cells are consistently more sensitive than the parent HCC827 cell line to SEQ ID NO: 180 (EZN-3920), but not to the control LNA oligomer SEQ ID NO: 265 (EZN-3046).

[49] FIG. 19 shows distinct characteristics between the gefitinib-resistant clones and parent cell line HCC827 based on protein expression profiling.

[50] FIG. 20 shows that R3 cells appear more sensitive to the combined treatment of SEQ ID NO: 180 (antisense HER3 oligomer; EZN3920) and SEQ ID NO: 254 (antisense PIK3CA oligomer; EZN4150) than to each agent alone, while no such enhanced effect was seen in HCC827 cells.

[51] FIG. 21A shows EZN-3920 (SEQ ID NO: 180 ) inhibits lapatinib-induced HER3 mRNA levels in BT474M1 breast cancer cells.

[52] FIG 2 IB shows EZN-3920 (SEQ ID NO: 180) inhibits lapatinib-induced HER3 protein expression in BT474M1 breast cancer cells.

[53] FIG. 21C shows EZN-3920 (SEQ ID NO: 180) potentiates the effect of lapatinib on the growth of BT474M1 breast cancer cells.

[54] FIG. 22 shows the effect of a combination of EZN-3920 (SEQ ID NO: 180) with lapatinib on mean tumor volume over time in a BT474M1 breast cancer xenograft model. [55] FIG. 23 shows the effect of a combination of EZN-3920 (SEQ ID NO: 180) with gefitinib on mean tumor volume over time in a BT474M1 breast cancer xenograft model.

[56] FIG. 24 shows the effects of a combination of EZN-3920 (SEQ ID NO: 180) with EZN-4150 (SEQ ID NO: 254, an LNA gapmer antisense antagonist of PIK3CA) on mean tumor volume over time in BT474M1 breast cancer xenograft model.

5. DETAILED DESCRIPTION

[57] In certain embodiments, the invention provides methods for modulating the expression of HER3, PIK3CA and optionally EGFR and/or HER2 in cells that are resistant to treatment with a protein tyrosine kinase inhibitor. In some embodiments, the resistant cells are cancer cells. In various embodiments, methods are provided for treating or preventing diseases associated with HER3 over-expression, such as cancers that are resistant to treatment with protein tyrosine kinase inhibitors, by administering antisense oligomers that specifically hybridize under intracellular conditions to nucleic acids encoding HER3, PIK3CA and optionally other targets..

[58] The term "HER3" is used herein interchangeably with the term "ErbB3".

5.1. Methods

[59] In various embodiments, the invention encompasses methods of inhibiting the expression and/or activity of HER3 in a cell, that is resistant to treatment with a protein tyrosine kinase inhibitor and/or HER2 or HER2 pathway inhibitor, comprising contacting the cell with an effective amount of an antisense oligomer(s) targeting HER3 (and optionally one or more of HER2 and EGFR) or a conjugate thereof and an antisense oligomer targeting PIK3CA or a conjugate thereof, so as to effect the inhibition (e.g., down-regulation) of HER3 and PIK3CA expression and/or activity in the cell. The cell may be a mammalian cell, such as a human cell. The cell may be a cancer cell or a non-cancerous cell.

[60] In certain embodiments, the contacting occurs in vitro. In other embodiments, the contacting is effected in vivo by administering compositions as described herein to a mammal, such as a human subject. In various embodiments, the invention provides a method of inhibiting (e.g., by down-regulating) the expression of HER3 protein and/or mRNA, the expression of PIK3CA protein and/or mRNA, and optionally the expression of HER2 and/or EGFR protein and/or mRNA in a cell. The sequence of the human HER2 mRNA is shown in SEQ ID NO: 199. In still further embodiments, the invention provides a method of inhibiting (e.g., by down-regulating) the expression of HER3 protein and/or mRNA in a cell, and the expression of EGFR protein and/or mRNA in a cell. The sequence of the human EGFR mR A is shown in SEQ ID NO: 198.

[61] As used interchangeably herein, the terms "protein tyrosine kinase inhibitor," "PTK inhibitor", and "tyrosine kinase inhibitor" refer to molecules that bind to and inhibit the activity of one or more tyrosine kinase domains. The protein tyrosine kinase inhibitor is not the oligomer targeting HER3 as described herein below. In some embodiments the protein tyrosine kinase inhibitor is a monoclonal antibody. In other embodiments the protein tyrosine kinase inhibitor is a small molecule, having a molecular weight of less than 1000 Da, such as between 300 - 700 Da.

[62] In certain embodiments, the PTK inhibitor is targeted to the tyrosine kinases of one or more EGFR family members. In various embodiments, the PTK inhibitor is targeted to the tyrosine kinases of one or more proteins that interact with or are regulated by one or more EGFR family members, e.g., proteins involved in one or more signaling cascades that originate with one or more EGFR family members. In some embodiments, the tyrosine kinase is a receptor tyrosine kinase, i.e., is an intra-cellular domain of a larger protein that has an extra-cellular ligand binding domain and is activated by the binding of one or more ligands. In certain embodiments, the protein tyrosine kinase is a non-receptor tyrosine kinase. Tyrosine kinase enzymes regulate the activities of other proteins in one or more signaling pathways by phosphorylating them.

[63] The term "protein tyrosine kinase inhibitor-resistant cancer" or "PTKI-resistant cancer" as used herein refers to a cancer whose growth progresses despite treatment with a protein tyrosine kinase inhibitor, for example, despite treatment with the clinically used dosage(s) or clinically used blood or tissue concentration(s) of the PTK inhibitor. This may include cancers whose growth and proliferation are not substantially reduced when contacted with a protein tyrosine kinase inhibitor. Without limitation, the growth or proliferation of a cancer may be considered resistant to treatment with a PTK inhibitor if, when contacted with the PTK inhibitor, the growth or proliferation of cancerous cells in the cancer is reduced by less than 30%, such as by less than 20%, such as less than by 10%, as compared to the reduction in the growth or proliferation of the same type of cancer cells that have not been previously contacted with the PTK inhibitor and lack such resistance. In some embodiments, resistant cancers are those that are inherently resistant to treatment with PTK inhibitors. In some embodiments, resistant cancers are cancers that have acquired resistance from prior exposure to a PTK inhibitor, either as a monotherapy or as part of a combination therapy with one or more additional agents, e.g., chemotherapeutic agents or antisense oligonucleotides; in other words, the cancer has become resistant to treatment with PTK inhibitor.

[64] The term "cancer is partially resistant to a protein tyrosine kinase inhibitor" as used herein refers to a cancer whose growth or proliferation is partially reduced when contacted with a protein tyrosine kinase inhibitor. As used herein, the growth or proliferation of a cancer may be considered partially resistant to treatment with a PTK inhibitor if, when contacted with the PTK inhibitor, the growth or proliferation of cancerous cells in the cancer is reduced by less than 60%, such as by less than 50%, such as less than by 40%, as compared to the growth or proliferation of same type of cancer cells that have not been contacted with the PTK inhibitor and lack such resistance. In some embodiments, partially resistant cancers are those that are inherently partially resistant to treatment with PTK inhibitors. In some embodiments, partially resistant cancers are cancers that have acquired partial resistance from prior exposure to a PTK inhibitor, either as a monotherapy or as part of a combination therapy with one or more additional agents, e.g., chemotherapeutic agents or antisense oligonucleotides; in other words, the cancer has become less responsive to treatment with PTK inhibitor.

[65] As used herein, a cell that is resistant to treatment with a protein tyrosine kinase inhibitor refers to a cell whose growth or proliferation is not substantially reduced when contacted with a protein tyrosine kinase inhibitor. As used herein, the growth or proliferation of a cell is resistant to treatment with a PTK inhibitor if, when contacted with the PTK inhibitor, the growth or proliferation is reduced by less than 30%, such as by less than 20%, such as less than by 10%, as compared to the growth or proliferation of same type of cell that has not been contacted with the PTK inhibitor and lacks such resistance. In some embodiments, resistant cells are those that are inherently resistant to treatment with PTK inhibitors. In some embodiments, resistant cells are cells that have acquired resistance from prior exposure to a PTK inhibitor, either as a monotherapy or as part of a combination therapy with one or more additional agents, e.g., chemotherapeutic agents or antisense oligonucleotides. Similarly, as used herein, a cell that is resistant to treatment with a HER2 inhibitor, or HER2 pathway inhibitor generally, refers to a cell whose growth or proliferation is not substantially reduced when contacted with such an inhibitor. As used herein, the growth or proliferation of a cell is resistant to treatment with a HER2 inhibitor or HER2 pathway inhibitor if, when contacted with the inhibitor, the growth or proliferation is reduced by less than 30%, such as by less than 20%, such as less than by 10%, as compared to the growth or proliferation of same type of cell that has not been contacted with the inhibitor and lacks such resistance. In some embodiments, resistant cells are those that are inherently resistant to treatment with a HER2 inhibitor or HER2 pathway inhibitor. In some embodiments, resistant cells are cells that have acquired resistance from prior exposure to a HER2 inhibitor or HER2 pathway inhibitor.

[66] In some embodiments, the cell has acquired resistance after having been exposed to a PTK inhibitor selected from gefitinib (ZD- 1839, Iressa®), imatinib (Gleevec®), erlotinib (OSI-1774, Tarceva™), canertinib (CI-1033), vandetanib (ZD6474, Zactima®), tyrphostin AG-825 (CAS 149092-50-2), lapatinib (GW-572016), sorafenib (BAY43-9006), AG-494 (CAS 133550-35-3), RG-13022 (CAS 149286-90-8), RG-14620 (CAS 136831-49-7), BIBW 2992 (Tovok), tyrphostin 9 (CAS 136831-49-7), tyrphostin 23 (CAS 1 18409-57-7), tyrphostin 25 (CAS 118409-58-8), tyrphostin 46 (CAS 122520-85-8), tyrphostin 47 (CAS 122520-86-9), tyrphostin 53 (CAS 122520-90-5), butein (l-(2,4-dihydroxyphenyl)-3-(3,4- dihydroxyphenyl)-2-propen-l-one 2',3,4,4'-Tetrahydroxychalcone; CAS 487-52-5), curcumin ((E,E)-l,7-bis(4-Hydroxy-3-methoxyphenyl)-l,6-heptadiene-3,5-dione; CAS 458-37-7), N4- (1 -Benzyl- lH-indazol-5-yl)-N6,N6-dimethyl-pyrido[3,4-d]pyrimidine-4,6-diamine (202272- 68-2), AG-1478, AG-879, Cyclopropanecarboxylic acid-(3-(6-(3-trifluoromethyl- phenylamino)-pyrimidin-4-ylamino)-phenyl)-amide (CAS 879127-07-8), N8-(3-Chloro-4- fluorophenyl)-N2-(l-methylpiperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, 2HC1 (CAS 196612-93-8), 4-(4-Benzyloxyanilino)-6,7-dimethoxyquinazoline (CAS 179248-61-4), N-(4-((3-Chloro-4-fluorophenyl)amino)pyrido[3,4-d]pyrimidin-6-yl)2-butynamide (CAS 881001-19-0), EKB-569, HKI-272, and HKI-357.

[67] In various embodiments, the cell has acquired resistance after having been exposed to a PTK inhibitor selected from gefitinib, imatinib, erlotinib, lapatinib, canertinib and sorafenib. In one variation, the cell has acquired resistance after having been exposed to gefitinib.

[68] In certain embodiments, the cell has acquired resistance after having been exposed to HER2 inhibitor such as a HER2 -binding and -inhibiting antibody or -binding and -inhibiting antibody fragment. In one variation, the cell has acquired resistance after having been exposed to trastuzumab and/or pertuzumab.

[69] In certain embodiments, the invention relates to a method of treating a disease in a patient, wherein the disease is resistant to treatment with a PTK inhibitor and/or HER2 or HER2 pathway inhibitor, comprising administering to a patient in need thereof a pharmaceutical composition comprising an effective amount of at least one oligomer, or a conjugate thereof, and optionally one or more pharmaceutically acceptable excipients. As used herein, the terms "treating" and "treatment" refer 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.

[70] In certain embodiments, the methods, pharmaceutical compositions and kits of the invention are useful for inhibiting proliferation of cells that are resistant to PTK inhibitor(s) and/or HER2 and/or HER2 pathway inhibitor(s). In various embodiments the antiproliferative effect is an at least 10% reduction, an at least 20% reduction, an at least 30% reduction, an at least 40% reduction, an at least 50% reduction, an at least 60% reduction, an at least 70% reduction, an at least 80% reduction, or an at least 90% reduction in cell proliferation as compared to a cell sample that is untreated. In other embodiments, the antiproliferative effect is an at least 10% reduction, an at least 20% reduction, an at least 30% reduction, an at least 40% reduction, an at least 50% reduction, an at least 60% reduction, an at least 70% reduction, an at least 80% reduction, or an at least 90% reduction in cell proliferation as compared to a cell sample that is treated with a small molecule protein tyrosine kinase inhibitor. In various embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is selected from a breast cancer cell, a prostate cancer cell, a lung cancer cell, and an epithelial carcinoma cell.

[71] Accordingly, the methods, pharmaceutical compositions and kits of the invention are useful for treating a hyperproliferative disease, such as cancer, which is resistant to treatment with a protein tyrosine kinase inhibitor and/or to treatment with a HER2 or HER2 pathway inhibitor. In some embodiments, the cancer, such as a resistant cancer, to be treated is selected from the group consisting of lymphomas and leukemias (e.g. non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma), colon carcinoma, rectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, cervical cancer, testicular cancer, non-small cell lung cancer, lung carcinoma, bladder carcinoma, melanoma, head and neck cancer, brain cancer, cancers of unknown primary site, neoplasms, cancers of the peripheral nervous system, cancers of the central nervous system, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, seminoma, embryonal carcinoma, Wilms' tumor, small cell lung carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma, heavy chain disease, metastases, or any disease or disorder characterized by uncontrolled or abnormal cell growth.

[72] In certain embodiments, the resistant cancer is selected from the group consisting of lung cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, epithelial carcinoma, and stomach cancer.

[73] In certain other embodiments, the lung cancer is non-small cell lung cancer. One such embodiment of the invention provides a method for the treatment of non-small cell lung cancer that includes administering to a mammal such as a human patient in need of treatment for said cancer, a therapeutically effective amount of at least one antisense oligomer or a conjugate thereof that reduces the expression of HER3, at least one oligomer targeted to PIK3CA or a conjugate thereof, and optionally one or more inhibitors of HER2 or the HER2 pathway. In one variation, the at least one oligomer or conjugate thereof that targets HER3 includes or is SEQ ID NO: 180 or a conjugate thereof. In one variation, the at least one oligomer or conjugate thereof that targets PIK3C includes or is SEQ ID NO: 254 or a conjugate thereof or SEQ ID NO: 257 or a conjugate thereof.

[74] In certain embodiments, the invention also provides for the use of the compounds or conjugates described herein for the manufacture of a medicament for the treatment of a PTK inhibitor-resistant, HER2 inhibitor-resistant or HER2 pathway inhibitor-resistant disorder as referred to herein, or for a method of the treatment of such a disorder as referred to herein.

[75] In various embodiments, the treatment of PTK inhibitor-resistant, HER2 inhibitor- resistant or HER2 pathway inhibitor-resistant disorders according to the invention may be combined with one or more other anti-cancer treatments, such as radiotherapy, chemotherapy or immunotherapy. [76] In certain embodiments, the PTK inhibitor-resistant disease is associated with a mutation in the HER3 gene (and/or the HER2 gene and/or the EGFR gene) or a gene whose protein product is associated with or interacts with HER3. In some embodiments, the mutated gene codes for a protein with a mutation in the tyrosine kinase domain. In various embodiments, the mutation in the tyrosine kinase domain is in the binding site of a small molecule PTK inhibitor and/or the ATP binding site. Therefore, in various embodiments, the target mRNA is a mutated form of the HER3 (and/or HER2 and/or EGFR) sequence; for example, it comprises one or more single point mutations, such as SNPs associated with cancer.

[77] In certain embodiments, the PTK inhibitor-resistant disease is associated with abnormal levels of a mutated form of HER3. In certain embodiments, the PTK inhibitor- resistant disease is associated with abnormal levels of a wild-type form of HER3. One aspect of the invention is directed to a method of treating a patient suffering from or susceptible to conditions associated with abnormal levels of HER3, comprising administering to the patient a therapeutically effective amount of an oligomer targeted to HER3 or a conjugate thereof and an oligomer targeted to PIK3CA or a conjugate thereof. In some embodiments, one or more, such as all, of the oligomers comprises one or more LNA units as described herein below.

[78] In various embodiments, the invention is directed to a method of treating a patient suffering from or susceptible to conditions associated with abnormal levels of a mutated form of HER2, or abnormal levels of a wild-type form of HER2, wherein the condition is resistant to treatment with a protein tyrosine kinase inhibitor, comprising administering to the mammal a therapeutically effective amount of an oligomer targeted to HER3 (and optionally to one or more of HER2 and EGFR) or a conjugate thereof and an oligomer targeted to PIK3CA or a conjugate thereof. In some embodiments, one or more, such as all, of the oligomers comprise one or more LNA units as described herein below.

[79] In still other embodiments, the invention is directed to a method of treating a patient suffering from or susceptible to conditions associated with abnormal levels of a mutated EGFR, or abnormal levels of a wild-type EGFR, wherein the condition is resistant to treatment with a protein tyrosine kinase inhibitor, comprising administering to the patient a therapeutically effective amount of an oligomer targeted to HER3 (and optionally to one or more of HER2 and EGFR) or a conjugate thereof and an oligomer targeted to PIK3CA or a conjugate thereof. In some embodiments, the oligomer comprises one or more LNA units as described herein below.

[80] In various embodiments, the invention described herein encompasses a method of preventing or treating a disease that is resistant to treatment with a protein tyrosine kinase inhibitor comprising administering to a human in need of such therapy a therapeutically effective amount a HER3 modulating oligomer (and optionally one or more of HER2 and EGFR) or a conjugate thereof and a PIK3CA modulating oligomer or a conjugate thereof.

[81] In various embodiments, the oligomer, or conjugate thereof, induces a desired therapeutic effect in humans through, for example, hydrogen bonding to a target nucleic acid. The oligomer causes a decrease (e.g., inhibition) in the expression of a target via hydrogen bonding (e.g., hybridization) to the mRNA of the target thereby resulting in a reduction in gene expression.

[82] It is highly preferred that the compounds of the invention are capable of hybridizing to the target nucleic acid, such as HER3 mRNA or PIK3CA mRNA, by Watson-Crick base pairing.

5.2. Oligomers

[83] Oligomeric compounds (referred to herein as oligomers), are provided that are useful, e.g., in modulating the function of nucleic acid molecules encoding mammalian HER3, such as the HER3 nucleic acid shown in SEQ ID No: 197, and naturally occurring allelic variants of such nucleic acid molecules encoding mammalian HER3, as well as mammalian PIK3CA and allelic variants thereof. The oligomers are composed of covalently linked monomers.

[84] 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.

[85] An "RNA monomer" is a nucleoside containing a ribose sugar and an unmodified nucleobase. [86] A "DNA monomer" is a nucleoside containing a deoxyribose sugar and an unmodified nucleobase.

[87] A "Locked Nucleic Acid monomer," "locked monomer," or "LNA monomer" is a nucleoside analogue having a bicyclic sugar, as further described herein below.

[88] 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.

[89] The terms "oligomer," "oligomeric compound," and "oligonucleotide" are used interchangeably in the context of the methods described herein, 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.

[90] 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.

[91] 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.

[92] 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.

[93] Thus, in various embodiments, oligomers for use in the methods of the invention include nucleoside monomers and at least one nucleoside analogue monomer, such as an LNA monomer, or other nucleoside analogue monomers.

[94] 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."

[95] In some embodiments, the oligomer consists of 10-50 contiguous monomers, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous monomers.

[96] In some embodiments, the oligomer consists of 10-25 monomers, preferably, 10-16 monomers, and more preferably, 12-16 monomers.

[97] In various embodiments, the oligomers comprise or consist of 10 -25 contiguous monomers, 10-24 contiguous monomers, 12 - 25 or 12-24 or 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.

[98] In various embodiments, the oligomers comprise or consist of 10-22 contiguous monomers, or 10-18, such as 12-18 or 13-17 or 12-16, such as 13, 14, 15 or 16 contiguous monomers.

[99] In some embodiments, the oligomers comprise or consist of 10-16 or 12-16 or 12-14 contiguous monomers. In other embodiments, the oligomers comprise or consist of 14-18 or 14-16 contiguous monomers.

[100] In various embodiments, the oligomers comprise or consist of 10, 11, 12, 13, or 14 contiguous monomers.

[101] In various embodiments, the oligomer consists of no more than 22 contiguous monomers, such as no more than 20 contiguous monomers, such as no more than 18 contiguous monomers, such as 15, 16 or 17 contiguous monomers. In certain embodiments, the oligomer comprises less than 20 contiguous monomers.

[102] In various embodiments, the oligomer does not comprise RNA monomers.

[103] It is preferred that the oligomers for use in the methods described herein are linear molecules or are linear as synthesized. The oligomer is, in such embodiments, a single stranded molecule, and typically does not comprise a short region 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 various embodiments, the oligomer is not substantially double-stranded, i.e., is not a siRNA.

[104] In some embodiments, the oligomer consists of a contiguous stretch of monomers, the sequence of which is identified by a SEQ ID NO. disclosed herein (see, e.g., Tables 1-4). 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.

5.3. Gapmer Design

[105] Typically, the oligomers for use in the methods of the invention are gapmers. However, other types of antisense oligomers may also be used according to the invention,

[106] A "gapmer" is an oligomer which comprises a contiguous stretch of monomers capable of recruiting an RNAse (e.g. 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.

[107] 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; and 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.

[108] 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.

[109] In certain embodiments, region B consists of or comprises 5, 6, 7, 8, 9, 10, 1 1 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.

[110] In certain 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.

[Ill] Further gapmer designs are disclosed in WO 2004/046160, which is hereby incorporated by reference.

[112] U.S. provisional application no. 60/977,409 and U.S. Pub. No. US 2010/249219 (Al), each hereby incorporated by reference, refer to "shortmer" gapmer oligomers. In some embodiments, oligomers presented here may be such shortmer gapmers.

[113] 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, 9, or 10 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. [114] 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 consists of 10 nucleoside monomers. In certain embodiments, region B comprises 1 - 10 DNA monomers, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 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 monomers. In certain embodiments, the number of monomers present in the A-B-C regions of the oligomers is selected from the group consisting of (nucleotide 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, 3-10-2, 2-10-3, and 3-10-3. In certain embodiments, the number of monomers present in the A-B-C regions of the oligomers described herein 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.

[115] In various embodiments, other gapmer designs include those where regions A and/or C consists of 3, 4, 5 or 6 nucleoside analogues, such as monomers containing a 2'-0- methoxyethyl-ribose sugar (2'MOE) or monomers containing a 2'-fluoro-deoxyribose sugar, and region B consists of 8, 9, 10, 1 1 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 2007/146511 A2, hereby incorporated by reference.

5.4. Linkage groups

[116] 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. [117] The terms "linkage group" or "internucleoside linkage" mean 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).

[118] Suitable linkage groups include those listed in WO 2007/031091, for example the linkage groups listed on the first paragraph of page 34 of WO 2007/031091 (hereby incorporated by reference).

[119] 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, permitting RNase-mediated antisense inhibition of expression of the target gene.

[120] 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.

[121] 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.

[122] In various embodiments, adjacent monomers of the oligomer are linked to each other by means of phosphorothioate groups.

[123] It is recognized 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 WO 2008/053314, hereby incorporated by reference.

[124] 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. [125] In some embodiments all the internucleoside linkage groups are phosphorothioate.

[126] When referring to specific gapmer oligonucleotide sequences, such as 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.

5.5. Target Nucleic Acid

[127] 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.

[128] In various embodiments, the term "target nucleic acid," as used herein, refers to the nucleic acid (such as DNA or RNA) encoding mammalian HER3 polypeptide (e.g., such as human HER3 mR A having the sequence in SEQ ID NO 197, or mammalian mRNAs having GenBank Accession numbers NM_001005915, NM_001982 and alternatively-spliced forms NP_001973.2 and NP 001005915.1 (human); NM_017218 (rat); NM_010153 (mouse); NM 001 103105 (cow); or predicted mRNA sequences having GenBank Accession numbers XM_001491896 (horse), XM OO 1169469 and XM_509131 (chimpanzee)).

[129] In various embodiments, "target nucleic acid" also includes a nucleic acid encoding a mammalian HER2 polypeptide (e.g., such mammalian mRNAs having GenBank Accession numbers NM_001005862 and NM_004448 (human); NM_017003 and NM_017218 (rat); NM_001003817 (mouse); NM_001003217 (dog); and NM_001048163 (cat)).

[130] In various embodiments, "target nucleic acid" also includes a nucleic acid encoding a mammalian EGFR polypeptide (e.g., such as mammalian mRNAs having GenBank Accession numbers NM_201284, NM_201283, NM_201282 and NM_005228 (human); NM_007912 and M_207655 (mouse); NM_031507 (rat); and M_214007 (pig)).

[131] It is recognized that the above-disclosed GenBank Accession numbers 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). [132] In various embodiments, "target nucleic acid" also includes HER3 (or HER2 or EGFR) encoding nucleic acids or naturally occurring variants thereof, and R A nucleic acids derived therefrom, preferably mRNA, such as pre-mRNA, although preferably mature mRNA. In various 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 target nucleic acids. The oligomers described herein are typically capable of hybridizing to the target nucleic acid.

[133] The term "naturally occurring variant thereof refers to variants of the HER3 (or HER2 or EGFR) polypeptide or nucleic acid sequence which exist naturally within the defined taxonomic group, such as mammalian, such as mouse, monkey, and preferably human. Typically, when referring to "naturally occurring variants" of a polynucleotide the term also may encompass any allelic variant of the HER3 (or HER2 or EGFR) encoding genomic DNA which is found at the Chromosome Chr 12: 54.76 - 54.78 Mb by chromosomal translocation or duplication, and the RNA, such as mRNA derived therefrom. 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.

[134] 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 "hybridization." 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.

[135] 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. [136] In determining the degree of "complementarity" between oligomers for use in the methods described herein (or regions thereof) and the target region of the nucleic acid which encodes mammalian HER3 (or HER2 or EGFR), 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 and the target region.

[137] 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.

[138] As will be understood, depending on context, "mismatch" refers to a nonidentity 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 HER3 encoding nucleic acids), or to noncomplementarity in sequence (as, for example, between an oligomer and the target region to which binds).

[139] Suitably, the oligomer (or conjugate, as further described, below) is capable of inhibiting (such as, by down-regulating) expression of the HER3 (or HER2 or EGFR) gene.

[140] In various embodiments, the oligomers described herein effect inhibition of HER3 (or HER2 or EGFR) mRNA expression of at least 10% as compared to the normal expression level, at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% as compared to the normal expression level. In various embodiments, the oligomers effect inhibition of HER3 (or HER2 or EGFR) protein expression of at least 10% as compared to the normal expression level, at least 20%, more preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% as compared to the normal expression level. In some embodiments, such inhibition is seen when using 1 nM of the oligomer or conjugate for use in the methods of the invention. In various embodiments, such inhibition is seen when using 25nM of the oligomer or conjugate.

[141] 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, the inhibition of protein 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.

[142] 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 inhibition when using an appropriate dosage, such as 1 and 25nM, is, in various embodiments, typically to a level of 10-20% of the normal levels in the absence of the compound.

[143] Modulation (i.e., inhibition or increase) of expression level may also be determined by measuring protein levels, e.g. by methods such as SDS-PAGE followed by western blotting using suitable antibodies raised against the target protein.

[144] In some embodiments, the invention provides oligomers that inhibit (e.g., down- regulate) the expression of one or more alternatively-spliced isoforms of HER3 mRNA and/or proteins derived therefrom. In some embodiments, the invention provides oligomers that inhibit expression of one or more of the alternatively-spliced protein isoforms of HER3 (GenBank Accession nos. NP_001973.2 and NP_001005915.1) and/or expression of the nucleic acids that encode the HER3 protein isoforms (GenBank Accession nos. NM_001982 and NM_001005915.1). In some embodiments, the mRNA encoding HER3 isoform 1 is the target nucleic acid. In other embodiments, the mRNA encoding HER3 isoform 2 is the target nucleic acid. In certain embodiments, the nucleic acids encoding HER3 isoform 1 and HER3 isoform 2 are target nucleic acids, for example, the oligomer having the sequence of SEQ ID NO: 180.

[145] In various embodiments, oligomers, or a first region thereof, have a base sequence that is complementary to the sequence of a target region in a HER3 nucleic acid, which oligomers down-regulate HER3 mRNA and/or HER3 protein expression and down-regulate the expression of mRNA and/or protein of one or more other ErbB receptor tyrosine kinase family members, such as HER2 and/or EGFR. Oligomers, or a first region thereof, that effectively bind to the target regions of two different ErbB receptor family nucleic acids (e.g., HER2 and HER3 mRNA) and that down-regulating the mRNA and/or protein expression of both targets are termed "bispecific." Oligomers, or a first region thereof, that bind to the target regions of three different ErbB receptor family members and are capable of effectively down-regulating all three genes are termed "trispecific". In various embodiments, an antisense oligonucleotide may be polyspecific, i.e. capable of binding to target regions of target nucleic acids of multiple members of the ErbB family of receptor tyrosine kinases and down-regulating their expression. As used herein, the terms "bispecific" and "trispecific" are understood not to be limiting in any way. For example, a "bispecific oligomer" may have some effect on a third target nucleic acid, while a "trispecific oligomer" may have a very weak and therefore insignificant effect on one of its three target nucleic acids.

[146] In various embodiments, bispecific oligomers, or a first region thereof, are capable of binding to a target region in a HER3 nucleic acid and a target region in a HER2 target nucleic acid and effectively down-regulating the expression of HER3 and HER2 mRNA and/or protein. In certain embodiments, the bispecific oligomers do not down-regulate expression of HER3 mRNA and/or protein and HER2 mRNA and/or protein to the same extent. In other preferred embodiments, the bispecific oligomers, or a first region thereof, are capable of binding to a target region in a HER3 target nucleic acid and a target region in an EGFR target nucleic acid and effectively down-regulating the expression of HER3 mRNA and/or protein and EGFR mRNA and/or protein. In various embodiments, the bispecific oligomers do not down-regulate expression of HER3 mRNA and/or protein and EGFR mRNA and/or protein to the same extent. In still other embodiments, trispecific oligomers, or a first region thereof, are capable of binding to a target region in a HER3 target nucleic acid, and to target regions in two other ErbB family of receptor tyrosine kinase target nucleic acids and effectively down-regulating the expression of HER3 mRNA and/or protein and mRNA and/or protein of the two other members of the ErbB family of receptor tyrosine kinases. In various preferred embodiments, the trispecific oligomers, or a first region thereof, are capable of effectively down-regulating the expression of HER3 mRNA and/or protein, the expression of HER2 mRNA and/or protein, and the expression of EGFR mRNA and/or protein. In various embodiments, the trispecific oligomers do not down-regulate expression of HER3 mRNA and/or protein, HER2 mRNA and/or protein and EGFR mRNA and/or protein to the same extent. [147] Antisense oligomers targeting mammalian PIK3CA that may be used according to the present invention include those disclosed in U.S. Pub. No. 2009/01921 10 (U.S. Application Serial No. 12/323,744, now U.S. Patent No. 7,863,437), which is incorporated by reference as if fully set forth herein.

[148] In various embodiments, the invention therefore provides a method of inhibiting (e.g., by down-regulating) the expression of HER3 protein and/or mRNA and PIK3CA protein and/or mRNA in a cancer cell which is expressing HER3 protein and/or mRNA and PIK3CA protein and/or mRNA, which is resistant to treatment with a protein tyrosine kinase inhibitor, the method comprising contacting the cell with an amount of an oligomer or conjugate as described herein effective to inhibit (e.g., to down-regulate) the expression of HER3 protein and/or mRNA in said cell and at least one antisense oligomer or conjugate thereof that down- modulates (reduces) the expression of PIK3CA. Suitably the cell is a mammalian cell, such as a human cell. The contacting may occur, in certain embodiments, in vitro. In other embodiments, the contacting may be effected in vivo, by administering the compound or conjugate described herein to a mammal. In various embodiments, the invention provides a method of inhibiting (e.g., by down-regulating) the expression of HER3 protein and/or mRNA, PIK3CA protein and/or mRNA and the expression of HER2 protein and/or mRNA in a cell that is resistant to treatment with a protein tyrosine kinase inhibitor. The sequence of the human HER2 mRNA is shown in SEQ ID NO: 199. In still further embodiments, the invention provides a method of inhibiting (e.g., by down-regulating) the expression of HER3 protein and/or mRNA and the expression of EGFR protein and/or mRNA in a cell that is resistant to treatment with a protein tyrosine kinase inhibitor. The sequence of the human EGFR mRNA is shown in SEQ ID NO: 198. In yet further embodiments, the invention provides a method of inhibiting (e.g., by down-regulating) the expression of HER3 PIK3CA and optionally HER2 and/or EGFR mRNA and/or protein in a cell that is resistant to treatment with a protein tyrosine kinase inhibitor.

[149] An oligomer as described herein may bind to a target region of the human HER3 and/or the human HER2 and/or the human EGFR mRNA, and as such, comprises or consists of a region having a base sequence that is complementary or partially complementary to the base sequence of e.g., SEQ ID NO 197, SEQ ID NO: 198 and/or SEQ ID NO: 199. In certain embodiments, the sequence of the oligomers described herein may optionally comprise 1, 2, 3, 4 or more base mismatches when compared to the sequence of the best- aligned target region of SEQ ID NOs: 197, 198 or 199. [150] In some embodiments, the oligomers described herein have sequences that are identical to a sequence selected from the group consisting of SEQ ID NOs: 200-227, 1-140 and 228-233 (see Table 1 herein below). In other embodiments, the oligomers have sequences that differ in one, two, or three bases when compared to a sequence selected from the group consisting of SEQ ID NOs: 200-227, 1-140 and 228-233. In some embodiments, the oligomers consist of or comprise 10-16 contiguous monomers. Examples of the sequences of oligomers consisting of 16 contiguous monomers are SEQ ID NOs: 1, 16, 17, 18, 19, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 74, 75, 76, 91, 92, 107, 122, 137, 138, 139, and 140. Shorter sequences can be derived therefrom, e.g., the sequence of the shorter oligomer may be identically present in a region of an oligomer selected from those having base sequences of SEQ ID NOs: 200-227, 1-140 and 228-233. Longer oligomers may include a region having a sequence of at least 10 contiguous monomers that is identically present in SEQ ID NOs: 200-227, 1-140 and 228-233.

[151] Further provided are target nucleic acids (e.g., DNA or mRNA encoding HER3), that contain target regions that are complementary or partially-complementary to one or more of the oligomers of SEQ ID NOs: 1-140, wherein the oligomers are capable of inhibiting expression (e.g., by down-regulation) of HER3 protein or mRNA. For example, target regions of human HER3 mRNA which are complementary to the antisense oligomers having sequences of SEQ ID NOs: 1, 16, 17, 18, 19, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 74, 75, 76, 91, 92, 107, 122, 137, 138, 139, and 140 are shown in Figure 1 (bold and underlined, with the corresponding oligomer SEQ ID NOs indicated above).

[152] In various embodiments, the oligomers have the base sequences shown in SEQ ID NOs: 141-168. In certain embodiments, the oligomers are LNA oligomers, for example, those having the sequences of SEQ ID NOS: 169-196 and 234, in particular those having the base sequences of SEQ ID NOs: 169, 170, 173, 174, 180, 181, 183, 185, 187, 188, 189, 190, 191, 192 and 194. In various embodiments, the oligomers are LNA oligomers such as those having base sequences of SEQ ID NOs: 169, 170, 172, 174, 175, 176 and 179. In some embodiments, the oligomers or a region thereof consist of or comprise a base sequence as shown in SEQ ID NOs: 169, 180 or 234. In some embodiments, conjugates include an oligomer having a base sequence as shown in SEQ ID NOs: 169, 180 or 234.

[153] In certain embodiments, the oligomer described herein may, suitably, comprise a region having a particular sequence, such as a sequence selected from SEQ ID NOs: 200- 227, that is identically present in a shorter oligomer. Preferably, the region comprises 10-16 monomers. For example, the oligomers having the base sequences of SEQ ID NOs: 200-227 each comprise a region wherein the sequence of the region is identically present in shorter oligomers having sequences of SEQ ID NOs: 1, 16, 17, 18, 19, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 74, 75, 76, 91, 92, 107, 122, 137, 138, 139, and 140, respectively. In some embodiments, oligomers which have fewer than 16 monomers, such as 10, 1 1, 12, 13, 14, or 15 monomers, have a region of at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14 or 15, contiguous monomers of which the sequence is identically present in oligomers having sequences of SEQ ID NOS: 1, 16, 17, 18, 19, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 74, 75, 76, 91, 92, 107, 122, 137, 138, 139, or 140. Hence, in various embodiments, the sequences of shorter oligomers are derived from the sequences of longer oligomers. In some embodiments, the sequences of oligomers having SEQ ID NOs disclosed herein, or the sequences of at least 10 contiguous monomers thereof, are identically present in longer oligomers. Typically an oligomer comprises a first region having a sequence that is identically present in SEQ ID NOs: 1, 16, 17, 18, 19, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 74, 75, 76, 91, 92, 107, 122, 137, 138, 139, or 140, and if the oligomer is longer than the first region that is identically present in SEQ ID NOs: 1, 16, 17, 18, 19, 34, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 74, 75, 76, 91, 92, 107, 122, 137, 138, 139, or 140, the flanking regions of the oligomer have sequences that are complementary to the sequences flanking the target region of the target nucleic acid. Two such oligomers are SEQ ID NO: 1 and SEQ ID NO: 54.

[154] In various embodiments, the HER3 antisense oligomer comprises or consists of a sequence of monomers which is fully complementary (perfectly complementary) to a target region of a target nucleic acid which encodes a mammalian HER3.

[155] However, in some embodiments, the sequence of the oligomer includes 1, 2, 3, or 4 (or more) mismatches as compared to the best-aligned target region of a HER3 target nucleic acid, and still sufficiently binds to the target region to effect inhibition of HER3 mRNA or protein expression. The destabilizing effect of mismatches on the 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.

[156] 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 HER3. [157] The base sequences of the oligomers described herein or of a region thereof are preferably at least 80% identical to a sequence selected from the group consisting of SEQ ID NOS: 200 - 227, 1 - 140 and 228 - 233, 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%, at least 97%, at least 98%, at least 99%, even 100% identical.

[158] The base sequences of the oligomers described herein or of a first region thereof are preferably at least 80% complementary to a sequence of a target region present in SEQ ID NOs: 197, 198 and/or 199 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%, at least 97%, at least 98%, at least 99%, even 100% complementary.

[159] In various embodiments, the sequence of the oligomer (or a first region thereof) is selected from the group consisting of SEQ ID NOs: 200 - 227, 1 - 140 and 228 - 233, or is selected from the group consisting of at least 10 contiguous monomers of SEQ ID NOs: 200 - 227, 1 - 140 and 228 - 233. In other embodiments, the sequence of the oligomer or a first region thereof optionally comprises 1 , 2 or 3 base moieties that differ from those in oligomers having sequences of SEQ ID NOs: 200 - 227, 1 - 140 and 228 - 233, or the sequences of at least 10 contiguous monomers thereof, when optimally aligned with said selected sequence or region thereof.

[160] In certain embodiments, the monomer 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 between 10-15, 12- 25, 12 -22, such as between 12-18 monomers. Suitably, in various embodiments, the region is of the same length as the oligomer.

[161] 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 5' end and/or 3' end of the oligomer, which are non-complementary to the sequence of the target region. In various embodiments, the oligomer comprises a region that is complementary to the target, which is flanked 5' and/or 3 ' by additional monomers. In various embodiments, the 3 ' end of the region is flanked by 1, 2 or 3 DNA or RNA monomers. 3 ' DNA monomers are frequently used during solid state synthesis of oligomers. In various embodiments, which may be the same or different, the 5' end of the oligomer is flanked by 1, 2 or 3 DNA or RNA monomers. In certain embodiments, the additional 5' or 3' monomers are nucleosides, such as DNA or RNA monomers. In various embodiments, the 5' or 3' monomers may represent region D as referred to in the context of gapmer oligomers herein.

[162] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:200, or according to a region thereof.

[163] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:201, or according to a region thereof.

[164] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:202, or according to a region thereof.

[165] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:203, or according to a region thereof.

[166] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:204, or according to a region thereof.

[167] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:205, or according to a region thereof.

[168] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:206, or according to a region thereof.

[169] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:207, or according to a region thereof.

[170] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:208, or according to a region thereof. [171] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:209, or according to a region thereof.

[172] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:210, or according to a region thereof.

[173] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:21 1, or according to a region thereof.

[174] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:212, or according to a region thereof.

[175] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:213, or according to a region thereof.

[176] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:214, or according to a region thereof.

[177] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:215, or according to a region thereof.

[178] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:216, or according to a region thereof.

[179] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:217, or according to a region thereof.

[180] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:218, or according to a region thereof. [181] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:219, or according to a region thereof.

[182] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:220, or according to a region thereof.

[183] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:221, or according to a region thereof.

[184] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:222, or according to a region thereof.

[185] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:223, or according to a region thereof.

[186] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:224, or according to a region thereof.

[187] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:225, or according to a region thereof.

[188] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:226, or according to a region thereof.

[189] In certain embodiments, the HER3 antisense oligomer consists of, or comprises, contiguous monomers having a nucleobase sequence according to SEQ ID NO:227, or according to a region thereof.

[190] Sequence alignments (such as those described above) can be used to identify regions of the nucleic acids encoding HER3, PIK3CA, HER2 or EGFR from human and one or more different mammalian species, such as monkey, mouse and/or rat, where there are sufficient stretches of nucleic acid identity between or among the species to allow the design of oligonucleotides which target (that is, which bind with sufficient specificity to inhibit expression of) both the human HER3, PIK3CA, HER2 or EGFR target nucleic acid and the corresponding nucleic acids present in the different mammalian species.

[191] In some embodiments, such oligomers consist of or comprise regions of at least 10, such as at least 12, such as at least 14, such as at least 16, such as at least 18, such as 1 1, 12, 13, 14, 15, 16, 17 or 18 contiguous monomers which are 100% complementary in sequence to the sequence of the target regions of the nucleic acid encoding HER3, PIK3CA, HER2 or EGFR from humans and of the nucleic acid(s) encoding HER3, PIK3CA, HER2 or EGFR from a different mammalian species.

[192] In some embodiments, the oligomer for use in the methods described herein comprises or consists of a region of contiguous monomers having a sequence that is at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% or 100% complementary to the sequence of the target regions of both the nucleic acid encoding human HER3, PIK3CA, HER2 or EGFR and a nucleic acid(s) encoding HER3, PIK3CA, HER2 or EGFR from a different mammalian species, such as the mouse nucleic acid encoding HER3, PIK3CA, HER2 or EGFR. It is preferable that the contiguous nucleobase sequence of the oligomer is 100% complementary to the target region of the human HER3, PIK3CA, HER2 or EGFR mRNA.

[193] In some embodiments, oligomers described herein bind to a target region of a HER3 target nucleic acid and down-regulate the expression of HER3 mRNA and/or protein. In various embodiments, oligomers described herein that bind to a target region of a HER3 nucleic acid have the sequences shown, for example, in SEQ ID NOs: 169-196 and 234.

[194] In some embodiments, a first region of a bispecific oligomer described herein binds to a target region of a HER 3 nucleic acid and a second region of the bispecific oligomer binds to a target region of a HER2 nucleic acid and said oligomer down-regulates the expression of HER3 and HER2. In various embodiments, the bispecific oligomer down-regulates the expression of HER 3 and HER2 to a different extent. In some embodiments, the first region and the second region of the oligomer are the same. In various embodiments, the first region and the second region of the oligomer overlap. In certain embodiments, the bispecific oligomers that bind to a target region of HER3 nucleic acid and a target region of HER2 nucleic acid have the sequences shown, for example, in SEQ ID NOs: 177 and 178. In still other embodiments, a bispecific oligomer binds to a target region of HER3 nucleic acid and to a target region of EGFR nucleic acid and down-regulates the expression of HER3 and EGFR. In some embodiments, bispecific oligomers that bind to a target region of HER3 nucleic acid and to a target region of EGFR nucleic acid have the sequences shown, for example, in SEQ ID NOs: 171 and 173. In some embodiments, a first region of a bispecific oligomer described herein binds to a target region of HER 3 nucleic acid and a second region of the bispecific oligomer binds to a target region of EGFR nucleic acid and said oligomer down-regulates the expression of HER3 and EGFR. In various embodiments, the bispecific oligomer down-regulates the expression of HER3 and EGFR to a different extent. In some embodiments, the first region and the second region of the oligomer are the same. In various embodiments, the first region and the second region of the oligomer overlap. In yet further embodiments, trispecific oligomers described herein bind to a target region of HER3 nucleic acid, to a target region of HER2 nucleic acid and to a target region of EGFR nucleic acid and down-regulate the expression of all three genes. In some embodiments, trispecific oligomers that bind to HER3, HER2 and EGFR have the sequences shown, for example, in SEQ ID NOs: 169, 170, 172, 174-176 and 179. In some embodiments, a first region of a trispecific oligomer binds to a target region of HER 3 nucleic acid, a second region of the trispecific oligomer binds to a target region of EGFR nucleic acid, and a third region of the trispecific oligomer binds to a target region of HER2 nucleic acid, and said oligomers down-regulate the expression of HER3, HER2 and EGFR. In various embodiments, the trispecific oligomer down-regulates the expression of HER3, HER2 and EGFR to different extents. In some embodiments, the first, second and third regions of the oligomer are the same. In various embodiments, the first, second and third regions of the oligomer overlap. In various embodiments, bispecific or trispecific oligomers have 1, 2, 3, 4, 5 or more mismatches when compared to the best-aligned target regions of, e.g., target nucleic acids having sequences shown in SEQ ID NO: 197, 198 and/or 199.

[195] Various oligonucleotides may be used to target different regions of human PIK3CA mRNA (phosphoinositide-3 -kinase, catalytic, alpha polypeptide) and inhibit its expression. (GenBank Accession number NM_006218, SEQ ID NO:250 [SEQ ID NO: l of U.S. Application Ser. No. 12/323,744 (U.S. Pub No. 2009/0192110, now U.S. Patent No. 7,863,437)] Exemplary PIK3CA antisense oligomers that may be used according to the present invention are found in the following table. Each of the listed PIK3CA antisense oligomers has previously demonstrated about 90% or greater inhibition of PIK3CA mRNA expression at 20 nM in PC3 and MCF7 cells.

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.

5.6. Nucleosides and nucleoside analogues

[196] 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.

[197] In various embodiments, at least one of the monomers present in the oligomer is a nucleoside analogue that contains a modified sugar.

[198] In some embodiments, the linkage between at least 2 contiguous monomers of the oligomer is other than a phosphodiester linkage. [199] 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.

[200] Specific examples of nucleoside analogues useful in the oligomers described herein 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

Phosphorthioate 2'-0-Methyl 2'-MOE 2'-Fluoro

2'-(3 -hydroxy )propyl

Boranophosphates

Scheme 1 [201] The oligomer may thus comprise or consist of a simple sequence of 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.

[202] Examples of suitable and preferred nucleoside analogues are described in WO 2007/031091, incorporated herein by reference in its entirety, or are referenced therein.

[203] In some embodiments, the nucleoside analogue comprises a sugar moiety modified to provide a 2'-substituent group, such as 2'-0-alkyl-ribose sugars, 2'-amino-deoxyribose sugars, and 2'-fluoro-deoxyribose sugars.

[204] In some embodiments, the nucleoside analogue comprises a sugar in which a bridged structure, creating a bicyclic sugar (LNA), is present, 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 as 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.

[205] 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 such affinity-enhancing nucleoside analogues allows the size of the oligomer to be reduced, and also reduce the upper limit to the size of the oligomer before non-specific or aberrant binding takes place.

[206] In certain embodiments, the oligomer comprises at least 2 nucleoside analogues. In some embodiments, the oligomer comprises from 3-8 nucleoside analogues, e.g. 6 or 1 nucleoside analogues. In preferred 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 nucleosides analogues are LNA monomers.

[207] It will be recognized 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 raises the duplex stability (T_m) of the oligomer/target region duplex (i.e. affinity enhancing nucleoside analogues).

[208] In various preferred embodiments, any mismatches (that is, noncomplementarities) between the base sequence of the oligomer and the base sequence of the target region, if present, are 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 B as referred to herein, and/or within region D as referred to herein, and/or in regions which are 5' or 3' to the region of the oligomer that is complementary to the target region.

[209] In some embodiments the nucleoside analogues present within the oligomer (such as in regions A and C mentioned herein) are independently selected from, for example: monomers containing 2'-0-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, Nucl. Acids. Res. 30: 4918-4925 (2002), hereby incorporated by reference, and monomers containing 2'MOE sugars. In certain embodiments, there is only one of the above types of nucleoside analogues present in the oligomer, or region thereof.

[210] In certain embodiments, the nucleoside analogues contain 2'-0-methoxyethyl-ribose sugars (2'MOE), or 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 certain embodiments, at least one of said nucleoside analogues contains a 2'-fluoro-deoxyribose sugar, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleoside analogues containing 2'-fluoro- deoxyribose sugars.

[211] In various embodiments, the oligomer as described herein 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 - 8 LNA monomers, or 3, 4, 5, 6 or 7 LNA monomers. In various embodiments, all of the nucleoside analogues are LNA monomers. In some 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 configuration, 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 certain 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 linked with modified linkage groups such as phosphorothioate.

[212] 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.

5.7. LNA

[213] 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.

[214] The LNA used in the oligonucleotide compounds of the invention preferably has the structure of the general formula I

wherein X is selected from -0-, -S-, -N(R^N*)-, -C(R⁶R^6*)-;

B is selected from hydrogen, optionally substituted Ci-4-alkoxy, optionally substituted Ci-4-alkyl, optionally substituted Ci-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 R^5*;

P* designates an internucleoside linkage to a preceding monomer, or a 3 '-terminal group;

R^4* and R^2* together designate a biradical consisting of 1-4 groups/atoms selected from -C(R^aR^b)-, -C(R^a)=C(R^b)-, -C(R^a)=N-, -0-, -Si(R^a)₂-, -S-, -S0₂-, -N(R^a)-, and >C=Z,

wherein Z is selected from -0-, -S-, and -N(R^a)-, and R^a and R^b each is independently selected from hydrogen, optionally substituted Ci-12-alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, Ci-12-alkoxy, C2-i2-alkoxyalkyl, C2-i2-alkenyloxy, carboxy, Ci-12-alkoxycarbonyl, C_1-12- alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_6- alkyl)amino, carbamoyl, mono- and di(Ci_6-alkyl)-amino-carbonyl, amino-Ci-6-alkyl- aminocarbonyl, mono- and di(Ci-6-alkyl)amino-Ci-6-alkyl-aminocarbonyl, Ci-6-alkyl- carbonylamino, carbamido, Ci_6-alkanoyloxy, sulphono, Ci_6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci-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 R^a and R^b together may designate optionally substituted methylene (=CH₂), and

each of the substituents R , R , R , R , R , R and R , which are present is independently selected from hydrogen, optionally substituted Ci-12-alkyl, optionally substituted C2-i2-alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, Ci-12-alkoxy, C_2-12- alkoxyalkyl, C2-i2-alkenyloxy, carboxy, Ci-12-alkoxycarbonyl, Ci-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci-6-alkyl)amino, carbamoyl, mono- and di(Ci- 6-alkyl)-amino-carbonyl, amino-Ci-6-alkyl-aminocarbonyl, mono- and di(Ci_6-alkyl)amino- Ci-6-alkyl-aminocarbonyl, Ci-6-alkyl-carbonylamino, carbamido, Ci_6-alkanoyloxy, sulphono, Ci-6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci_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 -0-, -S-, and -(NR^N)- where R^N is selected from hydrogen and Ci_4-alkyl, and where two adjacent (non-geminal) substituents may designate an additional bond resulting in a double bond; and R^N*, when present and not involved in a biradical, is selected from hydrogen and Ci-4-alkyl; and basic salts and acid addition salts thereof;

[215] In certain embodiments, R is selected from H, -CH₃, -CH₂-CH₃,- CH₂-0-CH₃, and - CH=CH₂.

[216] In various embodiments, R^4* and R^2* together designate a biradical selected from - C(R^aR^b)-0-, -C(R^aR^b)-C(R^cR^d)-0-, -C(R^aR^b)-C(R^cR^d)-C(R^eR^f)-0-, -C(R^aR^b)-0-C(R^cR^d)-, - C(R^aR^b)-0-C(R^cR^d)-0-, -C(R^aR^b)-C(R^cR^d)-, -C(R^aR^b)-C(R^cR^d)-C(R^eR^f)-, C(R^a)=C(R^b)-C(R^cR^d)-, -C(R^aR^b)-N(R^c)-, -C(R^aR^b)-C(R^cR^d)- N(R^e)-, -C(R^aR^b)-N(R^c)-0-, and -C(R^aR^b)-S-, -C(R^aR^b)-C(R^cR^d)-S-, wherein R^a, R^b, R^c, R^d, R^e, and R^f each is independently selected from hydrogen, optionally substituted Ci-12-alkyl, optionally substituted C_2-12- alkenyl, optionally substituted C2-i2-alkynyl, hydroxy, Ci-12-alkoxy, C2-i2-alkoxyalkyl, C_2-12- alkenyloxy, carboxy, Ci-12-alkoxycarbonyl, Ci-12-alkylcarbonyl, formyl, aryl, aryloxy- carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(Ci_6-alkyl)amino, carbamoyl, mono- and di(Ci_6- alkyl)-amino-carbonyl, amino-Ci-6-alkyl-aminocarbonyl, mono- and di(Ci_6-alkyl)amino-Ci_ 6-alkyl-aminocarbonyl, Ci-6-alkyl-carbonylamino, carbamido, Ci_6-alkanoyloxy, sulphono, Ci-6-alkylsulphonyloxy, nitro, azido, sulphanyl, Ci_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 R^a and R^b together may designate optionally substituted methylene (=CH₂),

[217] In further embodiments R^4* and R^2* together designate a biradical selected from -CH₂- 0-, -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)-0-, -CH2-O-CH2-, -CH(CH₃)-0-, -CH(CH₂-0-CH₃)- 0-.

[218] For all chiral centers, asymmetric groups may be found in either R or S orientation. [219] Preferably, the LNA monomer used in the oligomers described herein comprises at least one LNA monomer according to any of the formulas

wherein Y is -0-, -0-CH₂- ,-S-, -NH-, or N(R ); Z and Z* are independently selected among an intemucleoside linkage, a terminal group or a protecting group; B constitutes an unmodified base moiety or a modified base moiety that either occurs naturally in nucleic acids or does not occur naturally in nucleic acids, and R^H is selected from hydrogen and C_1-4- alkyl.

[220] Specifically preferred LNA monomers are shown in Scheme 2:

β-D-oxy-LNA

β-D-amino-LNA Scheme 2

[221] 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 the alpha-L- configuration.

[222] 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 Ci-4-alkyl. Amino-LNA can be in either the beta-D or the alpha-L- configuration.

[223] 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 the alpha-L- configuration.

[224] 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).

[225] In a preferred embodiment the LNA monomer is selected from a beta-D-oxy-LNA monomer, an alpha-L-oxy-LNA monomer, a beta-D-amino-LNA monomer and a beta-D- thio-LNA monomer, in particular a beta-D-oxy-LNA monomer.

[226] In the present context, the term "Cl-4-alkyl" 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.

5.8. RNAse H recruitment

[227] 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 described herein are capable of recruiting an endoribonuclease (RNase), such as RNase H.

[228] 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 certain embodiments, the region of the oligomer which is capable of recruiting RNAse, such as region B, consists of 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 monomers.

[229] EP 1 222 309 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability of the oligomers to recruit RNaseH. An oligomer is deemed capable of recruiting RNase H if, when contacted with the complementary target 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 Example 91 - 95 of EP 1 222 309, incorporated herein by reference.

[230] In various 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 Example 91 - 95 of EP 1 222 309.

[231] 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 Example 91 - 95 of EP 1 222 309.

[232] Typically, the region of the oligomer that forms a 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.

[233] In various embodiments, the oligomer comprises both nucleosides and nucleoside analogues, and is in the form of a gapmer as defined above, a headmer or a mixmer. [234] 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.

[235] 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 such 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 analogue monomers.

[236] 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.

[237] In some embodiments, in addition to enhancing affinity of the oligomer for the target region, some nucleoside analogues also mediate RNase (e.g., RNase H) binding and cleavage. Since a-L-LNA monomers recruit RNase activity to a certain extent, in some embodiments, gap regions (e.g., region B as referred to herein below) of oligomers containing a-L-LNA monomers consist of fewer monomers recognizable and cleavable by the RNase, and more flexibility in the mixmer construction is introduced.

5.9. Conjugates

[238] 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 moiety"). 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. WO 2007/031091 provides suitable moieties and conjugates, which are hereby incorporated by reference.

[239] 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 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 said oligomer. In any of the embodiments of the invention described herein, one or more of the antisense oligomers may be a conjugate of an antisense oligomer.

[240] In certain embodiments, the oligomer is conjugated to a moiety that increases the cellular uptake of oligomeric compounds.

[241] In various embodiments, conjugates may enhance the activity, cellular distribution or cellular uptake of the oligomers described herein. 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.

[242] In certain embodiments, the oligomers are conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.

[243] In certain embodiments, the conjugated moiety is a sterol, such as cholesterol.

[244] In various embodiments, the conjugated moiety comprises or consists of a positively charged polymer, such as a positively charged peptide 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 via a linker such as the releasable inker described in WO 2008/034123.

5.10. Activated oligomers

[245] The term "activated oligomer," as used herein, refers to an oligomer as described herein 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 in some embodiments is 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 NH₂ 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.

[246] In some embodiments, the functional moiety is self-cleaving. In other embodiments, the functional moiety is biodegradable. See e.g., U.S. Patent No. 7,087,229, which is incorporated by reference herein in its entirety.

[247] In some embodiments, oligomers 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 can be functionalized at the 3 ' end. In still other embodiments, oligomers can be functionalized along the backbone or on the heterocyclic base moiety. In yet other embodiments, oligomers can be functionalized at more than one position independently selected from the 5' end, the 3 ' end, the backbone and the base.

[248] In some embodiments, activated oligomers as described herein are synthesized by incorporating during the synthesis one or more monomers that is covalently attached to a functional moiety. In other embodiments, activated oligomers are synthesized with monomers that have not been functionalized, and the oligomer is functionalized upon completion of synthesis.

[249] In some embodiments, the oligomers are functionalized with a hindered ester containing an aminoalkyl linker, wherein the alkyl portion has the formula (C]¾)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 (-0-C(0)-(CH₂)_wNH).

[250] 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 (-0-C(0)-(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).

[251] 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 1 19: 143-152; and Zhao et al. (2005) Bioconjugate Chem. 16:758-766.

[252] In still other embodiments, the oligomers described herein are functionalized by introducing sulfhydryl, amino or hydroxyl groups into the oligomer by means of a functionalizing reagent substantially as described in U.S. Patent 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 have a functionalizing reagent coupled to a hydroxyl group on the backbone of the oligomer. In yet further embodiments, the oligomer is functionalized with more than one of the functionalizing reagents as described in U.S. Patent 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. Patent Nos. 4,962,029 and 4,914,210.

[253] 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.

[254] In various embodiments, the incorporation of monomers containing 2'-sugar modifications, such as a 2'-carbamate substituted sugar or a 2'-(0-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'-0-(e-phthalimidylaminopentyl)-2'-deoxyadenosine-3'— N,N- diisopropyl-cyanoethoxy phosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters, 1991, 34, 7171.

[255] In still further embodiments, the oligomers described herein 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 some embodiments, such functionalization may be achieved by using a commercial reagent that is already functionalized in the oligomer synthesis.

[256] Some functional moieties are commercially available, for example, heterobifunctional and homobifunctional linking moieties are available from the Pierce Co. (Rockford, III). 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).

5.11. Compositions

[257] In various embodiments, the oligomer as described herein is used in pharmaceutical formulations and compositions. Suitably, such compositions may comprise a pharmaceutically acceptable diluent, carrier, salt or adjuvant. WO2007/031091, which is hereby incorporated by reference, provides suitable and preferred pharmaceutically acceptable diluents, carriers and adjuvants. Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in WO2007/031091, 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.).

[258] In some embodiments, an oligomer described herein 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 as described herein 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 described herein 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 which is incorporated by reference herein.

[259] 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.

[260] The amount of the at least one oligomer that is effective for the treatment or prevention of a disease that is resistant to treatment with a PTK inhibitor can be determined by standard clinical techniques. Generally the dosage ranges can be estimated based on EC₅₀ found to be effective in in vitro and in vivo animal models. The precise doses to be employed will also depend on, e.g., the routes of administration and the seriousness of the disease, and can be decided according to the judgment of a practitioner and/or each patient's circumstances. In other examples thereof, variations will necessarily occur depending upon, inter alia, the weight and physical condition (e.g., hepatic and renal function) of the patient being treated, the affliction to be treated, the severity of the symptoms, the frequency of the dosage interval, and the presence of any deleterious side-effects.

[261] In various embodiments, the dosage of an oligomer is from about 0.01 μg to about 1 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 10 years or by continuous infusion for hours up to several months. In certain embodiments, repetition rates for dosing can be estimated based on measured residence times and concentrations of the active agent in bodily fluids or tissues. Following successful treatment, the patient may undergo maintenance therapy with the HER3 -targeted therapy, for example HER3 and PIK3CA targeted combination therapy, to prevent or reduce the risk of or time to recurrence of the disease state.

[262] One embodiment of the invention provides a pharmaceutical composition that includes an antisense HER3 oligomer or pharmaceutically acceptable salt thereof and an antisense PIK3CA oligomer or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutical excipients. The pharmaceutical composition may be for the treatment of PTKI-resistant, such as gefitinib-resistant, hyperproliferative disorder such as a cancer. The composition may, for example, further include a protein tyrosine kinase inhibitor such as but not limited to gefitinib, imatinib, erlotinib, lapatinib, canertinib or sorafenib. The composition may, for example, further include a HER2 inhibitor such as but not limited to trastuzumab or pertuzumab.

5.12. Combination with Other Antisense Oligomers and Chemotherapeutic

Agents

[263] In some embodiments, treatment with a combination of HER3 antisense oligomers and PIK3CA antisense oligomers is further supplemented by treatment with HER2 antisense oligomers and/or EGFR antisense oligomers. Thus, in some embodiments, the invention relates to methods of treating a disease that is resistant to treatment with a PTK inhibitor by administering not only HER3 antisense oligomers and PIK3CA antisense oligomers, but also further antisense oligomers, such as an antisense oligomer which targets either EGFR or HER2. In the methods described herein, such oligomers can be administered concurrently, or sequentially.

[264] In various embodiments the invention relates to methods of treating a PTK inhibitor- resistant disease by administering a pharmaceutical composition that comprises not only oligomers targeted to HER3 and PIK3CA, but also a further therapeutic agent which targets and down-regulates HER2 expression, such as an antisense oligomer which targets HER2 mRNA.

[265] In other embodiments, which may be the same or different, the invention relates to a method of treating a PTK inhibitor-resistant disease by administering a pharmaceutical composition comprising oligomers targeted to HER3 and PIK3CA, and a further therapeutic agent which targets and down-regulates EGFR expression, such as an antisense oligomer which target EGFR mRNA. [266] In some embodiments, oligomers that target HER2 and/or EGFR mRNA (or conjugates thereof), have the same designs (e.g., gapmers, headmers, tailmers) as the oligomers that target HER3 and PIK3CA. In various embodiments, oligomers that target HER2 and/or EGFR mRNA (or conjugates thereof), have different designs from oligomers that target HER3.

[267] In certain embodiments, the invention relates to a method of treating a PTK inhibitor- resistant disease by administering one or more oligomers as described herein and one or more additional chemotherapeutic agents, including but not limited to, alkylating agents, antimetabolites, epipodophyllotoxins, anthracyclines, vinca alkaloids, plant alkaloids and terpenoids, monoclonal antibodies, taxanes, topoisomerase inhibitors, and platinum compounds.

5.13. Kits

[268] The invention also provides methods of treating a hyperproliferative disease, such as one that is resistant to treatment with a protein tyrosine kinase inhibitor, or susceptible to such resistance, using a kit comprising a first component and a second component. In various embodiments, said first component comprises an antisense oligomer as described herein that is capable of inhibiting (e.g., by down-regulating) expression of HER3, or a conjugate and/or pharmaceutical composition thereof. In other embodiments, the second component comprises a second active ingredient. In some embodiments, the second component is a therapeutic agent that comprises an antisense oligomer that is capable of inhibiting (e.g., by down-regulating) expression of PIK3CA. In other embodiments, another therapeutic agent other than an oligonucleotide (e.g., a small molecule therapeutic agent such as taxol) or a protein tyrosine kinase inhibitor such as gefitinib is included in still another component of the kit. In some embodiments, kits described herein are used in methods of treating a hyperproliferative disorder, such as cancer which is resistant to treatment with a PTK inhibitor, which comprises administering to a patient in need thereof an effective amount of a first component and a second component of the kit. In various embodiments, the first and second components are administered simultaneously. In other embodiments, the first and second components are administered sequentially and in any order.

[269] In some embodiments, the kit comprises a first component that comprises an oligomer that is capable of inhibiting (e.g., by down-regulating) expression of HER3, or a conjugate and/or pharmaceutical composition thereof, as described herein, and a second component that is an antisense oligonucleotide capable of inhibiting (e.g., by down-regulating) the expression of PIK3CA expression as described herein, or a conjugate and/or pharmaceutical composition thereof, as described herein. The kit may be for the treatment of PTKI-resistant, such as gefitinib-resistant, hyperproliferative disorder such as a cancer. The kit may, for example, further include a protein tyrosine kinase inhibitor such as but not limited to gefitinib, imatinib, erlotinib, lapatinib, canertinib or sorafenib. The kit may, for example, further include a HER2 inhibitor such as but not limited to trastuzumab or pertuzumab.

6. EXAMPLES

6.1. Example 1 : Monomer synthesis

[270] The LNA monomer building blocks and derivatives thereof were prepared according to published procedures. See WO07/031081 and the references cited therein.

6.2. Example 2: Oligonucleotide synthesis

[271] Oligonucleotides were synthesized according to the method described in WO07/031081. Table 1 shows examples of antisense oligonucleotide motifs of the invention.

6.3. Example 3: Design of the oligonucleotides

[272] In accordance with the invention, a series of oligonucleotides were designed to target different regions of human EGFR (GenBank Accession number NM_005228, SEQ ID NO: 198) and human HER2 (GenBank Accession number NM_004448, SEQ ID NO: 199) in addition to human HER3 (GenBank Accession number NM_001982, SEQ ID NO: 197).

[273] Of the sequences shown in Table 1, below, SEQ ID NOs: 1-50, 53, 139 and 140 were designed to target human EGFR and human HER2 in addition to human HER3. The percentage of sequence homology with HER3, EGFR and HER2 is indicated. The sequences of the oligomers contain 0-2 mismatches when compared to the sequences of the best-aligned target regions of EGFR, and 1-2 mismatches when compared to the sequences of the best- aligned target regions of HER2.

Table 1

Antisense Oligonucleotide Sequences

Length Compl Compl

SEQ ID NO Sequence (5'-3') Target site HER3

(bases) EGFR HER2

SEQ ID NO: 1 GCTCCAGACATCACTC 16 2866 - 2881 100% 87.5% Table 1

Antisense Oligonucleotide Sequences

Length Compl Compl

SEQ ID NO Sequence (5'-3') Target site HER3

(bases) EGFR HER2

SEQ ID NO: 2 GCTCCAGACATCACT 15

SEQ ID NO: 3 CTCCAGACATCACTC 15

SEQ ID NO: 4 GCTCCAGACATCAC 14

SEQ ID NO: 5 CTCCAGACATCACT 14

SEQ ID NO: 6 TCCAGACATCACTC 14

SEQ ID NO: 7 GCTCCAGACATCA 13

SEQ ID NO: 8 CTCCAGACATCAC 13

SEQ ID NO: 9 TCCAGACATCACT 13

SEQ ID NO: 10 CCAGACATCACTC 13

SEQ ID NO: 1 1 GCTCCAGACATC 12

SEQ ID NO: 12 CTCCAGACATCA 12

SEQ ID NO: 13 TCCAGACATCAC 12

SEQ ID NO: 14 CCAGACATCACT 12

SEQ ID NO: 15 CAGACATCACTC 12

SEQ ID NO: 16 CTCCAGACATCACTCT 16 2865 - 2880 100% 93.8%

SEQ ID NO: 17 CAGACATCACTCTGGT 16 2862 - 2877 100% 93.8%

SEQ ID NO: 18 AGACATCACTCTGGTG 16 2861 - 2876 100% 93.8%

SEQ ID NO: 19 ATAGCTCCAGACATCA 16 2869 - 2884 93.8% 87.5%

SEQ ID NO: 20 ATAGCTCCAGACATC 15

SEQ ID NO: 21 TAGCTCCAGACATCA 15

SEQ ID NO: 22 ATAGCTCCAGACAT 14

SEQ ID NO: 23 TAGCTCCAGACATC 14

SEQ ID NO: 24 AGCTCCAGACATCA 14

SEQ ID NO: 25 ATAGCTCCAGACA 13

SEQ ID NO: 26 TAGCTCCAGACAT 13

SEQ ID NO: 27 AGCTCCAGACATC 13 Table 1

Antisense Oligonucleotide Sequences

Length Compl Compl

SEQ ID NO Sequence (5'-3') Target site HER3

(bases) EGFR HER2

SEQ ID NO: 28 GCTCCAGACATCA 13

SEQ ID NO: 29 ATAGCTCCAGAC 12

SEQ ID NO: 30 TAGCTCCAGACA 12

SEQ ID NO: 31 AGCTCCAGACAT 12

SEQ ID NO: 32 GCTCCAGACATC 12

SEQ ID NO: 33 CTCCAGACATCA 12

SEQ ID NO: 34 TCACACCATAGCTCCA 16 2876 - 2891 87.5% 93.8%

SEQ ID NO: 35 TCACACCATAGCTCC 15

SEQ ID NO: 36 CACACCATAGCTCCA 15

SEQ ID NO: 37 TCACACCATAGCTC 14

SEQ ID NO: 38 CACACCATAGCTCC 14

SEQ ID NO: 39 ACACCATAGCTCCA 14

SEQ ID NO: 40 TCACACCATAGCT 13

SEQ ID NO: 41 CACACCATAGCTC 13

SEQ ID NO: 42 ACACCATAGCTCC 13

SEQ ID NO: 43 CACCATAGCTCCA 13

SEQ ID NO: 44 TCACACCATAGC 12

SEQ ID NO: 45 CACACCATAGCT 12

SEQ ID NO: 46 ACACCATAGCTC 12

SEQ ID NO: 47 CACCATAGCTCC 12

SEQ ID NO: 48 ACCATAGCTCCA 12

SEQ ID NO: 49 CATCCAACACTTGACC 16 3025 - 3040 93.8% 93.8%

SEQ ID NO: 50 ATCCAACACTTGACCA 16 3024 - 3039 93.8% 93.8%

SEQ ID NO: 51 CAATCATCCAACACTT 16 3029 - 3044 87.5% 93.8%

SEQ ID NO: 52 TCAATCATCCAACACT 16 3030 - 3045 87.5% 93.8%

SEQ ID NO: 53 CATGTAGACATCAATT 16 3004 - 3019 87.5% 93.8% Table 1

Antisense Oligonucleotide Sequences

Length Compl Compl

SEQ ID NO Sequence (5'-3') Target site HER3

(bases) EGFR HER2

SEQ ID NO: 54 TAGCCTGTCACTTCTC 16 435 - 450 68.8% 75%

SEQ ID NO: 228 TAGCCTGTCACTTCT 15

SEQ ID NO: 229 AGCCTGTCACTTCTC 15

SEQ ID NO: 230 TAGCCTGTCACTTC 14

SEQ ID NO: 231 AGCCTGTCACTTCT 14

SEQ ID NO: 232 TAGCCTGTCACTT 13

SEQ ID NO: 233 TAGCCTGTCACT 12

SEQ ID NO: 55 AGATGGCAAACTTCCC 16 530 - 545 68.8% 68.8%

SEQ ID NO: 56 CAAGGCTCACACATCT 16 1 146 - 1 161 75% 68.8%

SEQ ID NO: 57 AAGTCCAGGTTGCCCA 16 1266 - 1281 75% 75%

SEQ ID NO: 58 CATTCAAGTTCTTCAT 16 1490 - 1505 75% 68.8%

SEQ ID NO: 59 CACTAATTTCCTTCAG 16 1529 - 1544 81.3% 68.8%

SEQ ID NO: 60 CACTAATTTCCTTCA 15

SEQ ID NO: 61 ACTAATTTCCTTCAG 15

SEQ ID NO: 62 CACTAATTTCCTTC 14

SEQ ID NO: 63 ACTAATTTCCTTCA 14

SEQ ID NO: 64 CTAATTTCCTTCAG 14

SEQ ID NO: 65 CACTAATTTCCTT 13

SEQ ID NO: 66 ACTAATTTCCTTC 13

SEQ ID NO: 67 CTAATTTCCTTCA 13

SEQ ID NO: 68 TAATTTCCTTCAG 13

SEQ ID NO: 69 CACTAATTTCCT 12

SEQ ID NO: 70 ACTAATTTCCTT 12

SEQ ID NO: 71 CTAATTTCCTTC 12

SEQ ID NO: 72 TAATTTCCTTCA 12

SEQ ID NO: 73 AATTTCCTTCAG 12 Table 1

Antisense Oligonucleotide Sequences

Length Compl Compl

SEQ ID NO Sequence (5'-3') Target site HER3

(bases) EGFR HER2

SEQ ID NO: 74 GCCCAGCACTAATTTC 16 1535 - 1550 75% 68.8%

SEQ ID NO: 75 CTTTGCCCTCTGCCAC 16 1673 - 1688 75% 75%

SEQ ID NO: 76 CACACACTTTGCCCTC 16 1679 - 1694 68.8% 75%

SEQ ID NO: 77 CACACACTTTGCCCT 15

SEQ ID NO: 78 ACACACTTTGCCCTC 15

SEQ ID NO: 79 CACACACTTTGCCC 14

SEQ ID NO: 80 ACACACTTTGCCCT 14

SEQ ID NO: 81 CACACTTTGCCCTC 14

SEQ ID NO: 82 CACACACTTTGCC 13

SEQ ID NO: 83 ACACACTTTGCCC 13

SEQ ID NO: 84 CACACTTTGCCCT 13

SEQ ID NO: 85 ACACTTTGCCCTC 13

SEQ ID NO: 86 CACACACTTTGC 12

SEQ ID NO: 87 ACACACTTTGCC 12

SEQ ID NO: 88 CACACTTTGCCC 12

SEQ ID NO: 89 ACACTTTGCCCT 12

SEQ ID NO: 90 CACTTTGCCCTC 12

SEQ ID NO: 91 CAGTTCCAAAGACACC 16 2345 - 2360 75% 68.8%

SEQ ID NO: 92 TGGCAATTTGTACTCC 16 2636 - 2651 75% 68.8%

SEQ ID NO: 93 TGGCAATTTGTACTC 15

SEQ ID NO: 94 GGCAATTTGTACTCC 15

SEQ ID NO: 95 TGGCAATTTGTACT 14

SEQ ID NO: 96 GGCAATTTGTACTC 14

SEQ ID NO: 97 GCAATTTGTACTCC 14

SEQ ID NO: 98 TGGCAATTTGTAC 13

SEQ ID NO: 99 GGCAATTTGTACT 13 Table 1

Antisense Oligonucleotide Sequences

Length Compl Compl

SEQ ID NO Sequence (5'-3') Target site HER3

(bases) EGFR HER2

SEQ ID NO: 100 GCAATTTGTACTC 13

SEQ ID NO: 101 CAATTTGTACTCC 13

SEQ ID NO: 102 TGGCAATTTGTA 12

SEQ ID NO: 103 GGCAATTTGTAC 12

SEQ ID NO: 104 GCAATTTGTACT 12

SEQ ID NO: 105 CAATTTGTACTC 12

SEQ ID NO: 106 AATTTGTACTCC 12

SEQ ID NO: 107 GTGTGTGTATTTCCCA 16 2848 - 2863 75% 68.8%

SEQ ID NO: 108 GTGTGTGTATTTCCC 15

SEQ ID NO: 109 TGTGTGTATTTCCCA 15

SEQ ID NO: 1 10 GTGTGTGTATTTCC 14

SEQ ID NO: 1 11 TGTGTGTATTTCCC 14

SEQ ID NO: 1 12 GTGTGTATTTCCCA 14

SEQ ID NO: 1 13 GTGTGTGTATTTC 13

SEQ ID NO: 1 14 TGTGTGTATTTCC 13

SEQ ID NO: 1 15 GTGTGTATTTCCC 13

SEQ ID NO: 1 16 TGTGTATTTCCCA 13

SEQ ID NO: 1 17 GTGTGTGTATTT 12

SEQ ID NO: 1 18 TGTGTGTATTTC 12

SEQ ID NO: 1 19 GTGTGTATTTCC 12

SEQ ID NO: 120 TGTGTATTTCCC 12

SEQ ID NO: 121 GTGTATTTCCCA 12

SEQ ID NO: 122 CCCTCTGATGACTCTG 16 3474 - 3489 68.8% 68.8%

SEQ ID NO: 123 CCCTCTGATGACTCT 15

SEQ ID NO: 124 CCTCTGATGACTCTG 15

SEQ ID NO: 125 CCCTCTGATGACTC 14 Table 1

Antisense Oligonucleotide Sequences

Length Compl Compl

SEQ ID NO Sequence (5'-3') Target site HER3

(bases) EGFR HER2

SEQ ID NO: 126 CCTCTGATGACTCT 14

SEQ ID NO: 127 CTCTGATGACTCTG 14

SEQ ID NO: 128 CCCTCTGATGACT 13

SEQ ID NO: 129 CCTCTGATGACTC 13

SEQ ID NO: 130 CTCTGATGACTCT 13

SEQ ID NO: 131 TCTGATGACTCTG 13

SEQ ID NO: 132 CCCTCTGATGAC 12

SEQ ID NO: 133 CCTCTGATGACT 12

SEQ ID NO: 134 CTCTGATGACTC 12

SEQ ID NO: 135 TCTGATGACTCT 12

SEQ ID NO: 136 CTGATGACTCTG 12

SEQ ID NO: 137 CATACTCCTCATCTTC 16 3770 - 3785 81.3% 81.3%

SEQ ID NO: 138 CCACCACAAAGTTATG 16 1067 - 1082 81.3% 68.8%

SEQ ID NO: 139 CATCACTCTGGTGTGT 16 2858 - 2873 93.8% 93.8%

SEQ ID NO: 140 GACATCACTCTGGTGT 16 2860 - 2875 93.8% 87.5%

[274] In Table 2, bold letters represent shorter sequences shown in Table 1.

Table 2

HER3 24mer Sequences

16mer SEQ IDs Corresponding 24mer sequence comprising 16mer 24mer SEQ ID

SEQ ID NO: 49 caatcatccaacacttgaccatca SEQ ID NO: 206

SEQ ID NO: 50 aatcatccaacacttgaccatcac SEQ ID NO: 207

SEQ ID NO: 51 tcatcaatcatccaacacttgacc SEQ ID NO: 208

SEQ ID NO: 52 ctcatcaatcatccaacacttgac SEQ ID NO: 209

SEQ ID NO: 53 tcaccatgtagacatcaattstac SEQ ID NO: 210

SEQ ID NO: 54 sacatagcctgtcacttctc aaat SEQ ID NO: 21 1

SEQ ID NO: 55 acgaagatggcaaacttcccatcg SEQ ID NO: 212

SEQ ID NO: 56 cccacaaeectcacacatcttsas SEQ ID NO: 213

SEQ ID NO: 57 casaaagtccaeettecccassat SEQ ID NO: 214

SEQ ID NO: 58 gt gacattc aa2ttcttc at gate SEQ ID NO: 215

SEQ ID NO: 59 ccagcactaatttccttca2ggat SEQ ID NO: 216

SEQ ID NO: 74 atac2ccca2cactaatttccttc SEQ ID NO: 217

SEQ ID NO: 75 cacacttt2ccctct2ccacgcag SEQ ID NO: 218

SEQ ID NO: 76 gggtcacacacttt2ccctctgcc SEQ ID NO: 219

SEQ ID NO:91 tgcaca2ttccaaa2acacccgag SEQ ID NO: 220

SEQ ID NO:92 ccctt22caattt2tactccccag SEQ ID NO: 221

SEQ ID NO: 107 tctg2t2t2t2tatttcccaaagt SEQ ID NO: 222

SEQ ID NO: 122 atgcccctct2at2actct2atgc SEQ ID NO: 223

SEQ ID NO: 137 tattcatactcctcatcttcatct SEQ ID NO: 224

SEQ ID NO: 138 tgatccaccacaaa2ttat2ggga SEQ ID NO: 225

SEQ ID NO: 139 cagacatcactct22t2t2tgtat SEQ ID NO: 226

SEQ ID NO: 140 tcca2acatcactct22t2tgtgt SEQ ID NO: 227

[275] In SEQ ID NOs: 141-168 shown in Table 3, uppercase letters indicate nucleoside analogue monomers and the subscript "s" represents a phosphorothioate linkage. Lowercase letters represent DNA monomers. The absence of "s" between monomers (if any) indicates a phosphodiester linkage. Table 3

HER3 Oligonucleotide Ga mers

SEQ ID NO Sequence (5 '-3')

GC T c c ag a c at c aC T C

SEQ ID NO: 141

s s s s s s s s s ss s s s s

C T C c ag ac at c ac T C T

SEQ ID NO: 142

s s s s s s s s ss s s s s s

C A G ac at c ac t c t GG T

SEQ ID NO: 143

s s s s s ss s s ss ss s s

A GA c at c ac t c t g GT G

SEQ ID NO: 144

s s s s ss s s ss ss s s s

A TA g c t c c ag a c aT C A

SEQ ID NO: 145

s s s s ss s s s s s s s s s

T C A c ac c at ag c t C C A

SEQ ID NO: 146

s s s s s s s ss s s ss s s

C A T c c aac ac tt gA C C

SEQ ID NO: 147

s s s s s s s s s sss s s s

A T C c aac ac tt g aC C A

SEQ ID NO: 148

s s s s s s s s sss s s s s

C A At c at c c aac aC T T

SEQ ID NO: 149

s s ss s ss s s s s s s s s

T C A at c at c c aac A C T

SEQ ID NO: 150

s s s ss s ss s s s s s s s

C A T gt ag ac at c aA T T

SEQ ID NO: 151

s s s ss s s s s ss s s s s

TA Gc c t gt c ac tt C T C

SEQ ID NO: 152

s s s s ss ss s s sss s s

A GA tg g c aaac tt C C C

SEQ ID NO: 153

s s ss s s s s s s sss s s

C A A g g c tc ac ac a T C T

SEQ ID NO: 154

s s s s s ss s s s s s s s s

AA Gtc c ag gtt g c C C A

SEQ ID NO: 155

s s ss s s s s sss s s s s

C A Tt c aagtt c ttC A T

SEQ ID NO: 156

s s ss s s s sss sss s s

C A C t aattt c c ttC A G

SEQ ID NO: 157

s s ss s ssss s sss s s

GC C c ag c ac t aatT T C

SEQ ID NO: 158

s s s s s s s s ss s ss s s Table 3

HER3 Oligonucleotide Ga mers

SEQ ID NO Sequence (5 '-3')

C T Tt g c c c t c t g c C A C

SEQ ID NO: 159

s s ss s s s ss ss s s s s

C A C ac a cttt g c c C T C

SEQ ID NO: 160

s s s s s s ssss s s s s s

C A Gtt c c aaag ac A C C

SEQ ID NO: 161

s s sss s s s s s s s s s s

T G Gc aattt gt ac T C C

SEQ ID NO: 162

s s s s s ssss ss s s s s

GT Gt gt gt attt c C C A

SEQ ID NO: 163

s s ss ss ss ssss s s s

C C C t c t g at g ac t C T G

SEQ ID NO: 164

s s ss ss s ss s s ss s s

C A T ac t c c t c at c T T C

SEQ ID NO: 165

s s s s ss s ss s ss s s s

C C A c c a c aaag ttA T G

SEQ ID NO: 166

s s s s s s s s s s sss s s

C A T c ac t c t g gt g T G T

SEQ ID NO: 167

s s s s s ss ss s ss s s s

GA C atc ac t c t g g T G T

SEQ ID NO: 168

s s s ss s s ss ss s s s s

6.4. Example 4: In vitro model: Cell culture

[276] 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, or 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 chosen cell type.

[277] 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. [278] 15PC3 : The human prostate cancer cell line 15PC3 was cultured in DMEM (Sigma) + 10% fetal bovine serum (FBS) + 2 mM Glutamax I + gentamicin (25μg/ml).

[279] HUH7: The human hepatocarcinoma cell line was cultured in DMEM (Sigma) + 10% fetal bovine serum (FBS) + 2 mM Glutamax I + gentamicin (25μg/ml) + lx Non Essential Amino Acids.

6.5. Example 5: In vitro model: Treatment with antisense oligonucleotides

[280] The cells were treated with oligonucleotides 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 ranged from 1 nM to 25 nM final concentration. Formulation of oligomer-lipid complexes was 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 medium was added. After oligomer treatment, cells were allowed to recover for 20 hours before they were harvested for RNA analysis.

6.6. Example 6: In vitro model: Extraction of RNA and cDNA synthesis

[281] 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.

[282] For each sample 0.5 μg total RNA was adjusted to (10.8 μΐ) with RNase free H₂0 and mixed with 2 μΐ random decamers (50 μΜ) and 4 μΐ 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 μΐ lOx Buffer RT, 1 μΐ MMLV Reverse Transcriptase (100 U/μΙ) and 0.25 μΐ RNase inhibitor (10 U/μΙ) was 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 the sample to 4 °C. 6.7. Example 7: In vitro model: Analysis of Oligonucleotide Inhibition of HER3, EGFR and HER2 Expression by Real-time PCR

[283] Antisense modulation of HER3, EGFR and HER2 expression can be assayed in a variety of ways known in the art. For example, HER3, EGFR and HER2 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.

[284] Real-time quantitative (PCR) can be conveniently accomplished using the commercially available Multi-Color Real Time PCR Detection System, available from Applied Biosystem.

Real-time Quantitative PCR Analysis of HER3. EGFR and HER2 mRNA Levels

[285] The sample content of human HER3, EGFR and HER2 mRNA was quantified using the human HER3, EGFR and HER2 ABI Prism Pre-Developed TaqMan Assay Reagents (Applied Biosystems cat. no. Hs00951444_ml (HER3), Hs00193306_ml (EGFR) and Hs00170433_ml (HER2) according to the manufacturer's instructions.

[286] Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) mRNA quantity was used as an endogenous control for normalizing any variance in sample preparation. The sample content of human GAPDH mRNA was quantified using the human GAPDH ABI Prism Pre-Developed TaqMan Assay Reagent (Applied Biosystems cat. no. 4310884E) according to the manufacturer's instructions.

[287] Real-time Quantitative PCR is a technique well known in the art and is taught in for example in Heid et al. Real time quantitative PCR, Genome Research (1996), 6: 986-994.

Real time PCR

[288] The cDNA from the first strand synthesis performed as described in Example 5 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 x Taqman Fast Universal PCR master mix (2x) (Applied Biosystems Cat.# 4364103) and added to 4 μΐ cDNA to a final volume of 10 μΐ. Each sample was analysed in duplicate. Assaying 2-fold dilutions of a cDNA that had been prepared on material purified from a cell line expressing the RNA of interest generated standard curves for the assays. Sterile H₂0 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 sequence 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.

6.8. Example 8: In vitro analysis: Antisense Inhibition of Human HER3, EGFR and HER2 Expression by oligonucleotide compounds.

[289] Oligonucleotides presented in Table 4 were evaluated for their potential to down- regulate HER3, EGFR and HER2 mRNA at concentrations of 1, 5 and 25 nM in 15PC3 cells (or HUH-7 as indicated by *) (see Figures 2, 3, 4 and 5). SEQ ID NOs: 235 and 236 were used as scrambled controls.

[290] The data in Table 4 are presented as percentage down-regulation of mRNA relative to mock transfected cells at 25 nM. Lower-case letters represent DNA monomers, bold, uppercase letters represent β-D-oxy-LNA monomers. All cytosines in LNA monomers are 5- methylcytosines. Subscript "s" represents a phosphorothioate linkage.

Table 4

Inhibition of human HER3, EGFR and HER2 expression by antisense oligonucleotides

Test substance Sequence (5'-3') HER3 EGFR HER2

51.1% 0% 63.4%

C AA tc at c c aac aC T T

SEQIDNO: 177

s s ss s ss s s s s s s s s

76.7% 0% 88.6%

T C A atc at c c aac A C T

SEQIDNO: 178

s s s ss s ss s s s s s s s

70.5% 52.6% 75.6%

C A T g t ag ac at c aA T T

SEQIDNO: 179

s s s ss s s s s ss s s s s

92.8% N.D. N.D.

TA G c c tgt c ac tt C T C

SEQIDNO: 180

s s s s ss ss s s sss s s

90.6% N.D. N.D.

A GA tg g c aaac ttC C C

SEQIDNO: 181

s s ss s s s s s s sss s s

74.6% N.D. N.D.

C AA g g c tc ac ac aT C T

SEQIDNO: 182

s s s s s ss s s s s s s s s

85.9% N.D. N.D.

A A Gtc c ag gttg c C C A

SEQIDNO: 183

s s ss s s s s sss s s s s

81.1% N.D. N.D.

C A T tc a ag tt c tt C A T

SEQIDNO: 184

s s ss s s s sss sss s s

89.1% N.D. N.D.

C A C taattt c c tt C A G

SEQIDNO: 185

s s ss s ssss s sss s s

79.9% N.D. N.D.

G C C c ag c ac t aat T T C

SEQIDNO: 186

s s s s s s s s ss s ss s s

90.4% N.D. N.D.

C T Tt g c c c t c t g c C A C

SEQIDNO: 187

s s ss s s s ss ss s s s s

96.1% N.D. N.D.

C A C ac ac ttt g c c C T C

SEQIDNO: 188

s s s s s s ssss s s s s s

88.9% N.D. N.D.

C A Gtt c c aaag ac A C C

SEQIDNO: 189

s s sss s s s s s s s s s s

95.7% N.D. N.D.

T G G c aattt gt ac T C C

SEQIDNO: 190

s s s s s ssss ss s s s s

97.7% N.D. N.D.

G T Gtg tgt attt c C C A

SEQIDNO: 191

s s ss ss ss ssss s s s

92.3% N.D. N.D.

C C C tc t g at g ac t C T G

SEQIDNO: 192

s s ss ss s ss s s ss s s

64% N.D. N.D.

C A T ac t c c t c at c T T C

SEQIDNO: 193

s s s s ss s ss s ss s s s

87.5% N.D. N.D.

C C A c c ac aaag ttA T G

SEQIDNO: 194

s s s s s s s s s s sss s s Table 4

Inhibition of human HER3, EGFR and HER2 expression by antisense

oligonucleotides

Test substance Sequence (5'-3') HER3 EGFR HER2

64.4%* N.D. N.D.

C A T c ac tc t g gt g T GT

SEQIDNO: 195

s s s s s ss ss s ss s s s

77.0%* N.D. N.D.

GA C atc ac t c t g g T G T

SEQIDNO: 196

s s s ss s s ss ss s s s s

TA g c c t gt c aC T T

SEQ ID NO: 234

s s s s ss ss s s s s

C GT c ag tat g c gA A T c

SEQ ID NO: 235

s s s s s ss ss s s s s s s

C GC A g att ag aaA C C t

SEQ ID NO: 236

s s s s s sss s s s s s s s

TA G c c ttt g ac c t C T C

SEQ ID NO: 249

s s s s ssss s s s ss s s

[291] As shown in Table 4, oligonucleotides having the sequences shown in SEQ ID NOs: 169, 170, 173, 174, 180, 181, 183, 185, 187, 188, 189, 190, 191, 192 and 194 demonstrated about 85% or greater inhibition of HER3 mRNA expression at 25 nM in 15PC3 cells in these experiments, and are therefore preferred.

[292] Also preferred are oligonucleotides based on the illustrated antisense oligomer sequences, for example varying the length (shorter or longer) and/or monomer content (e.g., the type and/or proportion of nucleoside analogue monomers), which also provide good inhibition of HER3 expression.

6.9. Example 9: Apoptosis induction by LNA oligonucleotides

[293] HUH7 cells were seeded in 6-well culture plates (NUNC) the day before transfection at a density of 2.5 x 10⁵ cells/well. The cells were treated with oligonucleotides using the cationic liposome formulation LipofectAMINE 2000 (Gibco) as transfection vehicle when 75-90% confluent. The oligomer concentrations used were 5 nM and 25 nM (final concentration in well). Formulation of oligomer-lipid complexes was 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. After washing with Optimem, 300 μΐ of trypsin was added to each well until the cells detached from the wells. The trypsin was inactivated by adding 3ml HUH7 culture medium to the well and a single cell suspension was made by gently pipetting the cell suspension up and down. The scrambled oligomer SEQ ID NO: 235 was used as control.

[294] Following this, 100 μΐ of the cell suspension was added to each well of a white 96- well plate from Nunc (cat #136101) (four plates were prepared, for measurement at different time points). The plates were then incubated at 37°C, 95 % humidity and 5 % CO₂ until the assays were performed.

[295] Caspase assay; The activities of apoptosis-specific caspases 3 and 7 were measured using a luminogenic Caspase-Glo 3/7-substrate assay (Cat#G8091 from Promega). The plate to be analyzed was equilibrated to room temperature for 15 min. The Caspase-Glo ® 3/7 buffer was mixed with the Caspase-Glo ® 3/7 substrate to form a Caspase-Glo ® working solution which was equilibrated to room temperature. Then, 100 μΐ of the Caspase-Glo ® working solution was carefully added to the medium in each well of the 96-well plate (avoiding bubbles and contamination between wells). The plate was carefully shaken for 1 min, after which it was incubated at room temperature for lh, protected from light. The caspase activity was measured as Relative Light Units per second (RLU/s) in a Luminoscan Ascent instrument (Thermo Labsystems). Data were correlated and plotted relative to an average value of the mock samples, which was set to 1. See Figure 6.

6.10. Example 10: In vitro inhibition of proliferation using LNA

oligonucleotides

[296] HUH7 cells were transfected and harvested into a single cell suspension as described in Example 9. SEQ ID NO: 235 served as a scrambled control. Following harvesting, 100 μΐ of the cell suspension was added to each well of a 96-well plate ("Orange Scientific") for MTS assay (four plates were prepared, for measurement at different time points). The plates were then incubated at 37°C, 95 % humidity and 5 % CO₂ until the assays were performed.

Measurement of proliferating viable cells (MTS assay)

[297] For the proliferation assay, 10 μΐ CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, G3582) were added to the medium of each well of the 96-well plate, the plate was carefully shaken, and incubated at 37°C, 95 % humidity and 5 % CO₂ for lh before measurement. The absorbance was measured at 490 nm in a spectrophotometer and background for the assay was subtracted from wells containing only medium. The absorbance at 490 nm is proportional to the number of viable cells and was plotted over time for the mock transfected cells and for cells transfected with oligomers. See Figure 7.

6.11. Example 11: Evaluation of target mRNA knockdown in vivo

[298] To evaluate the knockdown efficacy of the HER3 oligomeric compounds in vivo, the female nude mice bearing 15PC3 xenografts developed by subcutaneous injection of 5 x 10⁶ cells/mouse into the right axillary flank, were injected intravenously with the oligomers at various doses and injection schedules (i.e. single dose, qd, q3d, q4d). Scrambled oligomer SEQ ID NO: 236 served as a negative control. 24 hours after the last injection, the mice were euthanized and liver and tumor tissues were collected in RNAlater solution (Ambion). Total RNA was purified from the tissues and the levels of HER3 mRNA were determined by quantitative reverse transcription-real time PCR (qRT-PCR) using the QuantiTect Probe RT- PCR kit (Cat#: 204443; Qiagen). GAPDH mRNA served as an internal control.

[299] Mouse HER3: probe: cca cac ctg gtc ata gcg gtg a, primer- 1 : ctg ttt agg cca age aga gg, primer-2: att ctg aat cct gcg tec ac.

Human HER3 : probe: cat tgc cca acc tec gcg tg, primer- 1 : tgc agt gga ttc gag aag tg, primer-2: ggc aaa ctt ccc ate gta ga.

Human GAPDH: probe: act ggc get gec aag get gt, primer- 1 : cca ccc aga aga ctg tgg at, primer-2: ttc age tea ggg atg acc tt.

Mouse GAPDH: probe: age tgt ggc gtg atg gec gt, primer- 1 : aac ttt ggc att gtg gaa gg, primer-2: gga tgc agg gat gat gtt ct

200 ng of total RNA was used in the PCR reaction. The data analyses were performed by using the AB 1-7500 PCR Fast System included software. See Table 5.

[300] Data in Table 5 are presented as % HER3 mRNA levels relative to saline treated controls in liver and tumor samples after i.v. dosing of animals on 5 consecutive days with oligonucleotides in the doses indicated.

Table 5

Inhibition of HER3 mRNA in mouse liver and tumor

Dosage HER3 mRNA

LNA ID (mg/kg, i.v., Liver (% of Sal

Tumor (%)

Ctrl)

qdx5)

76.3 78 ± 17 100 ± 10.5

SEQ ID 60 86.5; J 9.9 95.5 ± 12.7

NO: 236 30 87.6 ± 19 101.2 ± 21.1

22.9 81.4 ± 6.5 1 19.3 ± 24.9

Table 5

Inhibition of HER3 mRNA in mouse liver and tumor

Dosage HER3 mRNA

LNA ID (mg/kg, i.v., Liver (% of Sal

Tumor (%)

Ctrl)

qdx5)

85.3 1 ± 0.3 25.8 ± 4.1

66 6 ± 5.3 32.3 ± 9.7

SEQ ID

31.3 1.6 ± 0.3 37 ± 5.8

NO: 180

25.6 3 ± 0.3 65 ± 20.2

19.8 1.7 ± 0.6 83.1 ± 19.5

SEQ ID 37.7 20.7 ± 9.8 77 ± 10

NO: 169 1 1.3 10.2 ± 5.5 ND

SEQ ID 32.4 7.4 ± 5.2 78.1 ± 15.3

NO: 172 9.7 12.2 ± 5.9 ND

6.12. Example 12: Evaluation of Tumor growth inhibition

[301] The ability of the HER3 specific LNAs to inhibit tumor growth in vivo was evaluated in nude female mice bearing 15PC3 xenografts. 15PC3 human prostate tumor model was developed by subcutaneous ly injection of 5 x 10⁶ cells/mouse into the right axillary flank. The tumor volume was determined by measuring two dimensions with callipers and calculated using the formula: tumor volume = (length x width²)/2). When the tumors reached an average volume of 70-100 mm³, the mice bearing tumors were divided into treatment and control groups. The mice were injected intravenously with 25 and 50 mg/kg of SEQ ID NO: 180 respectively, with a q3d xlO schedule. Saline or scrambled oligonucleotide having SEQ ID NO: 236 served as a control. The body weights and tumor sizes of the mice were measured twice weekly. The toxicity was estimated by clinical observation, clinical chemistry and histopathological examination. Tumor HER3 mRNA was measured by QPCR as described in Example 1 1. See Figure 8A and 8B.

6.13. Example 13: Inhibition of HER3 mRNA in mouse liver

[302] NMRI mice were dosed i.v. with 1 or 5 mg/kg oligonucleotides on three consecutive days (group size of 5 mice). The antisense oligonucleotides (SEQ ID NO: 180 and SEQ ID NO: 234) were dissolved in 0.9% saline (NaCl). Animals were sacrificed 24h after last dosing and liver tissue was sampled and stored in RNA later (Ambion) until RNA extraction and QPCR analysis. Total RNA was extracted and HER3 mRNA expression in liver samples was measured by QPCR as described in Example 7 using a mouse HER3 QPCR assay (cat. no. MmOl 159999_ml, Applied Biosystems). Results were normalized to mouse GAPDH (cat. no. 4352339E, Applied Biosystems) and plotted relative to saline treated controls (see Figure 9)·

6.14. Example 14: Preparation of conjugates of oligomers with polyethylene glycol

[303] The oligomers having sequences shown as SEQ ID NO: 141 or SEQ ID NO: 152 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):

O II

O— — O— G_sC_sT_s¾¾a_sg_sa_sc_sa_st_sCsa_sC_sT_sC— OH

°^"(IA)

(IB)

wherein the bold uppercase letters represent nucleoside analogue monomers, lowercase letters represent DNA monomers, and the subscript "s" represents a phosphorothioate linkage.

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

(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.

[305] 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: 141 and 152, respectively as show in formulas (IIIA) and (IIIB):

wherein each of the oligomers of SEQ ID NOs: 141 and 152 is attached to a PEG polymer having average molecular weight of 12,000 via a releasable linker.

[306] Chemical structures of PEG polymer conjugates that can be made with oligomers having sequences shown in SEQ ID NOs: 169, 180 and 234 using the process described above are respectively shown in formulas (IV A), (IVB) and (TVC):

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.

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

(VB)

(VC)

6.15. Example 15: Evaluation of target mRNA knockdown in vivo with different dosing cycle

[308] The knockdown efficacy of oligomers was evaluated in vivo in nude mice bearing xenograft tumors derived from 15PC3 cells or A549 cells (NSCLC) or N87 cells (gastric carcinoma) using a similar protocol to the one described above in Example 1 1. Oligomers were administered by injection every third day in 2-4 doses. Tissues were harvested 3 or 4 days after the last injection.

[309] Data in Tables 6 and 7 are presented as % HER3 mRNA or HIF-1 alpha mRNA relative to saline treated controls in liver and tumor samples after i.v. dosing of animals with the indicated oligomers.

Table 6

Inhibition of ErbB3 mRNA in mouse liver and xenograft tumor derived from 15PC3 cell (3-5 mice/group) Treatment Dosage Tumor Liver (mg/kg)

HER3 (%) HiflA (%) HER3 (%)

Saline 0x4 100 ± 10 100 ±8 100 ± 18

SEQ ID No: 76.3 x 4 106 ±6.6 101 ± 13.8 115.9 ± 26.3

236

SEQ ID No: 37.7x4 81.6 ± 12.7 94.6 ± 19.6 39 ±4.6

169

SEQ ID No: 32.4x4 107.3 ± 17 100.3 ±7.5 44.3 ± 10.6

172

SEQ ID No: 60.2 x 2 or 3 47.1 ±2.2 101 ±7.3 6.9 ±3.6

180

60.2 x 4 54.2 ±9.1 ND 31.8 ± 5

[310] The observed knockdown effects of the oligomers having the sequences of SEQ ID NO: 169 and SEQ ID NO: 180 are not unique to 15PC3 tumor cells, since similar effects were observed in the tumors derived from A549 (NSCLC) and N87 (gastric carcinoma) cells. See Table 7, below.

A549 Saline 0 x 3 100 ± 20.9 100 ± 4.8

SEQ ID No: 35, q4d x 3 87.6 ± 1 1.9 97.5 ± 21.2

236

SEQ ID No: 35, q4d x 3 54.6 ± 15.2 31.8 ± 5.7

180

N87 Saline 0 x 5 100 ± 8.2 100 ± 9.4

SEQ ID No: 25, q3d x 5 99.0 ± 8.9 123 ± 4.5

249

SEQ ID No: 25, q3d x 5 46.6 ± 13.4 24.7 ± 3.1

180

6.16. Example 16: Generation of a gefitinib-resistant cell line

[311] HCC827 lung adenocarcinoma cells (ATCC CRL-2868) were maintained at 37°C in a humidified atmosphere of 5% CO₂ and 95% air in RPMI medium supplemented with 10% fetal bovine serum. To generate gefitinib resistance, cells were treated with increasing amounts of gefitinib (up to 500 nM) for a period of 3 months. At the end of the 3 month period, cell proliferation was tested comparing both the parental and HCC827R gefitinib resistant cells using an MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results show that HCC827R cells are resistant to gefitinib even at the highest concentration tested (10 μΜ). (Figure 10)

6.17. Example 17: Characterization of gefitinib-resistant cell line

[312] Expression levels and phosphorylation status of receptor tyrosine kinases ("RTK") in HCC827 and the gefitinib-resistant cells, HCC827R, were profiled using the RTK Antibody Array kit (R&D Systems, Inc., Minneapolis, MN). Briefly, cells were solubilized in the lysis buffer and total protein concentration in the cell lysates was determined. 500 ug of total protein was diluted in the array incubation buffer, incubated with the array membrane, and processed according to the protocol provided by the manufacturer. The final imaging result (Figure 11) shows that phosphorylated EGFR levels in the HCC827R cells were much lower than those of the parent. Western blot analysis

[313] HCC827 and gefitinib-resistant clones (R2, R3, and R5) were cultured in medium with ("+") or without ("-") 1 μΜ of gefitinib for 24 h. Cell lysates were then prepared and total protein concentration was determined. Approximately 15 μg/lane of protein were electrophoresed in 8% SDS-PAGE gels and transferred to PVDF using a BioRad liquid transfer apparatus. The western analysis was performed with the appropriate horseradish peroxidase-conjugated secondary antibodies (Transduction Labs) and enhanced chemiluminescence reagents (SuperSignal, Pierce). The primary antibodies (Abs) used include: anti-Met monoclonal Ab (25H2), anti-phosphor-Met(Y1234) rabbit monoclonal Ab (D26), and anti-phosphor-ErbB3(Y1289) rabbit monoclonal Ab (21D3), from Cell Signaling; anti-ErbB3 Ab (sc285) from Santa Crutz; anti-phosphor-Met(Y1349) Ab (Ab47606R), anti- phosphor-EGFR rabbit monoclonal Ab (Ab40815), and anti-EGFR Ab, from Abeam; and a horseradish peroxidase-conjugated anti-tubulin Ab for loading control.

[314] Data show that levels of unphosphorylated and phosphorylated EGFR are significantly reduced in HCC827 gefitinib-resistant clones, either in the presence ("+") or absence ("-") of gefitinib, as compared to the levels of unphosphorylated and phosphorylated EGFR in untreated ("-") parent cells. In contrast, the levels of ErbB3 or MET, which are also involved in the EGFR signaling pathway, are not significantly decreased in the resistant clones compared to the parent cells. These findings indicate that down-regulation of EGFR may be a mechanism by which some cancer cells acquire resistance to gefitinib.

6.18. Example 18: Effect of oligomer on gefitinib-resistant cells

[315] HCC287 and HCC287R cells were plated in duplicate at 200 cells/well of a 6-well plate and incubated for 24 hours. Cells were treated with 1 μΜ of ON 180 (SEQ ID NO: 180) and incubated for 10 days, after which cells were stained with MTT and the number of colonies counted. Percent of control was calculated for both HCC827 and HCC727R cells. Results shown in Figure 13 indicate that oligonucleotide ON 180 is significantly more effective in down-regulating gefitinib-resistant cells (greater than 80% reduction in cell growth as compared to the untreated control) than in down-regulating growth of HCC287 gefitinib-sensitive cells (about 50% reduction in cell growth as compared to the untreated control).

[316] Still further aspects and embodiments of the invention are illustrated with respect to FIGS. 14-16.. [317] Figure 14 shows that HER3 expression-reducing LNA oligomer, but not trastuzumab, is able to prevent feedback upregulation of HER3 and P-HER3 expression by lapatinib in three human breast cancer cell lines, BT474, SKBR3 and MDA453. The expression level of HER3, P-HER3 (Y1197) and P-HER3 (Y1289) is shown at 0, 1, 4, 24 and 48 hours as indicated for lapitinib-only treated cells (1), lapatinib plus trastuzumab-treated cells (2), lapitinib plus SEQ ID NO: 180-treated cells (3) and SEQ ID NO: 180-only treated cells (4). Lapatinib was used at a concentration of 1 μΜ, trastuzumab at a concentration of 10 μg/ml, and SEQ ID NO: 180 at a concentration of 5 μΜ.

[318] Figure 15 shows that synergistic promotion of apoptosis in three human breast cancer cell lines is greater for a combination of lapatinib and a HER3 expression-reducing LNA oligomer than for a combination of lapatinib and trastuzumab. The figure shows the results of an ApoBrdU apoptosis assay performed for each of the three cells lines (same lines as in Figure 14). Cells were treated at 48 hours with lapatinib and/or trastuzumab. At 72 hours, the cells were serum starved and treated with SEQ ID NO: 180 or a randomized control oligomer. For each of the cell lines, treatments were randomized oligonucleotide control-only (1), SEQ ID NO: 180-only (2), trastuzumab-only (3), lapatinib-only (4), lapatinib plus SEQ ID NO: 180 (5), and lapatinib plus trastuzumab (6). Lapatinib was used at a concentration of 1 μΜ, trastuzumab at a concentration of 10 μg/ml, and SEQ ID NO: 180 at a concentration of 5 μΜ.

[319] Figure 16 shows that SEQ ID NO: 180 inhibits tumor growth in an in vivo mouse xenograft model of the human non-small cell lung cancer using the HCC827 human cell line. Mean tumor volume was reduced 65.5% vs. saline control for treatment with 30 mg/kg SEQ ID NO: 180 i.v. (intravenous) at approximately 31 days and was reduced 81.3% vs. saline control for treatment with 45 mg/kg SEQ ID NO: 180 i.v. at approximately 31 days. N=6.

6.19. Example 19: Effect of combined PIK3CA and HER3 inhibition in

gefitinib-resistant cell lines

[320] Rl, 2, 3, 4, and R5 are gefitinib-resistant subclones of the lung cancer cell line

HCC827 (ATCC CRL-2868) that had been chronically adapted to grow in culture medium containing increasing concentrations of gefitinib, up to 250 nM. To determine the effect of gefitinib on the growth of cells, parent HCC827 and the resistant cells were cultured in medium containing varying concentrations of gefitinib for 6 days, after which viable cell populations were determined by MTT assay. Shown in FIG. 17, HCC827 was sensitive to gefitinib as indicated by a concentration-dependent reduction in viable cells. In contrast, growth of R1-R5 cells was not affected by gefitinib up to 10 uM, the highest concentration tested. These cells had been propagated in medium without gefitinib for over 30 passages and retain the drug-resistance property.

Although the R1-R5 are resistant to gefitinib, they are equally or somehow more sensitive to SEQ ID NO: 180 (EZN3920) mediated growth inhibition as their parent HCC827, in cell proliferation assays. The gefitinib-resistant clones and parent HCC827 were treated with varying concentrations of SEQ ID NO: 180 or a scrambled control oligonucleotide SEQ ID NO: 265 (C_sG_sC_sA_sg_sa_st_st_sa_sg_saga_sA_sC_sC_st; EZN-3046) for 6 days. Cell proliferation was determined by MTT assay. Shown in FIG. 18 are representative data of multiple independent assays. Rl, R3, R4, and R5 were consistently more sensitive than the parent HCC827 to SEQ ID NO: 180 (EZN3920), but not to the control LNA compound, SEQ ID NO: 265 (EZN- 3046).

[321] Based on protein expression profiling, distinct characteristics were revealed between the resistant clones and HCC827. For example, SEQ ID NO: 180 (EZN3920) treatment resulted in down-modulation of pAkt level in HCC827, indicating the compound blocked PI3K/Akt signal pathway. But in SEQ ID NO: 180 treated R3 cells, although confirmed reduced level of ErbB3 expression, Akt was persistently phosphorylated (FIG. 19), suggesting an active PI3K/Akt pathway controlled by an ErbB3-independent mechanism. This suggested that direct down-regulation of both ErbB3 and PIK3CA could be an effective mean of control proliferation of gefitinib-resistant cells, such as R3.

[322] The effect of combinational treatment with SEQ ID NO: 180 (EZN3920) and SEQ ID NO: 254 (EZN4150) in R3 and HCC827 cells was evaluated. The gefitinib-resistant clones and parent HCC827 were treated with varying concentrations of SEQ ID NO: 180 at 0-4 uM or SEQ ID NO: 254 (5'-A_sG_sC_sc_{s s}t_sc_sa_st_st_sc_sc_sA_sC_sC-3 '; EZN4150) at 0-20 uM alone, or in combination (a ratio of SEQ ID NO: 180 to SEQ ID NO: 254 of 1 :5), for 6 days. Cell proliferation was determined by MTT assay. Shown in FIG. 20, R3 cells were 3-4-fold more sensitive to SEQ ID NO: 180, or SEQ ID NO: 254 than the parent HCC827 cells. In addition, R3 cells appeared more sensitive to the combined treatment of SEQ ID NO: 180 and SEQ ID NO: 254 than to each agent alone, while no such enhanced effect was seen in HCC827 cells.

[323] To determine whether the combined inhibitory effect of SEQ ID NO: 180 and SEQ ID NO: 254 in R3 cells is additive or synergistic, a formal quantitative analysis of the data was used to calculate the combination index (CI) based on a multiple drug-effect equation (Ref: Reynolds CP, Maurer BJ. Evaluating response to antineoplastic drug combinations in tissue culture methods. Mol Med. 2005; 110: 173-183). Based on the dose-titration curves (FIG. 20, R3 cells), the overall CI values (FIG. 20, CI plot) were determined between 0.5 and 0.8, indicating a synergistic effect by SEQ ID NO: 180 and SEQ ID NO: 254 combination in R3 cells. These results suggest down-regulation of HER3/PIK3CA signaling pathway using anti- sense approach as an effective therapeutic approach to gefitinib-resistant lung cancers.

[324] FIGS. 21A-C show that EZN-3920 greatly potentiates the effect of lapatinib in BT474M1 breast cancer cells. BT474M1 cells were cultured in 12-well plates and treated with EZN-3920 (SEQ ID NO: 180) or a scrambled control oligo ("EZN-SCR") for 72 hrs. On day 4, lapatinib was added to the cell culture. After 24 hr treatment, cells were harvested to determine the level of HER3.

[325] FIG. 21 A shows EZN-3920 (SEQ ID NO: 180) inhibited lapatinib-induced HER3 mRNA in BT474M1 breast cancer cells. * p<0.05 vs lapatinib group.

[326] FIG 2 IB shows EZN-3920 (SEQ ID NO: 180) inhibited lapatinib-induced HER3 protein expression in BT474M1 breast cancer cells. * p<0.05 vs lapatinib group.

[327] FIG. 21C shows EZN-3920 (SEQ ID NO: 180) potentiates the effect of lapatinib on the growth of BT474M1 breast cancer cells. Cells were plated in 96-well plates and treated with EZN-3920 or EZN-SCR for 72 hrs prior to lapatinib treatment. Cell growth was determined after additional 72 hrs using MTS assay. * p<0.05 vs lapatinib group.

[328] FIG. 22 shows the effect of a combination of EZN-3920 (SEQ ID NO: 180) with lapatinib on mean tumor volume over time in a BT474M1 breast cancer xenograft model, versus control treatments. Mice were administered 30 mg kg EZN-3920 (q3.5dx9, i.v.) (n = 8 per group) and 100 mg/kg lapatinib (qdxl5, p.o.). * p<0.05 vs saline group. ** p<0.05 combo group ("E+L") vs EZN-3920 group.

[329] FIG. 23 shows the effect of a combination of EZN-3920 (SEQ ID NO: 180) with gefitinib on mean tumor volume over time in a BT474M1 breast cancer xenograft model, versus control treatments. Mice were administered 30 mg/kg EZN-3920 (q3.5dxl0, i.v.) (n = 8 per group) and 15 mg/kg gefitinib (qdxlO, p.o.). * p<0.05 vs saline group. ** p<0.05 combo group ("E+G") vs gefitinib group.

[330] FIG. 24 shows the effects of a combination of EZN-3920 (SEQ ID NO: 180) with EZN-4150 (SEQ ID NO: 254; an LNA gapmer antisense antagonist of PIK3CA) on mean tumor volume over time in BT474M1 breast cancer xenograft model, versus control treatments. Mice were administered 30 mg/kg EZN-3920 (q3.5dx8, i.v.) (n = 8 per group) and 100 mg/kg lapatinib (qdxlO, i.p.). * p<0.05 vs saline group. ** p<0.05 combo group vs EZN-3920, or EZN-4150 group.

SPECIFIC EMBODIMENTS, CITATION OF REFERENCES

[331] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications within the scope of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying figures. Moreover, features described in connection with one embodiment of the invention may be used in conjunction with other embodiments, even if not explicitly stated above.

[332] While the invention has been described with respect to its application to various cancers and related embodiments, the invention also provides corresponding embodiments including medical methods, uses in treatment, uses in the manufacture of medicaments, pharmaceutical compositions and pharmaceutical kits for the prevention of cancers, for the treatment of precancerous conditions and for the treatment of hyper-proliferative conditions including cancerous and non-cancerous hyper-proliferative conditions, in mammals such as humans in need thereof.

[333] Various references, including patent applications, patents, and scientific publications, are cited herein; the disclosure of each such reference is hereby incorporated herein by reference in its entirety.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a cancer in a mammal, comprising:

administering a protein tyrosine kinase inhibitor to the mammal; administering to the mammal at least one antisense oligomer or conjugate thereof that reduces the expression of HER3; and administering to the mammal at least one antisense oligomer or conjugate thereof that reduces the expression of PIK3CA, wherein the inhibitory activity of the protein tyrosine kinase inhibitor and the reduction of expression of HER3 and PIK3CA are temporally overlapping.

2. The method of claim 1, wherein the cancer is breast cancer.

3. The method of claim 1, wherein the cancer is at least partially resistant to treatment with the protein tyrosine kinase inhibitor and said resistance is at least partially reversed by administering the at least one antisense oligomer or conjugate thereof that reduces the expression of HER3.

4. The method of claim 3, wherein the cancer is breast cancer.

5. The method of any one of the preceding claims, wherein the protein tyrosine kinase inhibitor is selected from the group consisting of gefitinib, imatinib, erlotinib, lapatinib, canertinib and sorafenib.

6. The method of any one of the preceding claims, wherein the at least one antisense oligomer or conjugate thereof that reduces the expression of HER3 comprises

5'-T_sA_sG_sc_sc_st_sgst_sc_sa_sc_st_st_s ^MeC_sT_s ^MeC -3' (SEQ ID NO: 180), 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, or a conjugate thereof.

7. The method of any one of the preceding claims, wherein the at least one antisense oligomer or conjugate thereof that reduces expression of PIK3CA comprises an oligomer selected from the group consisting of:

5' A_sG_s ^MeC_sc_sa_st_st_sc_sa_st_st_sc_scA^MeC_s ^MeC-3' (SEQ ID NO: 254);

5'- T_sT_sA_st_st_sg_st_sg_sc_sa_st_sc_st_s ^MeC_sA_sG -3' (SEQ ID NO: 257), 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, or a conjugate thereof.

8. Combination use of at least one antisense oligomer or a conjugate thereof that reduces the expression of HER3 and at least one antisense oligomer or a conjugate thereof that reduces the expression of PIK3CA and, optionally, a protein tyrosine kinase inhibitor and/or a HER2 inhibitor, in the treatment of a hyperproliferative disorder such as cancer in a mammal, preferably a protein tyrosine kinase inhibitor-resistant cancer in a mammal or a cancer susceptible to developing resistance to a protein tyrosine kinase inhibitor in a mammal or a cancer partially resistant to a protein tyrosine kinase inhibitor in a mammal.

9. Use of at least one antisense oligomer or a conjugate thereof that reduces the expression of HER3 and at least one antisense oligomer or a conjugate thereof that reduces the expression of PIK3CA and, optionally, a protein tyrosine kinase inhibitor and/or a HER2 inhibitor, in the preparation of a medicament for the treatment of hyperproliferative disorder such as cancer in a mammal, preferably a protein tyrosine kinase inhibitor-resistant cancer in a mammal or a cancer susceptible to developing resistance to a protein tyrosine kinase inhibitor in a mammal or a cancer partially resistant to a protein tyrosine kinase inhibitor, in a mammal.

10. The use of claim 8 or 9, wherein the at least one antisense oligomer or conjugate thereof that reduces the expression of HER3 comprises a therapeutically effective amount of

5'-T_sA_sG_sc_sc_st_sgst_sc_sa_sc_st_st_s ^MeCJ_s ^MeC -3' (SEQ ID NO: 180), 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, or a conjugate thereof.

11. The use of any one of claims 8-10, wherein the at least one antisense oligomer or conjugate thereof that reduces expression of PIK3CA comprises an oligomer selected from the group consisting of:

5' A_sG_s ^MeC_sc_sa_st_st_sc_sa_st_st_sc_scA^MeC_s ^MeC-3' (SEQ ID NO: 254);

5'- T_sT_sA_st_st_sg_st_sg_sc_sa_st_sc_st_s ^MeC_sA_sG -3' (SEQ ID NO: 257), 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, or a conjugate thereof.

12. A method for treating a cancer in a mammal, comprising: administering a HER2 inhibitor to the mammal;

administering to the mammal at least one antisense oligomer or conjugate thereof that reduces the expression of HER3; and administering to the mammal at least one antisense oligomer or conjugate thereof that reduces the expression of PIK3CA,

wherein the inhibitory activity of the HER2 inhibitor and the reduction of expression of HE 3 and PIK3CA are temporally overlapping.

13. The method of claim 12, wherein the cancer is breast cancer.

14. The method of claim 12, wherein the cancer is at least partially resistant to treatment with the HER2 inhibitor and said resistance is at least partially reversed by administering the at least one antisense oligomer or conjugate thereof that reduces the expression of HER3.

15. The method of claim 14, wherein the cancer is breast cancer.

16. The method of any one of claims 12-15, wherein the HER2 inhibitor is selected from the group consisting of trastuzumab and pertuzumab.

17. The method of any one of claims 12-16, wherein the at least one HER3 antisense oligomer or conjugate thereof comprises:

5'-T_sA_sG_sc_sc_st_sgst_sc_sa_sc_st_st_s ^MeC_sT_s ^MeC -3' (SEQ ID NO: 180), 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, or a conjugate thereof.

18. The method of any one of claims 12-17, wherein the at least one antisense oligomer or conjugate thereof that reduces expression of PIK3CA comprises an oligomer selected from the group consisting of:

5' AsGs^CsCsasWsCsaststsCscA^Cs^C-S' (SEQ ID NO: 254); and

5'- T_sT_sA_st_st_sg_st_sg_sc_sa_st_sc_st_s ^MeC_sA_sG -3' (SEQ ID NO: 257),

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, or a conjugate thereof.

19. Combination use of a HER2 inhibitor; at least one antisense oligomer or conjugate thereof that reduces the expression of HER3; and at least one antisense oligomer or conjugate thereof that reduces the expression of PIK3CA,

in the treatment of cancer in a mammal.

20. The use of claim 19, wherein the cancer is resistant to treatment with the HER2 inhibitor or is susceptible to developing resistance to the HER2 inhibitor.

21. Any one of the preceding claims, wherein the cancer is selected from the group consisting of non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, colon carcinoma, rectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, cervical cancer, testicular cancer, non-small cell lung cancer, lung carcinoma, bladder carcinoma, melanoma, head and neck cancer, brain cancer, cancers of unknown primary site, neoplasms, cancers of the peripheral nervous system, cancers of the central nervous system, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, seminoma, embryonal carcinoma, Wilms' tumor, small cell lung carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma.

22. A pharmaceutical composition comprising: at least one antisense oligomer or conjugate thereof that reduces the expression of HER3; and

at least one antisense oligomer or conjugate thereof that reduces the expression of PIK3CA.

23. The pharmaceutical composition of claim 22, further comprising a pharmaceutically acceptable excipient.

24. A kit comprising: at least one antisense oligomer or conjugate thereof that reduces the expression of HER3; and

at least one antisense oligomer or conjugate thereof that reduces the expression of PIK3CA.

25. The kit of claim 24, further comprising a protein tyrosine kinase inhibitor.

26. The kit of claim 25, wherein the protein tyrosine kinase inhibitor is selected from the group consisting of gefitinib, imatinib, erlotinib, lapatinib, canertinib and sorafenib.

27. The kit of claim 24, further comprising: a HER2 inhibitor.

28. The kit of claim 27, wherein the HER2 inhibitor is selected from the group consisting of trastuzumab and pertuzumab.

29. The pharmaceutical composition according to claim 22 or 23, wherein said pharmaceutical composition further comprises a protein tyrosine kinase inhibitor and/or a HER2 inhibitor.

30. A composition comprising at least one antisense oligomer or a conjugate thereof that reduces the expression of HER3 and at least one antisense oligomer or a conjugate thereof that reduces the expression of PIK3CA for use in the treatment of a hyperproliferative disease in a mammal.

31. The composition according to claim 30 wherein said hyperproliferative disease is cancer.

32. The composition according to claim 31 wherein said cancer is a protein tyrosine kinase inhibitor-resistant cancer.

33. The composition according to claim 31 wherein said cancer is a cancer susceptible to developing resistance to a protein tyrosine kinase inhibitor.

34. The composition according to claim 31 wherein said cancer is partially resistant to a protein tyrosine kinase inhibitor.

35. The composition according to any one of claims 30 to 34, wherein the cancer is selected from the group consisting of non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, colon carcinoma, rectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, cervical cancer, testicular cancer, non-small cell lung cancer, lung carcinoma, bladder carcinoma, melanoma, head and neck cancer, brain cancer, cancers of unknown primary site, neoplasms, cancers of the peripheral nervous system, cancers of the central nervous system, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, seminoma, embryonal carcinoma, Wilms' tumor, small cell lung carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma.

36. The composition of any one of claims 30 to 35, wherein the at least one antisense oligomer or conjugate thereof comprises a therapeutically effective amount of

5'-T_sAsG_sc_sc_st_sgst_sc_sa_sc_st_st_s ^MeC_sT_s ^MeC -3 ' (SEQ ID NO: 180), 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, or a conjugate thereof.

37. The composition of any one of claims 30 to 36, wherein the at least one antisense oligomer or conjugate thereof that reduces expression of PIK3CA comprises an oligomer selected from the group consisting of:

5' A_sG_s ^MeC_sc_{s s}t_sc_sa_st_st_sc_sc_sA_s ^MeC_s ^MeC-3' (SEQ ID NO: 254);

5'- T_sT_sA_st_st_sgst_sgsc_sa_st_sc_st_s ^MeC_sA_sG -3' (SEQ ID NO: 257), 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, or a conjugate thereof.

38. The composition according to any one of claims 30 to 37, wherein said composition further comprises a protein tyrosine kinase inhibitor and/or a HER2 inhibitor.

39. The composition according to any one of claims 30 to 38, wherein said composition further comprises a pharmaceutical acceptable diluent, carrier, salt or adjuvant.