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Número de publicaciónWO2011107100 A1
Tipo de publicaciónSolicitud
Número de solicitudPCT/DK2011/050062
Fecha de publicación9 Sep 2011
Fecha de presentación2 Mar 2011
Fecha de prioridad3 Mar 2010
Número de publicaciónPCT/2011/50062, PCT/DK/11/050062, PCT/DK/11/50062, PCT/DK/2011/050062, PCT/DK/2011/50062, PCT/DK11/050062, PCT/DK11/50062, PCT/DK11050062, PCT/DK1150062, PCT/DK2011/050062, PCT/DK2011/50062, PCT/DK2011050062, PCT/DK201150062, WO 2011/107100 A1, WO 2011107100 A1, WO 2011107100A1, WO-A1-2011107100, WO2011/107100A1, WO2011107100 A1, WO2011107100A1
InventoresMidtjylland Region, Suzette SØRENSEN, Trine ØSTERGAARD, Boe S. SØRENSEN, Jørgen Kjems
SolicitanteAarhus Universitet
Exportar citaBiBTeX, EndNote, RefMan
Enlaces externos:  Patentscope, Espacenet
Methods and compositions for regulation of herv4
WO 2011107100 A1
Resumen
Compositions are provided comprising a binding member for the specific downregulation of the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 isoform polypeptide and/or upregulation of the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 isoform polypeptide. Methods for use of such compositions are also provided, as well as the use thereof as a medicament, for example for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. Also splice-switching oligonucleotides targeting HER4 pre-m RNA are provided, which are capable of modulating the alternative splicing of HER4, and thereby favor the production of HER4 CYT-2 polypeptide over HER4 CYT-1 polypeptide. Moreover, a diagnostic method and a method for screening candidate drugs for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder are provided.
Reclamaciones  (El texto procesado por OCR puede contener errores)
Claims
A composition comprising at least one binding member capable of binding to a HER4 polypeptide and/or a HER4 RNA, thereby
a. specifically downregulating the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 polypeptide and/or
b. specifically upregulating the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 polypeptide,
for use as a medicament.
The composition according to claim 1 , where said binding member specifically binds
a. HER4 CYT-1 polypeptide or part thereof, and not HER4 CYT-2 polypeptide, and/or
b. HER4 RNA in a region comprising exon 26 and/or in any region affecting the splicing of exon 26.
The composition according to any of the preceding claims, wherein at least one of said binding members is at least one splice-switching oligonucleotide.
The composition according to claim 3, wherein said at least one splice- switching oligonucleotide specifically binds an RNA species encoding a HER4 polypeptide.
The composition according to claim 4, wherein said at least one splice- switching oligonucleotide is capable of modulating the splicing of HER4 exon 26.
The composition according to claim 5, wherein said at least one splice- switching oligonucleotide is capable of promoting splicing of HER4 exon 26, thereby favouring the production of HER4 CYT-2 polypeptide over HER4 CYT-1 polypeptide.
The composition according to any of claims 3 to 6, wherein said at least one splice-switching oligonucleotide comprises a sequence selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 -3, or the complement thereof.
8. The composition according to any of claims 3 to 7, wherein at least one said splice-switching oligonucleotide comprises or consists of 5'- TUACUCCUGACAUGG-3' and/or 5'-TUTCACUCTAATAGG-3'.
9. The composition according to any of claims 3 to 8, wherein said at least one splice-switching oligonucleotide comprises one or more modified nucleotides.
10. The composition according to any one of claims 1 and 2, wherein at least one of said binding members is an siRNA and/or a microRNA
1 1 . The composition according to claim 10, wherein at least one of said binding members is an siRNA comprising a sequence selected from any region of a HER4 encoding nucleic acid sequence.
12. The composition according to claim 1 1 , wherein said at least one siRNA
comprises a sequence selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 -3, or the complement thereof.
13. The composition according to any one of claims 1 and 2, wherein said at least one binding member is selected from the group consisting of oligonucleotides, nucleic acid aptamers, antibodies, antigen binding fragments, polypeptides, peptides, peptide fragments, peptide aptamers, nucleic acid aptamers, small molecules, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immonoglobulin binding members.
14. The composition according to claim 13, wherein said binding member is an antibody and/or an antigen binding fragment thereof.
15. The composition according to claim 14, wherein said antibody and/or an antigen binding fragment thereof is specific for HER4 CYT-1 , said antibody and/or an antigen binding fragment thereof being capable of downregulating the level or activity of HER4 CYT-1 polypeptide.
16. The composition according to any of claims 14 to 15, wherein said antibody and/or an antigen binding fragment thereof is specific for an epitope selected from SEQ ID NO: 4, such as an epitope comprising or consisting of 3-7 consecutive amino acids selected from the group consisting of 1030-1080, 1046-1061 (exon 26), 1000-1030, 1031 -1040, 1035-1046, 1040-1050, 1045- 1055, 1050-1061 , 1055-1065, 1058-1065, 1058-1070 and 1065-1080 of SEQ ID NO: 4.
17. The composition according to any of claims 14 to 16, comprising a mixture at least two antibodies and/or antigen binding fragments.
18. The composition according to according to any of claims, said composition further comprising
a. HER4 CYT-2 polypeptide or a functional homolog or fragment thereof, and/or
b. nucleic acid encoding HER4 CYT-2 polypeptide or a functional homolog or fragment thereof.
19. The composition according to any of the preceding claims, said composition comprising a mixture of at least two binding members.
20. A splice-switching oligonucleotide comprising or consisting of 5-100
nucleotides, which specifically binds an RNA species encoded by a HER4 gene and/or encoding a HER4 polypeptide.
21 . The splice-switching oligonucleotide of claim 20, said splice-switching
oligonucleotide comprising a sequence selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 -3, or the complement thereof.
22. The splice-switching oligonucleotide of claim 21 , comprising a nucleic acid sequence selected from SEQ ID NO: 1 or the complement thereof.
23. The splice-switching oligonucleotide of claim 21 , said splice-switching
oligonucleotide comprising a nucleic acid sequence selected from any region of the complementary sequence to SEQ ID NO: 1 .
24. The splice-switching oligonucleotide of claim 21 , said splice-switching
oligonucleotide comprising a nucleic acid sequence consisting of 15-25 consecutive nucleic acids selected from any region of the nucleic acid sequences selected from the group consisting of the complementary sequence of nucleotides 1 -33496, 32645-32720, 32645-32675, 32645-32668, 32660- 32680, 32670-32700, 32680-33710 and 32690-33720 of SEQ ID NO: 1 .
25. The splice-switching oligonucleotide any one of claims 20 to 24, wherein said splice-switching oligonucleotide comprises or consists of 5'- TUACUCCUGACAUGG-3' and/or 5'-TUTCACUCTAATAGG-3'.
26. The splice-switching oligonucleotide of any of claims 20 to 25, wherein said splice-switching oligonucleotide is capable of modulating the splicing of HER4 exon 26.
27. The splice-switching oligonucleotide of claim 26, wherein said at least one
splice-switching oligonucleotide is capable of promoting splicing of HER4 exon 26, thereby favouring the production of HER4 CYT-2 polypeptide over HER4 CYT-1 polypeptide.
28. A composition as defined in any of claims 1 to 18 and/or a splice-switching oligonucleotides as defined in any of claims 20 to 27 for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.
29. The composition according to claim 28, wherein said cancer is growth factor dependent.
30. The composition according to claim 28, wherein cells derived from said cancer expresses the HER4 CYT-1 polypeptide or a functional homolog or fragment thereof and/or RNA encoding said HER4 CYT-1 polypeptide or functional homolog or fragment thereof.
31 . The composition according to any one of claims 28 to 30, wherein said cancer is selected from the group consisting of breast carcinoma, bladder carcinoma, anaplastic large cell lymphoma, B-cell lymphoma, chronic lymphoblastic leukemia, multiple myeloma, testicular tumor, astrocytoma, acute myelogenous leukemia, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, esophageal squamous cell carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma and urothelial carcinoma.
32. The composition according to claim 31 , wherein said cancer is wherein said cancer is breast carcinoma or bladder carcinoma.
33. The composition according to any one of claims 28 to 32, wherein said
treatment is combined with another treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or is combined with another medicament for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.
34. The composition according to any one of claims 28 to 33, wherein medicaments of the combined treatment are provided for separate, successive or
simultaneous administration.
35. The composition according to any one of claims 28 to 34, wherein said
treatment is prophylactic treatment.
36. A method for
assisting in diagnosing cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or
diagnosing cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or
determining cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or
determining a predisposition for cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder,
in an individual, said method comprising the steps of
a. determining the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide in a sample from said individual, and/or b. determining in said sample the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide, and
c. comparing the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide with the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide.
37. The method according to claim 36, wherein the presence of HER4 CYT-1
polypeptide and/or RNA encoding HER4 CYT-1 polypeptide is indicative of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or predisposition of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or wherein the presence of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is indicative of a reduced risk of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or a reduced predisposition for cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.
38. The method according to claim 37, wherein a higher level of HER4 CYT-1
polypeptide and/or RNA encoding HER4 CYT-1 polypeptide relative to HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is indicative of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or predisposition of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.
39. The method according to claim 36, wherein the level of HER4 CYT-1
polypeptide and/or RNA encoding HER4 CYT-1 polypeptide and/or the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is determined by using a binding member as defined in any of the preceding claims 1 to 19.
40. The method according to any of the preceding claims 36 to 39, wherein said cancer is as defined in any of claims 29 to 32.
41 . The method according to any of the preceding claims 36 to 40, wherein said sample is derived from tumor tissue, and/or comprises a tumour cell and/or a neuronal cell.
42. A method for identifying a compound for the treatment and/or diagnosis of
cancer or neuropsyciatric disorders, such as schizophrenia or bipolar disorder comprising
a. providing a sample
b. bringing at least one compound into contact with said sample, and c. determining the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide in said sample in the presence and absence of said at least one compound
d. comparing the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide in said sample in the presence and absence of said at least one compound.
43. The method according to claim 42, wherein a reduced level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide in the presence of said at least one compound compared with the absence is indicative of a compound for the treatment and/or diagnosis of cancer or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.
44. The method according to claim 43, further comprising the step of determining in said sample the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide in the presence and absence of said at least one compound.
45. The method according to claim 43, wherein an increased level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide in the presence of said at least one compound compared with the absence is indicative of a compound for the treatment and/or diagnosis of cancer or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.
46. The method according to any one of claims 44 and 45, said method further comprising the step of comparing the level of HER4 CYT-1 polypeptide and/or
RNA encoding HER4 CYT-1 polypeptide with the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide.
47. The method according to claim 46, wherein a higher level of HER4 CYT-1
polypeptide and/or RNA encoding HER4 CYT-1 polypeptide relative to HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is indicative of a compound for the treatment and/or diagnosis of cancer or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.
48. The method according to any one of claims 42 to 47, wherein the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide and/or the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is determined by using a binding member as defined in any of the preceding claims 1 to 19.
49. The method according to any of the preceding claims 42 to 48, wherein said cancer is as defined in any of claims 29 to 32.
50. The method according to any of the preceding claims 42 to 49, wherein said sample is derived from tumor tissue, and/or or consists of one or more cancer cells, tumor cells and/or one or more neuronal cells.
51 . The method according to claim 50, wherein said sample comprises or consists of the human lung cancer cell line NCI358 and/or the human breast cancer cell line MDA-MB-231 .
52. A compound identified by a method as defined in any one of claims 42 to 50 for use as a medicament.
53. The use according to claim 52 for the treatment of cancer and/or
neuropsyciatric disorders, such as schizophrenia or bipolar disorder.
54. The use according to claim 53, wherein said cancer is as defined in any of claims 29 to 32.
Descripción  (El texto procesado por OCR puede contener errores)

Methods and compositions for regulation of HER4 Field of invention

The present invention relates to compositions and methods relating to HER4.

Compositions are provided comprising binding member for the specific downregulation of the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 isoform polypeptide and/or upregulation of the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 isoform polypeptide, for use as a medicament, for example for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. Also splice-switching oligonucleotides targeting

HER4 pre-mRNA are provided, which are capable of modulating the alternative splicing of HER4, and thereby favor the production of HER4 CYT-2 polypeptide over HER4 CYT-1 polypeptide. Moreover, a diagnostic method and a method for screening candidate drugs for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder are provided.

Background of invention

Human epidermal growth factor (EGF) receptor 4 (HER4) belongs to the EGF family of receptors and ligands, which is known to be involved in development and growth of many types of cancers. HER4, however, is controversial since overexpression of this receptor in some cases can inhibit cell growth and be beneficial for the survival of cancer patients. HER4 has been implicated in cancer and schizophrenia and bipolar disorders; cf. WO009103072 and WO2002 18444.

Because of the high frequency of which the EGF system is involved in cancer, therapies have been made to specifically target this system. Especially, the two receptors HER1 and HER2 have been used as specific targets because of the general agreement that these two receptors are indicators of poor patient outcome (9;10). Also HER4 has been suggested as a possible target (1 1 ). However, since HER4 can be found not only to indicate poor patient outcome, but in some cases actually indicate a better patient survival, a total inhibition of this receptor is not desired.

HER4 is alternatively spliced into the two isoforms CYT-1 and CYT-2. Skipping of exon 26 results in a receptor lacking 16 amino acids (CYT-2) compared to the full-length receptor (CYT-1 ). The skipped exon encodes a protein fragment that comprises a binding site for, among others, PI3K (8). Although HER4 has been implicated in different cancer forms, the CYT-1 isoform has not previously been reported to be specifically associated with cancer or other disorders. In contrast, a paper from Shealton Earps Ill's laboratory reports rapid cellular proliferation of mammary epithelial cells, which express the CYT-2 isoform, and growth inhibition of cells expressing CYT-1 ; cf. Muraoka-Cook et al., 2009.

Summary of invention

The present invention relates to HER4 gene, and provides compounds and

compositions relating to HER4 for medical use. The invention specifically relates to compounds and compositions, as well as methods for the use thereof, which distinguish between the CYT-1 and CYT-2 isoforms of HER4.

According to the present invention, the reason for the dual function of HER4 is the presence of the two splicing variants CYT-1 and CYT-2 and it will therefore be desirable to target only the CYT-1 isoform that causes the poor patient outcome while maintaining or increasing the expression and functionality of CYT-2. A switch in alternative splicing of mRNA is often associated with carcinogenesis, and the present invention provides means and methods for restoring normal splicing patterns in cells, where splicing have switched in favour of generating the CYT-1 isoform of HER4.

In one aspect, the present invention relates to a composition comprising at least one binding member capable of binding to a HER4 polypeptide and/or a HER4 RNA, thereby

a. specifically downregulating the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 isoform polypeptide and/or

b. upregulating the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 isoform polypeptide,

for use as a medicament.

The binding member preferably binds specifically: a. HER4 CYT-1 polypeptide or part thereof, and not HER4 CYT-2 polypeptide, and/or b. HER4 RNA in a region comprising exon 26 and/or in any region affecting the splicing of exon 26. In a preferred

embodiment, the at least one binding member is a splice-switching oligonucleotide, which, preferably, specifically binds an RNA species encoding a HER4 polypeptide. The at least one splice-switching oligonucleotide is in one embodiment capable of modulating the splicing of HER4 exon 26, and is preferably capable of promoting splicing of HER4 exon 26, thereby favouring the production of HER4 CYT-2

polypeptide over HER4 CYT-1 polypeptide.

The at least one splice-switching oligonucleotide of the composition preferably comprises a sequence selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 -3, or the complement thereof. In a most preferred embodiment, the at least one said splice-switching oligonucleotide comprises 5'-TUACUCCUGACAUGG-3' (SEQ ID NO: 6) and/or 5'- TUTCACUCTAATAGG-3' (SEQ ID NO: 7), or is 5'-TUACUCCUGACAUGG-3' (SEQ ID NO: 6) and/or 5'-TUTCACUCTAATAGG-3' (SEQ ID NO: 7). The splice-switching oligonucleotides of the invention in one embodiment comprise one or more modified nucleotides, such as LNA or methylated nucleotides. For example, the at least one said splice-switching oligonucleotide comprises or is 5'-(l)T (m)U (l)A (m)C (m)U (l)C (m)C (m)U (l)G (m)A (m)C (l)A (m)U (l)G (l)G-3' and/or 5'-(l)T (m)U (l)T (m)C (m)A (l)C (m)U (m)C (l)T (m)A (m)A (l)T (m)A (l)G (l)G-3', wherein I = LNA and m = methylated nucleotides.

However, in another embodiment, the at least one of said binding member of the composition is an siRNA and/or a microRNA. For example, the binding member is an siRNA comprising a sequence selected from any region of a HER4 encoding nucleic acid sequence. Thus, the at least one siRNA comprises in one embodiment a sequence selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 -3, or the complement thereof.

The at least one binding member of the composition of the present invention is selected from any suitable compound or substance available in the art. For example, the at least one binding member is selected from the group consisting of

oligonucleotides, nucleic acid aptamers, antibodies, antigen binding fragments, polypeptides, peptides, peptide fragments, peptide aptamers, nucleic acid aptamers, small molecules, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immonoglobulin binding members. In a specific embodiment, the binding member is an antibody and/or an antigen binding fragment thereof, such as an antibody and/or an antigen binding fragment thereof, which is specific for HER4 CYT-1 , and/or wherein the antibody and/or an antigen binding fragment thereof is capable of downregulating the level or activity of HER4 CYT-1 polypeptide.

In a more specific embodiment, the antibody and/or an antigen binding fragment thereof is specific for an epitope selected from SEQ ID NO: 4, such as an epitope comprising or consisting of 3-7 consecutive amino acids selected from the group consisting of 1030-1080, 1046-1061 (exon 26), 1000-1030, 1031 -1040, 1035-1046, 1040-1050, 1045-1055, 1050-1061 , 1055-1065, 1058-1065, 1058-1070 and 1065-1080 of SEQ ID NO: 4. The composition may comprise a mixture of one or more binding members, such as a mixture at least two antibodies and/or antigen binding fragments. In one embodiment, the composition of the invention further comprises: a) HER4 CYT- 2 polypeptide or a functional homolog or fragment thereof, and/or b) nucleic acid encoding HER4 CYT-2 polypeptide or a functional homolog or fragment thereof.

In another aspect, the present invention relates to a splice-switching oligonucleotide, which specifically binds an RNA species encoded by a HER4 gene and/or encoding a HER4 polypeptide. The splice-switching oligonucleotides comprises or consists of 5- 100 nucleotides, such as preferably 5-25, for example 13-20, such as 14-18

nucleotides, for example the splice-switching oligonucleotide comprises a sequence selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 -3, or the complement thereof, and in particular the splice-switching oligonucleotides comprises a sequence selected from any region of SEQ ID NO: 1 or the complement thereof. In a particularly preferred embodiment, the splice-switching oligonucleotide comprises a nucleic acid sequence selected from any region of the complementary sequence to SEQ ID NO: 1 . In a more specific

embodiment, the splice-switching oligonucleotide comprises a nucleic acid sequence consisting of 15-25 consecutive nucleic acids selected from any region of the nucleic acid sequences selected from the group consisting of the complementary sequence of nucleotides 1 -33496, 32645-32720, 32645-32675, 32645-32668, 32660-32680, 32670- 32700, 32680-33710 and 32690-33720 of SEQ ID NO: 1 . Specifically preferred embodiments of the splice-switching oligonucleotides comprises or consists of 5'- TUACUCCUGACAUGG-3' and/or 5'-TUTCACUCTAATAGG-3'.

The splice-switching oligonucleotides of the invention in one embodiment comprise one or more modified nucleotides, such as LNA or methylated nucleotides. For example, the at least one said splice-switching oligonucleotide comprises or consists of 5'-(l)T (m)U (l)A (m)C (m)U (l)C (m)C (m)U (l)G (m)A (m)C (l)A (m)U (l)G (l)G-3' and/or 5'-(l)T (m)U (l)T (m)C (m)A (l)C (m)U (m)C (l)T (m)A (m)A (l)T (m)A (l)G (l)G-3\ wherein I = LNA and m = methylated nucleotides.

Functionally, the splice-switching oligonucleotide of the present invention is capable of modulating the splicing of HER4 exon 26, and more specifically the splice-switching oligonucleotide of claim 26, wherein said at least one splice-switching oligonucleotide is capable of promoting splicing of HER4 exon 26, thereby favouring the production of HER4 CYT-2 polypeptide over HER4 CYT-1 polypeptide.

In another aspect, the present invention relates a composition of the invention as described above and/or a splice-switching oligonucleotides of the invention as described above for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. In one embodiment, the cancer is growth factor dependent, and in another embodiment, cells derived from cancer or tumour tissue expresses HER4 CYT-1 polypeptide or a functional homolog or fragment thereof and/or RNA encoding said HER4 CYT-1 polypeptide or functional homolog or fragment thereof. The cancer is in one embodiment selected from the group consisting of breast carcinoma, bladder carcinoma, anaplastic large cell lymphoma, B-cell lymphoma, chronic lymphoblastic leukemia, multiple myeloma, testicular tumor, astrocytoma, acute myelogenous leukemia, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, esophageal squamous cell carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma,

neuroblastoma, non-Hodgkin lymphoma, lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, and urothelial carcinoma.

In a preferred embodiment, the cancer is breast carcinoma or bladder carcinoma.

In one embodiment, the composition for treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder of the present invention is combined with another treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or is combined with another medicament for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. The compositions and/or medicaments of the combined treatment are provided for separate, successive or simultaneous administration.

The composition of the invention is in one embodiment claimed for use in a curative treatment, and in another embodiment for ameliorating treatment and in yet another embodiment for prophylactic treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. In a further aspect, the present invention relates to a method for assisting in diagnosing cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or diagnosing cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or determining cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or determining a predisposition for cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder in an individual, said method comprising the steps of

a. determining the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT- 1 polypeptide in a sample from said individual, and/or

b. determining in said sample the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide, and

c. comparing the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide with the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide.

In the preferred embodiment of the method, the presence of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide is indicative of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or predisposition of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or the presence of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is indicative of a reduced risk of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or a reduced predisposition for cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. In one

embodiment a higher level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide relative to HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is indicative of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or predisposition of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. In another embodiment, the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide and/or the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is determined by using a binding member of the present invention. The cancer determined or diagnosed according to the method of the invention is in one

embodiment a growth factor dependent cancer, and in another embodiment, cells derived from cancer or tumour tissue expresses HER4 CYT-1 polypeptide or a functional homolog or fragment thereof and/or RNA encoding said HER4 CYT-1 polypeptide or functional homolog or fragment thereof. The cancer is in one embodiment selected from the group consisting of breast carcinoma, bladder carcinoma, anaplastic large cell lymphoma, B-cell lymphoma, chronic lymphoblastic leukemia, multiple myeloma, testicular tumor, astrocytoma, acute myelogenous leukemia, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, esophageal squamous cell carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, and urothelial carcinoma.

In a preferred embodiment, the cancer is breast carcinoma or bladder carcinoma. The sample wherein HER4 polypeptide or RNA is determined is preferably derived from cancer or tumor tissue.

The present invention in a further aspect relates to a screening method, such as a method for identifying a compound for the treatment and/or diagnosis of cancer or neuropsyciatric disorders, such as schizophrenia or bipolar disorder comprising a. providing a sample

b. bringing at least one compound into contact with said sample, and

c. determining the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT- 1 polypeptide in said sample in the presence and absence of said at least one compound, and

d. comparing the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide in said sample in the presence and absence of said at least one compound.

In one embodiment of the screening method, a reduced level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide in the presence of said at least one compound compared with the absence is indicative of a compound for the treatment and/or diagnosis of cancer or neuropsyciatric disorders, such as

schizophrenia or bipolar disorder.

The screening method in one embodiment further comprises the step of determining in said sample the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide in the presence and absence of said at least one compound. For example, an increased level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide in the presence of said at least one compound compared with the absence of said at least one compound is indicative of a compound for the treatment and/or diagnosis of cancer or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.

The method also in a further embodiment comprises the further step of comparing the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide with the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide. A higher level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide relative to HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is in a preferred embodiment indicative of a compound for the treatment and/or diagnosis of cancer or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.

The level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide and/or the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide are, for example, determined by using a binding member of the present invention as elsewhere defined herein.

The compounds identified according to the screening method of the present invention are preferably claimed for the treatment of a cancer, such as a growth factor dependent cancer, and in another embodiment, cancer types, which expresses HER4 CYT-1 polypeptide or a functional homolog or fragment thereof and/or RNA encoding said HER4 CYT-1 polypeptide or functional homolog or fragment thereof. The cancer is in one embodiment selected from the group consisting of breast carcinoma, bladder carcinoma, anaplastic large cell lymphoma, B-cell lymphoma, chronic lymphoblastic leukemia, multiple myeloma, testicular tumor, astrocytoma, acute myelogenous leukemia, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, esophageal squamous cell carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, and urothelial carcinoma. In a preferred embodiment, the cancer is breast carcinoma or bladder carcinoma.

The sample is preferably derived from cancer or tumor tissue. In yet another aspect, the present invention relates to a compound identified by the scrrening methods defined above for use as a medicament. In particuilar, the compound is provided for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. Relevant cancer forms are for example described elsewhere herein.

Description of Drawings

Figure 1 A) Inclusion of exon 26 in HER4 pre-mRNA generates the CYT1 isoform. Skipping of exon 26 generates the CYT2 isoform. B) Part of the HER4 mRNA sequence showing the 3'-end of intron 25, exon 26 (green) and the 5'-end of intron 26. The sequence of 15 nucleotides annealing with SSOe26 is underlined (red). The numbers indicate the nucleotide number of the first and the last nucleotide in exon 26 according to NM_005235.2, NCBI C) Sequence and modifications of SSOe26 and SSOsc. (I) = LNA, (m) = methylation.

Figure 2 mRNA expression levels of CYT1 and CYT2. Cells were treated with fresh media (Control), transfection reagent only (Lipo) or increasing amounts of SSOe26 as indicated. Expressions are the ratio of CYT1 or CYT2 expression to the β-actin expression.

Figure 3 Growth inhibition of NCI358 and MDA-MB-231 cells by SSOe26 treatment. Cells were treated with fresh media containing lipofectamin (green), control oligo

SSOsc (100 nM) (red) or SSOe26 (100 nM) (blue). Shown are the mean of triplicates. Proliferation was measured as the cell index using a Real-Time Cell Analyzer (RTCA) station.

Figure 4 Growth inhibition of NCI358 cells by increasing concentrations of SSOe26. Cells were treated with fresh media (light blue, top) fresh media containing lipofectamin (purple, 2nd line from top), 1 nM SSOe26 (red, 3rd line from top), 20 nM SSOe26 (green, 4th from top), 50 nM SSOe26 (blue, 5th from top) or 100 nM SSOe26 (pink, 6th from top). Shown are the mean of triplicates. Proliferation was measured as the cell index using a Real-Time Cell Analyzer (RTCA) station.

Figure 5 Growth inhibition of MCF7 A) and T47D B) cells by SSOe26 treatment. Cells were treated with fresh media (red, top line), media containing lipofectamin (green, 2nd line from top), control oligo SSOsc (100 nM) (pink, 3rd line from top) or SSOe26 (100 nM) (blue, 4th line from top). Shown are the mean of triplicates. Proliferation was measured as the cell index using a Real-Time Cell Analyzer (RTCA) station.

Normalization of cell index to the time just after treatment Figure 6 Growth inhibition of MCF7 A) and T47D B) cells by increasing concentrations of SSOe26. Cells were treated with 10 nM SSOe26 (pink, top line), 25 nM SSOe26 (blue, 2nd line from top), 50 nM SSOe26 (green, 3rd line from top) or 100 nM SSOe26 (red, 4th line from top). Shown are the mean of triplicates. Proliferation was measured as the cell index using a Real-Time Cell Analyzer (RTCA) station. Normalization of cell index to the time just after treatment.

Figure 7 A) MMT assay on NCI-358 lung cancer cells treated with fresh media, media containing lipofectamin, control oligo SSOsc (100 nM) or SSOe26 (100 nM) as indicated. B) Protein expression of PARP in NCI-358 lung cancer cells treated with fresh media, media containing lipofectamin, control oligo SSOsc (100 nM) or SSOe26 (100 nM) as indicated. PARP cleavage is indicative of apoptosis.

Detailed description of the invention

The present invention relates to any means, compound, drug, composition, binding member or method, which is capable of a. downregulating the level of HER4 CYT-1 isoform polypeptide and/or mRNA encoding a HER4 CYT-1 isoform polypeptide and/or b. upregulating the level of HER4 CYT-2 isoform polypeptide and/or mRNA encoding a HER4 CYT-2 isoform polypeptide. Such means, compound, drug, composition, or binding member is particularly claimed in the present invention for use as a

medicament, or for use for the manufacture of a medicament. Preferably, the medicament is claimed for the treatment of cancer or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, but also for the treatment of any disorder associated with HER4. Human epidermal growth factor (EGF) receptor 4 (HER4) exists in two isoforms, CYT-1 and CYT-2 due to alternative splicing. The CYT-2 isoform lacks 16 amino acids compared to the full-length receptor, CYT1 , due to skipping of exon 26. The two isoforms are presumably functionally different since the skipped exon 26 encodes a protein fragment that comprises several binding sites, for example a PI3K binding site. The present invention provides methods and means for modulating alternative splicing of HER4, and thereby offers a methodology for the functional regulation of HER4, because the ligand binding sites comprised in the polypeptide region encoded by exon 26 may be excluded by alternative splicing of the HER4 pre-mRNA. Elements of the EGF system including HER4 are involved in different clinical conditions, including cancers and neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and therefore, the means and methods of the present invention are applicable for use in pharmaceutical and diagnostic methods for the diagnosis and/or treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.

The influence of HER4 on the outcome of cancer patients is generally unclear, since HER4 has been found not only to indicate poor patient outcome, but in some cases actually indicate a better patient survival. Therefore, a total inhibition of the HER4 receptor is not desired. According to the present invention, the reason for the dual function of HER4 is the presence of the two splicing variants CYT-1 and CYT-2.

According to the present invention, only the CYT-1 isoform that causes the poor patient outcome is targeted by the compounds, compositions and methods of the present invention, while the expression and functionality of CYT2 is maintained or even increased. For example, the present invention provides means and methods for restoring normal splicing patterns in cells, where splicing have switched in favour of generating the CYT-1 isoform of HER4. Thus, the present invention relates to any means or method which can be used to modulate alternative splicing of HER4.

Definitions

To facilitate the understanding of the invention, some definitions of important terms are provided herein below. The term "modulate" encompasses an increase or a decrease, a stimulation, inhibition, or blockage in the measured activity when compared to a suitable control. "Modulation" of expression levels includes increasing the level and decreasing the level of an mRNA or polypeptide encoded by a polynucleotide of the invention when compared to a control lacking a specific compound or binding member. "Modulation" of splicing refers to changes in the choices of splice sites in the presence of a modulating agent, for example a splice-switching oligonucleotide. Modulation of splicing for example encompass a change in the splicing patern of a given transcript, and thereby also in the expression profile of a gene, because an alternative polypeptide is generated. The term "gene product" as used herein refers to any transcriptional or translational product of a gene. A transcriptional product comprises any RNA-species, which is transcribed from the specific gene, such as pre-mRNA, mRNA, tRNA, miRNA, spliced and nonspliced RNA. The transcript may be bound by RNA-binding proteins and packaged into a ribonucleoprotein (RNP), for example an mRNP molecule. A translational gene product of the present invention comprises any peptide or polypeptide encoded by the gene or a fragment thereof. Thus, a "polypeptide encoded by a gene of the present invention" is comprised in the terms "gene product", or "translational gene product". A translational gene product of the present invention comprises any polypeptide-species encoded by a nucleic acid sequence of the present invention. For example, a translational gene product of the present invention comprises any polypeptide-species encoded by a sequence selected from any of SEQ ID NO: 1 -3, or the complement thereof or part thereof. The terms "increase" or "decrease" as used herein in respect of the level of a transcriptional and/or translational gene product refers to a rise or reduction, respectively, of said transcriptional and/or translational gene product; i.e. the level of transcriptional and/or translational gene product is lower compared to the average level for example in an animal, tissue, and/or population of cells before or after a given treatment, e.g. administration of a composition, a binding member or a compound of the present invention. In respect of a transcriptional product of the present invention, the level of transcript may for example be determined by quantitative or

semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). The level of transcript may be normalized according to an endogenous transcript. A decreased activity of a transcriptional product is for example observed by a reduction or decrease of the level of a specific RNA transcript, as determined for example by RT-PCR. In a preferred embodiment the level of RNA is determined by RT-PCR.

Decrease of the activity of a translational product comprises both a reduction in the amount/level of polypeptide, such as reporter polypeptide, and/or reduced enzymatic activity of said polypeptide and/or reduced ability of the polypeptide to interact with other polypeptides and signal cascades. The level of polypeptide may be determined by any suitable method available to those of skill in the art, for example by western blotting, or ELISA. The expression is in one embodiment increased by at least 10 %, such as at least 20%, such at least 30%, such at least 40%, such at least 50%, such at least 60%, such at least 70%, such at least 80%, such at least 90%, such at least 100%, such at least 200%, such at least 300%, such at least 400%, such at least 500%, such at least 600%, such at least 700%, such at least 800%, such at least 900%, such at least 1000% in the presence of an agent of the present invention compared with the absence of said agent. In another embodiment, the expression is decreased to less than 95%, such as less than 90%, such as less than 80%, such as less than 70%, such as less than 60%, such as less than 50%, such as less than 40%, such as less than 30%, such as less than 20%, such as less than 10%, such as less than 9%, such as less than 8%, such as less than 7%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% in the presence of an agent of the present invention compared with the absence of said agent.

The level of a translational or transcriptional gene product may in the present invention also be determined by the relative amount of one splice variant of a gene product compared with an alternative splice variant of said gene product.

The term "sample" as used herein refers to any suitable biological sample comprising genetic material, such as RNA or DNA, and/or proteins. The sample is in a preferred embodiment, isolated from the subject, such as a pig, mouse, or another mammal. In a preferred embodiment the sample is a tissue sample selected from the group consisting of skin, epidermis, dermis, hypodermis, breast, fat, thymus, gut, small intestine, large intestine, stomach, muscle, pancreas, heart muscle, skeletal muscle, smooth muscle, liver, lung, brain, cornea and tumours, ovarian tissue, uterine tissue, colon tissue, prostate tissue, lung tissue, renal tissue, thymus tissue, testis tissue, hematopoietic tissue, bone marrow, urogenital tissue, expiration air, stem cells, including cancer stem cell, and body fluids, such as sputum, urine, blood and/or sweat. The most convenient sample type is a blood sample; however, the choice of sample depends on the specific disorder or clinical condition as well as detection method and will be evident for those of skill in the art. In one embodiment, the sample is derived from tumorigenic tissue, such as cancer tissue, tumor tissue or malignant tissue. In one embodiment, the sample comprises cancer cells.

The term "complementary region" or "complement" refers to a region of a nucleic acid or mimetic that is or is at least 60% complementary to the relevant sequence, such as SEQ ID NO: 1 , 2, 3, or a fragment thereof.. The complementary region is or is at least 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.1 , 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein. With single polynucleotide sequences, there may be a hairpin loop structure as a result of chemical bonding between the polynucleotide binding member and the

complementary region.

Sequence identity

The term "sequence identity" indicates a quantitative measure of the degree of homology between two amino acid sequences or between two nucleic acid sequences of equal length. If the two sequences to be compared are not of equal length, they must be aligned to give the best possible fit, allowing the insertion of gaps or, alternatively, truncation at the ends of the polypeptide sequences or nucleotide sequences. The sequence identity can be calculated, wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences, preferably sequence identity is calculated over the full length reference as provided herein. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Ndif=2 and Nref=8). A gap is counted as non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (Ndif =2 and Nref =8).

With respect to all embodiments of the invention relating to nucleotide sequences, the percentage of sequence identity between one or more sequences may also be based on alignments using the clustalW software (http:/www.ebi. ac.uk/clustalW/index. html) with default settings. For nucleotide sequence alignments these settings are:

Alignment=3Dfull, Gap Open 10.00, Gap Ext. 0.20, Gap separation Dist. 4, DNA weight matrix: identity (IUB).

The following terms are used to describe the sequence relationships between two or more polynucleotides: "predetermined sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity".

A "predetermined sequence" is a defined sequence used as a basis for a sequence comparison; a predetermined sequence may be a subset of a larger sequence, for example, as a segment of a full-length DNA or gene sequence given in a sequence listing, such as a polynucleotide sequence of SEQ ID NO: 1 , 2, or 3, or may comprise a complete DNA or gene sequence. Generally, a predetermined sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Likewise, the predetermined seequence is that of the

polypeptides of the invention.

Homologs of the disclosed polypeptides are typically characterised by possession of at least 94% sequence identity counted over the full length alignment with the disclosed amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Alternatively, one may manually align the sequences and count the number of identical amino acids. This number divided by the total number of amino acids in your sequence multiplied by 100 results in the percent identity.

As used herein, "nucleic acid" or "polynucleotide" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Polynucleotides can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., (alpha-enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.

Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term "polynucleotide" also includes so-called "peptide nucleic acids," which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. The term "complement" in terms of a nucleic acid sequence refers to a polynucleotide having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.

"Complementary DNA (cDNA)" is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription. Those skilled in the art also use the term "cDNA" to refer to a double- stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand. The term "cDNA" also refers to a clone of a cDNA molecule synthesized from an RNA template.

The term 'nucleotides' as used herein refers to both natural nucleotides and non- natural nucleotides, which are capable of being incorporated into an oligonucleotide, such as a splice-switching oligonucleotide. Nucleotides may differ from natural nucleotides by having a different phosphate moiety, sugar moiety and/or base moiety. Nucleotides may accordingly be bound to their respective neighbour(s) in a template or a complementing template by a natural bond in the form of a phosphodiester bond, or in the form of a non-natural bond, such as e.g. a peptide bond as in the case of PNA (peptide nucleic acids).

The term 'oligonucleotide' is used herein interchangebly with polynucleotide. As used herein the term "oligonucleotide" refers to a single stranded or double stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, such as modified oligonucleotide sequences, for example LNA or methylated residues. The term includes oligonucleotides composed of naturally- occurring bases, sugars and covalent internucleoside linkages (e.g., backbone) as well as oligonucleotides having non-naturally-occurring portions which function similarly to respective naturally-occurring portions (see disclosed in U.S. Pat. Nos. 4,469,863; 4,476,301 ; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;

5,286,717; 5,321 ,131 ; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;

5,476,925; 5,519,126; 5,536,821 ; 5,541 ,306; 5,550,1 1 1 ; 5,563,253; 5,571 ,799;

5,587,361 ; and 5,625,050). The term oligonucleotide thus also refers to any

combination of oligonucleotides of natural and non-natural nucleotides. The natural and/or non-natural nucleotides may be linked by natural phosphodiester bonds or by non-natural bonds. Preferred oligonucleotides comprise only natural nucleotides linked by phosphodiester bonds. Oligonucleotide is used interchangeably with polynucleotide. The oligomer or polymer sequences of the present invention are formed from the chemical or enzymatic addition of monomer subunits. The term "oligonucleotide" as used herein includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleotides, ribonucleotides, anomeric forms thereof, peptide nucleic acid monomers (PNAs), locked nucleotide acid monomers (LNA), and the like, capable of specifically binding to a single stranded polynucleotide tag by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g. 3-4, to several tens of monomeric units, e.g. 40-60. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5' - 3' order from left to right and the "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted. The oligonucleotides of the invention may comprise the four natural nucleotides; however, they splice-switching oligonucleotides of the invention preferably comprise methylated or non-natural nucleotide analogs. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers (Tetrahedron Lett., 22, 1859-1862, 1981 ), or by the triester method according to Matteucci, et al. (J. Am. Chem. Soc, 103, 3185, 1981 ), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPS.TM. technology. When oligonucleotides are referred to as "double-stranded," it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical configuration typically associated with, for example, DNA. In addition to the 100% complementary form of double- stranded oligonucleotides, the term "double-stranded" as used herein is also meant to refer to those forms which include such structural features as bulges and loops. For example as described in US 5.770.722 for a unimolecular double-stranded DNA. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed, e.g. where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required. When nucleotides are conjugated together in a string using synthetic procedures, they are always referred to as oligonucleotides.

A plurality of individual nucleotides linked together in a single molecule may form a polynucleotide. Polynucleotide covers any derivatized nucleotides such as DNA, RNA, PNA, LNA etc. Any oligonucleotide is also a polynucleotide, but every polynucleotide is not an oligonucleotide.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. Functional equivalents, variants or homologs are used interchangeably herein. In one preferred embodiment of the invention there is also provided binding members specifically binding a functional fragment or homolog of HER4 CYT-1 . When being polypeptides, variants are determined on the basis of their degree of identity or their homology with a predetermined amino acid sequence, said predetermined amino acid sequence being of SEQ ID NO: 4, when the variant is a fragment, a fragment of any of the aforementioned amino acid sequences, respectively.

In addition to full-length HER4, substantially full-length HER4, such as HER4 CYT-2, C- terminal peptides, N-terminal peptides and truncated forms of HER4, the present invention provides for functional fragments and homologs of the polypeptides. A HER4 polypeptide or fragment is functional if it exhibits an activity of naturally occurring HER4. It is to be understood that the invention also relates to substantially purified HER4 CYT-2. One HER4 activity of a functional fragment or homolog of HER4 is the ability to compete with naturally occurring HER4 in a receptor-binding assay.

Functionale fragments and homologs may also be defined with reference to one or more of the other in vitro and/or in vivo biological assays described in the examples.

One or more amino acid residues of the polypeptides of the present invention are in one embodiment modified so as to preferably improve the resistance to proteolytic degradation and stability or to optimize solubility properties or to render the polypeptide more suitable as a therapeutic agent. Thus, the polypeptide may comprise amino acid residues other than naturally occurring L-amino acid residues. For example, the polypeptides may comprise D-amino acid residues. However, the polypeptides may also comprise non-naturally occurring, synthetic amino acids. As used herein "a functional homolog" refers to polypeptides or proteins which corresponds to the basic protein, which is suitably HER4 (such as CYT-1 or CYT-2, SEQ ID NO.: 4 or 5), but which differs from the base sequence from which they are derived in that one or more amino acids within the sequence are substituted for other amino acids. Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. Non- conservative substitutions are where amino acids are replaced with amino acids of a different type. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide.

Suitably fragments of homologs are of HER4 CYT-2 has at least 80% sequence identity, such as preferably at least 81 % sequence identity, more preferably e.g. at least 82% sequence identity, such as more preferably at least 83% sequence identity, e.g. more preferably at least 84% sequence identity, more preferably such as at least 85% sequence identity, more preferably e.g. at least 86% sequence identity, more preferably such as at least 87% sequence identity, more preferably e.g. at least 88% sequence identity, more preferably such as at least 89% sequence identity, more preferably e.g. at least 90% sequence identity, more preferably such as at least 91 % sequence identity, more preferably e.g. at least 92% sequence identity, such as at least 93% sequence identity, more preferably e.g. at least 94% sequence identity, more preferably such as at least 95% sequence identity, more preferably e.g. at least 96% sequence identity, more preferably such as at least 97% sequence identity, more preferably e.g. at least 98% sequence identity, more preferably such as at least 99% sequence identity, more preferably e.g. at least 99.5% sequence identity identity with the predetermined sequence of HER4 CYT-2, identified as SEQ ID No: 5.

A fragment of the invention, wherein the fragment has a stretch of at least 50 contiguous amino acids contains less than 410 consecutive amino acid residues of SEQ ID NO: 4 or 5, such as less than 400 consecutive amino acid residues, such as less than 395 consecutive amino acid residues, e.g. less than 390 consecutive amino acid residues, such as less than 385 consecutive amino acid residues, e.g. less than 380 consecutive amino acid residues, such as less than 370 consecutive amino acid residues, e.g. less than 360 consecutive amino acid residues, such as less than 350 consecutive amino acid residues, e.g. less than 345 consecutive amino acid residues, such as less than 340 consecutive amino acid residues, e.g. less than 335 consecutive amino acid residues, such as less than 330 consecutive amino acid residues, e.g. less than 325 consecutive amino acid residues, such as less than 300 consecutive amino acid residues, e.g. less than 295 consecutive amino acid residues, such as less than 290 consecutive amino acid residues, e.g. less than 285 consecutive amino acid residues, such as less than 280 consecutive amino acid residues, e.g. less than 275 consecutive amino acid residues, such as less than 270 consecutive amino acid residues, e.g. less than 265 consecutive amino acid residues, such as less than 260 consecutive amino acid residues, such as less than 255 consecutive amino acid residues, e.g. less than 250 consecutive amino acid residues, such as less than 245 consecutive amino acid residues, e.g. less than 240 consecutive amino acid residues, such as less than 235 consecutive amino acid residues, e.g. less than 230 consecutive amino acid residues, such as less than 225 consecutive amino acid residues, such as less than 220 consecutive amino acid residues, such as less than 215 consecutive amino acid residues, e.g. less than 210 consecutive amino acid residues, such as less than 205 consecutive amino acid residues, e.g. less than 200 consecutive amino acid residues, such as less than 195 consecutive amino acid residues, e.g. less than 190 consecutive amino acid residues, such as less than 185 consecutive amino acid residues, e.g. less than 180 consecutive amino acid residues, such as less than 175 consecutive amino acid residues, e.g. less than 170 consecutive amino acid residues, such as less than 165 consecutive amino acid residues, e.g. less than 160 consecutive amino acid residues, such as less than 155 consecutive amino acid residues, e.g. less than 150 consecutive amino acid residues, such as less than 145 consecutive amino acid residues, e.g. less than 140 consecutive amino acid residues, such as less than 135 consecutive amino acid residues, e.g. less than 130 consecutive amino acid residues, such as less than 125 consecutive amino acid residues, e.g. less than 120 consecutive amino acid residues, such as less than 1 15 consecutive amino acid residues, e.g. less than 1 10 consecutive amino acid residues, such as less than 105 consecutive amino acid residues, e.g. less than 100 consecutive amino acid residues, such as less than 95 consecutive amino acid residues, e.g. less than 90 consecutive amino acid residues, such as less than 85 consecutive amino acid residues, e.g. less than 80 consecutive amino acid residues, such as less than 75, e.g. less than 60 consecutive amino acid residues of SEQ ID NO: 4 or 5.

In addition, the protein may comprise a protein tag to allow subsequent purification and optionally removal of the tag using an endopeptidase. The tag may also comprise a protease cleavage site to facilitate subsequent removal of the tag. Non-limiting examples of affinity tags include a polyhis tag, a GST tag, a HA tag, a Flag tag, a C- myc tag, a HSV tag, a V5 tag, a maltose binding protein tag, a cellulose binding domain tag. Preferably for production and purification, the tag is a polyhistag. Preferably, the tag is in the C-terminal portion of the protein. An isolated HER4 CYT-2 polypeptide may operably be fused to an affinity tag, such as a His-tag.

The term "fragment thereof" may refer to any portion of the given amino acid sequence. Fragments may comprise more than one portion from within the full-length protein, joined together. Suitable fragments may be deletion or addition mutants. The addition of at least one amino acid may be an addition of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids. Fragments may include small regions from the protein or combinations of these.

Suitable fragments may be deletion or addition mutants. The addition or deletion of at least one amino acid may be an addition or deletion of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids. The deletion and/or the addition may - independently of one another - be a deletion and/or an addition within a sequence and/or at the end of a sequence.

It is preferred that functional homologues of HER4 comprises at the most 450, more preferably at the most 400, even more preferably at the most 300, yet more preferably at the most 200, such as at the most 175, for example at the most 160, such as at the most 150 amino acids, for example at the most 140 amino acids.

HER4

HER4 (Human epidermal growth factor (EGF) receptor 4) belongs to the EGF family, which comprise of receptors and ligands. HER4 is alternatively spliced into the two polypeptide isoforms CYT-1 and CYT-2. Skipping of exon 26 results in a receptor lacking 16 amino acids (CYT-2) compared to the full-length receptor (CYT-1 ). The present invention relates to HER4 gene, and provides compounds binding members, and compositions for medical use, as well as methods employing those, which distinguish between the CYT-1 and CYT-2 isoforms. The invention specifically relates to compounds and compositions, as well as methods for the use thereof, which distinguish between the CYT-1 and CYT-2 isoforms of HER4.

The present invention relates to binding members and compounds specific for CYT-1 , as well as compositions comprising at least one such binding member capable of binding to a HER4 polypeptide and/or a HER4 RNA, thereby

a. specifically downregulating the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 isoform polypeptide and/or

b. upregulating the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 isoform polypeptide.

The HER4 gene is relative large, extending over 1 .162.910 nucleotides. The complete HER4 genomic DNA sequence is retrievable from the NCBI database, cf.

http://www.ncbi.nlm.nih.goV/nuccore/NC_000002.1 1 ?&from=212240441 &to=21340335 1 &report=genbank&strand=true.

NCBI Reference Sequence: NC_000002.1 1 , Gl:22458981 1

The HER4 exons are located in the genomic sequence as indicated below:

1 ..179 (EXON 1 ),413724..413875 (EXON 2),59101 1 ..591 197 (EXON

3),750468..750602 (EXON 4), 787923.-787988 (EXON 5), 813433..813551 (EXON 6), 816093..816234 (EXON 7), 824979..825092 (EXON 8), 826451 ..826577 (EXON 9),

833236..833309 (EXON 10), 834433.-834523 (EXON 1 1 ), 836461 ..836660 (EXON 12), 859443.-859575 (EXON 13), 865370-865463 (EXON 14), 873150-873304 (EXON 15), 880799-880873 (EXON 16), 908033-908165 (EXON 17), 914583-914705 (EXON 18), 919352-919450 (EXON 19), 976539-976724 (EXON 20), 1 107527-1 107682 (EXON 21 ), 1 1 10144-1 1 10219 (EXON 22), 1 1 14326-1 1 14472 (EXON 23), 1 1 16523-1 1 16620 (EXON 24), 1 1 18016-1 1 18186 (EXON 25), 1 150635-1 150682 (EXON

26), 1 151477..1 151774, (EXON 27), 1 154567..1 162910 (EXON 28)

gene="ERBB4"/product="v-erb- aerythroblasticleukemiaviraloncogenehomolog4(avian),transcriptvariantJM-a/CVT-1 "

Thus, intron 25 is located from 1 1 18187-1 150634, exon 26 from 1 150635-1 150682, and intron 26 from 1 150683-1 151476. The specific sequence of intron 25-exon 26- intron 26 and some of the flanking sequences is provided in SEQ ID NO: 1 .

The database content related to any nucleic acids and genes designated by an accession number or a database submission are incorporated herein by reference as of the filing date of this application. Binding member

In a main aspect, the present invention relates to a composition for use as a medicament, wherein the composition comprises at least one binding member capable of binding to a HER4 polypeptide and/or a HER4 RNA. The function of the binding member is to a) specifically downregulate the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 isoform polypeptide and/or b) upregulate the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 isoform polypeptide.

The binding member is preferably specific for binding the HER4 CYT-1 polypeptide or a biologically active fragment or homolog thereof. Thus, the binding member does not bind HER4 CYT-2 polypeptide. The binding member may also further bind HER4 RNA in a region comprising exon 26 and/or in any region affecting the splicing of exon 26. in one embodiment, the binding member specifically binds the HER4 CYT-1 polypeptide or a biologically active fragment or homolog thereof and HER4 RNA in a region comprising exon 26 and/or in any region affecting the splicing of exon 26. The at least one binding member of the composition of the present invention is selected from any suitable compound or substance available in the art. For example, the at least one binding member is selected from the group consisting of

oligonucleotides, nucleic acid aptamers, antibodies, antigen binding fragments, polypeptides, peptides, peptide fragments, peptide aptamers, nucleic acid aptamers, small molecules, natural single domain antibodies, affibodies, affibody-antibody chimeras, and non-immonoglobulin binding members.

In a specific embodiment, the binding member is an antibody and/or an antigen binding fragment thereof, such as an antibody and/or an antigen binding fragment thereof, which is specific for HER4 CYT-1 , and/or wherein the antibody and/or an antigen binding fragment thereof is capable of downregulating the level or activity of HER4 CYT-1 polypeptide.

In a more specific embodiment, the antibody and/or an antigen binding fragment thereof is specific for an epitope selected from SEQ ID NO: 4, such as an epitope comprising or consisting of 3-7 consecutive amino acids selected from the group consisting of 1030-1080, 1046-1061 (exon 26), 1000-1030, 1031 -1040, 1035-1046, 1040-1050, 1045-1055, 1050-1061 , 1055-1065, 1058-1065, 1058-1070 and 1065-1080 of SEQ ID NO: 4. The composition may comprise a mixture of one or more binding members, such as a mixture at least two antibodies and/or antigen binding fragments. In one embodiment, the composition of the invention further comprises: a) HER4 CYT- 2 polypeptide or a functional homolog or fragment thereof, and/or b) nucleic acid encoding HER4 CYT-2 polypeptide or a functional homolog or fragment thereof. In a most preferred embodiment, the at least one binding member of the composition is a splice-switching oligonucleotide. The splice-switching oligonucleotides preferably specifically bind an RNA species encoding a HER4 polypeptide, thereby modulating the splicing of exon 26 of the HER4 transcript. Splice-switching oligonucleotides

According to the present invention the reason for the dual function of HER4 is the presence of the two splicing variants CYT-1 and CYT-2 and therefore to target only the CYT-1 isoform that causes the poor patient outcome while maintaining or even increasing the expression and functionality of CYT2.

Splice switching oligonucleotides (SSOs) can be used to manipulate alternative splicing pathways by hybridizing to pre-mRNA sequences important for splicing, thereby altering the expression of targeted genes, both in cell culture and in vivo. SSOs can repair aberrant splicing or alter normal splicing to abrogate the expression of disease- causing genes, and even lead to production of autoinhibitors.

In a preferred embodiment of the composition of the present invention, the at least one binding member is a splice-switching oligonucleotide, which, preferably, specifically binds an RNA species encoding a HER4 polypeptide. The at least one splice-switching oligonucleotide is in one embodiment capable of modulating the splicing of HER4 exon 26, and is preferably capable of promoting splicing of HER4 exon 26, thereby favouring the production of HER4 CYT-2 polypeptide over HER4 CYT-1 polypeptide. The present invention also in one aspect relates to a splice-switching oligonucleotide, which specifically binds an RNA species encoded by a HER4 gene and/or encoding a HER4 polypeptide. The splice-switching oligonucleotides can be designed to target any positive splicing regulatory signal, including intron splicing enhancers, ISE and exon splicing enhancer, ESE, in the HER4 RNA transcript, in particular in intron and exon 26.

The splice-switching oligonucleotide preferably comprises a sequence selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 -3, or the complement thereof. For example, the splice- switching oligonucleotides comprise a sequence corresponding to exon 26, and its two flanking introns. In a most preferred embodiment, the at least one said splice-switching oligonucleotide comprises or consists of 5'-TUACUCCUGACAUGG-3' (SEQ ID NO: 6) and/or 5'-TUTCACUCTAATAGG-3' (SEQ ID NO: 7). In one embodiment, the splice- switching oligonucleotide of the invention is at least 70% identical, such as at least 75% for example at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 100% identical to any one of SEQ ID NO: 6 or 7.

In another embodiment, the splice-switching oligonucleotide comprises or consists of a nucleic acid sequence, such as a nucleic acid sequence of 5-100, such as 10-80, for example 10-70, such as 10-60, such as 10-50, such as 10-40, such as 10-35, such as 10-30, such as 15-30 consecutive nucleotides selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 or the complement thereof, as well as for example selected from any such region which is at least 70% identical, such as at least 75% for example at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as at least 100% identical to SEQ ID NO: 1 or the complement thereof.

It is understood that the splice-switching oligonucleotides of the present invention are preferably ribonucleotides or modified nucleotides such as modified ribonucleotides. Thus, when a splice-switching oligonucleotide is described as identical or derived from a DNA sequence, the relevant splice-switching oligonucleotide sequence also comprise the corresponding ribonucleotide sequence; i.e. wherein thymine of a DNA sequence is replaced with uracil in the splice-switching oligonucleotide ribonucleotide sequence.

The splice-switching oligonucleotides typically comprise one or more modified nucleotides to escape intracellular degradation, such as RNAse H mediated decay and/or to increase target specificity. Non-limiting examples of modified nucleotides are LNA (locked nucleic acid), ALN (a-L-LNA), a-L-LNA, HM (4'-C-hydroxymethyl-DNA), ADA (2'-N-adamantylmethylcarbonyl-2'amino-LNA), PYR (2'-N-pyren-1 -ylmethyl-2'- amino-LNA), EA (2'-aminoethyl), GE (2'-guanidinoethyl), CE (2'-cyanoethyl), AP (2'- aminopropyl), OX (oxetane-locked nucleic acid), CLNA (2',4'-carbocyclic-LNA-locked nucleic acid), CENA (2',4'-carbocyclic-ENA-locked nucleic acid), AENA (2'-deoxy-2'- N,4'-C-ethylene-locked nucleic acid ), OMe (2'-0-methyl), F (2'-fluoro) (22), ANA (altritol nucleic acid), HNA (hexitol nucleic acid), AEM (2'-aminoethoxymethyl) and APM (2'-aminopropoxymethyl). The nucleotide is preferably LNA and/or methylated nucleotides. For example, the at least one said splice-switching oligonucleotide is 5'- (l)T (m)U (l)A (m)C (m)U (l)C (m)C (m)U (l)G (m)A (m)C (l)A (m)U (l)G (l)G-3' and/or 5'- (l)T (m)U (l)T (m)C (m)A (l)C (m)U (m)C (l)T (m)A (m)A (l)T (m)A (l)G (l)G-3', wherein I = LNA and m = methylated nucleotides.

The splice-switching oligonucleotides may in a further modification comprise a 51-end nuclear localization signal to increase nuclear localization of the nucleotide. The nuclear localization signal is for example composed of a 2,2,7-trimethylguanosine cap (m3G-CAP).

The splice-switching oligonucleotides of the invention comprises or consists of 5-100 nucleotides, such as preferably 5-25, for example 13-20, such as 14-18 nucleotides, for example the splice-switching oligonucleotide comprises a sequence selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 -3, or the complement thereof, and in particular the splice-switching oligonucleotides comprises a sequence selected from any region of SEQ ID NO: 1 or the complement thereof. In a particularly preferred embodiment, the splice-switching oligonucleotide comprises a nucleic acid sequence selected from any region of the complementary sequence to SEQ ID NO: 1 . In a more specific embodiment, the splice- switching oligonucleotide comprises a nucleic acid sequence consisting of 15-25 consecutive nucleic acids selected from any region of the nucleic acid sequences selected from the group consisting of the complementary sequence of nucleotides 1 - 33496, 32645-32720, 32645-32675, 32645-32668, 32660-32680, 32670-32700, 32680-33710 and 32690-33720 of SEQ ID NO: 1 . Specifically preferred embodiments of the splice-switching oligonucleotides comprises or consists of 5'- TUACUCCUGACAUGG-3' (SEQ ID NO: 6) and/or 5'-TUTCACUCTAATAGG-3' (SEQ ID NO: 7). In a particularly preferred embodiment, the residues of the splice-switching oligonucleotides are modified to include LNA and/or methylated residues, such as (5'-(l)T (m)U (l)A (m)C (m)U (l)C (m)C (m)U (l)G (m)A (m)C (l)A (m)U (l)G (l)G-3' or

5'-(l)T (m)U (l)T (m)C (m)A (l)C (m)U (m)C (l)T (m)A (m)A (l)T (m)A (l)G (l)G-3\ wherein (I = LNA, m = methylated))

Functionally, the splice-switching oligonucleotide of the present invention is capable of modulating the splicing of HER4 exon 26, and more specifically the splice-switching oligonucleotide is capable of promoting splicing of HER4 exon 26, thereby favouring the production of HER4 CYT-2 polypeptide over HER4 CYT-1 polypeptide. siRNA and microRNA

In one preferred embodiment, the at least one binding member of the composition is an siRNA and/or a microRNA.

siRNA is a class of double-stranded RNA molecules that inter alia is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. The length of an siRNA is typically 18-25 nucleotides, such as 18-22 nucleotides. In one embodiment, the binding member is an siRNA which promotes the

downregulation of HER4 CYT-1 transcript and/or polypeptide. Design of siRNAs is wellknown in the art, and any known principle or algorithm may be employed for design of effective siRNA binding member species. microRNAs are short nucleotide RNA sequences of approximately 22 nucleotides, such as between 19 and 25 nucleotides, such as 19, 20, 21 , 22, 23, 24, or 25 nucleotides, that bind to complementary sequences of a target mRNA, which leads to silencing of the targeted gene. Therefore, microRNAs may be used as a binding member of the present invention to specifically downregulate the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 isoform polypeptide and/or upregulate the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 isoform polypeptide. In one embodiment, the siRNA or microRNA of the present invention comprises a sequence selected or derived from any region of a HER4 encoding nucleic acid sequence or the complement thereof. Thus, the at least one siRNA or microRNA comprises in one embodiment a sequence selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 -3, or the complement thereof. siRNA or microRNA preferably mediate knock down of HER4

CYT-1 by targeting sequences in exon 26 or junction sequences at the border/junction of exon 25-exon 26 and exon 26- exon 27, including up to 20 nucleotides upstream ans/or downstream of the exon-exon junctions. The siRNA or microRNA of the present invention comprises or consists of 5-50 nucleotides, such as preferably 5-30, for example 13-20, such as preferably 18-22 nucleotides. The siRNA or microRNA, for example, comprises a sequence selected from any region of a HER4 gene or the complement thereof, such as selected from any region of SEQ ID NO: 1 -3, or the complement thereof, and in particular the siRNA or microRNA comprises a sequence selected from any region of SEQ ID NO: 1 or the complement thereof. In a more specific embodiment, the siRNA comprises a nucleic acid sequence consisting of 15-25 such as preferably 18-22 consecutive nucleic acids selected from any region of the nucleic acid sequences selected from the group consisting of nucleotides 1 -33496, 32645-32720, 32645-32675, 32645-32668, 32660- 32680, 32670-32700, 32680-33710 and 32690-33720 of SEQ ID NO: 1 . In a pecifically preferred embodiment, the siRNA or microRNA comprises or consists of the sequence 5'-TUACUCCUGACAUGG-3' and/or 5'-TUTCACUCTAATAGG-3'. In a particularly preferred embodiment, the residues of the siRNA or microRNA are modified to include LNA and/or methylated residues, such as

(5'-(l)T (m)U (l)A (m)C (m)U (l)C (m)C (m)U (l)G (m)A (m)C (l)A (m)U (l)G (l)G-3' or 5'-(l)T (m)U (l)T (m)C (m)A (l)C (m)U (m)C (l)T (m)A (m)A (l)T (m)A (l)G (l)G-3\ wherein (I = LNA, m = methylated))

Composition

In a main aspect, the present invention relates to a composition for use as a medicament, said composition comprising at least one binding member capable of binding to a HER4 polypeptide and/or a HER4 RNA, thereby

a. specifically downregulating the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 isoform polypeptide and/or

b. upregulating the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 isoform polypeptide.

The compostion of the present invention is claimed for medical use, and the composition is thus in a preferred embodiment a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprise

a) a binding member of the invention and/or

b) a nucleic acid encoding a HER4 CYT-1 polypeptide and/or

c) an expression vector of the invention,

and a pharmaceutical acceptable carrier and/or diluent or excipient.

Pharmaceutical formulation

Whilst it is possible for the composition of the present invention to be administered as the raw composition, it is preferred to present it in the form of a pharmaceutical formulation. Accordingly, the present invention further provides a pharmaceutical formulation, for medicinal application, which comprises a composition of the present invention or a pharmaceutically acceptable salt thereof, as herein defined, and a pharmaceutically acceptable carrier therefore. Pharmaceutical compositions containing the binding members, splice-switching oligonucleotides, polypeptides, polynucleotides or vectors of the present invention may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa. The compositions may appear in conventional forms, for example capsules, tablets, aerosols, solutions, suspensions or topical applications. The present invention provides pharmaceutical compositions for treating, ameliorating and/or preventing a cancer form as described herein and/or a neuropsychiatric disorder such as bipolar disorder or schizophrenia, wherein the composition comprises at least one binding member capable of binding to a HER4 polypeptide and/or a HER4 RNA, thereby

a. specifically downregulating the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 isoform polypeptide and/or

b. upregulating the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 isoform polypeptide.

In one aspect the present invention relates to a pharmaceutical composition. The pharmaceutical composition may be formulated in a number of different manners, depending on the purpose for the particular pharmaceutical composition. For example the pharmaceutical composition may be formulated in a manner so it is useful for a particular administration form. Preferred administration forms are described herein below.

In one embodiment the pharmaceutical composition is formulated so it is a liquid. For example the composition may be a protein solution or the composition may be a protein suspension. Said liquid may be suitable for parenteral administration, for example for injection or infusion. The liquid may be any useful liquid, however it is frequently preferred that the liquid is an aqueous liquid. For many purposes, in particular when the liquid should be used for parenteral administration, it is furthermore preferred that the liquid is sterile. Sterility may be conferred by any conventional method, for example filtration, irradiation or heating. Furthermore, it is preferred that the liquid has been subjected to a virus reduction step, in particular if the liquid is formulated for parenteral administration.

Virus reduction may for example be performed by nanofiltration or virus filtering over a suitable filter, such as a Planova filter consisting of several layers. The Planova filter may be any suitable size for example 75N, 35N, 20N or 15N or filters of different size may be used, for example Planova 20N. Virus reduction may also comprise a step of prefiltering with another filter, for example using a filter with a pore size of the the range of 0.01 to 1 μηι, such as in the range of 0.05 to 0.5 μηι, for example around 0.1 μηι. Virus reductions may also include an acidic treatment step. The pharmaceutical composition may be packaged in single dosage units, which may be more convenient for the user. Hence, pharmaceutical compositions for bolus injections may be packages in dosage units of for example at the most 10 ml, preferably at the most 8 ml, more preferably at the most 6 ml, such as at the most 5 ml, for example at the most 4 ml, such as at the most 3 ml, for example around 2 ml. The pharmaceutical composition may be packaged in any suitable container. In one example a single dosage of the pharmaceutical composition may be packaged in injection syringes or in a container useful for infusion.

In another embodiment of the present invention the pharmaceutical composition is a dry composition. The dry composition may be used as such, but for most purposes the composition is a dry composition for storage only. Prior to use the dry composition may be dissolved or suspended in a suitable liquid composition, for example sterile water.

The pharmaceutical composition according to the present invention preferably comprises a binding member as described herein above, in a particularly preferred embodiment a splice-switching oligonucleotides, as described herein. The splice- switching oligonucleotides preferably comprise a sequence derived from the HER4 gene; in particular a sequence derived from exon 26 and/or its flanking introns. Thus, it is preferred that the binding member specifically downregulates the CYT-1 polypeptide and not the CYT-2 polypeptide, while maybe also maintaining or even increasing the level of CYT-2 polypeptide, for example in a human being or even in a specific human tissue, such as a cancer tissue or tumor.

It is also comprised within the invention that the pharmaceutical composition may be applied topically to the site of the site, for example in the form of a lotion, a creme, an ointment, a spray, such as an aerosol spray or a nasal spray, rectal or vaginal suppositories, drops, such as eye drops or nasal drops, a patch, an occlusive dressing or the like.

Pharmaceutically acceptable additives

The pharmaceutical compositions may be prepared by any conventional technique, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa. The pharmaceutically acceptable additives may be any conventionally used

pharmaceutically acceptable additive, which should be selected according to the specific formulation, intended administration route etc. For example the

pharmaceutically acceptable additives may be any of the additives mentioned in Nema et al, 1997. Furthermore, the pharmaceutically acceptable additive may be any accepted additive from FDA's "inactive ingredients list", which for example is available on the internet address http://www.fda.gov/cder/drug/iig/default.htm. In some embodiments of the present invention it is desirable that the pharmaceutical composition comprises an isotonic agent. In particular when the pharmaceutical composition is prepared for administration by injection or infusion it is often desirable that an isotonic agent is added. Accordingly, the composition may comprise at least one pharmaceutically acceptable additive which is an isotonic agent.

The pharmaceutical composition may be isotonic, hypotonic or hypertonic. However it is often preferred that a pharmaceutical composition for infusion or injection is essentially isotonic, when it is administrated. Hence, for storage the pharmaceutical composition may preferably be isotonic or hypertonic. If the pharmaceutical

composition is hypertonic for storage, it may be diluted to become an isotonic solution prior to administration. The isotonic agent may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate. Examples of ionic isotonic agents include but are not limited to NaCI, CaCI2, KCI and MgCI2. Examples of non-ionic isotonic agents include but are not limited to mannitol and glycerol. However, in other embodiments of the invention the pharmaceutical composition may comprise no buffer at all or only micromolar amounts of buffer.

In a preferred embodiment the buffer is TRIS. TRIS buffer is known under various other names for example tromethamine including tromethamine USP, THAM, Trizma, Trisamine, Tris amino and trometamol. The designation TRIS covers all the

aforementioned designations. The buffer may furthermore for example be selected from USP compatible buffers for parenteral use, in particular, when the pharmaceutical formulation is for parenteral use. For example the buffer may be selected from the group consisting of monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic, dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric, polybasic acids such as citric and phosphoric and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.

The pharmaceutical compositions may comprise at least one pharmaceutically acceptable additive which is a stabiliser. For example the stabiliser may be selected from the group consisting of poloxamers, Tween-20, Tween-40, Tween-60, Tween-80, Brij, metal ions, amino acids, polyethylene glycol, Triton, EDTA, ascorbic acid, Triton X- 100, NP40 or CHAPS. The pharmaceutical composition according to the invention may also comprise one or more cryoprotectant agents. In particular, when the composition comprises freeze-dried protein or the composition should be stored frozen, it may be desirable to add a cryoprotecting agent to the pharmaceutical composition. The cryoprotectant agent may be any useful cryoprotectant agent, for example the cryoprotectant agent may be selected from the group consisting of dextran, glycerin, polyethylenglycol, sucrose, trehalose and mannitol. Accordingly, the pharmaceutically acceptable additives may comprise one or more selected from the group consisting of isotonic salt, hypertonic salt, buffer and stabilisers. Furthermore, the pharmaceutically acceptable additives may comprise one or more selected from the group consisting of isotonic agents, buffer, stabilisers and cryoprotectant agents. For example, the pharmaceutically acceptable additives comprise glucosemonohydrate, glycine, NaCI and polyethyleneglycol 3350. Oral administration

The compositions of the present invention may be formulated in a wide variety of oral administration dosage forms. The pharmaceutical compositions and dosage forms may comprise the compositions of the invention or its pharmaceutically acceptable salt or a crystal form thereof as the active component. The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material. Preferably, the composition will be about 0.5% to 75% by weight of a composition or compositions of the invention, with the remainder consisting of suitable pharmaceutical excipients. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

In powders, the carrier is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from 1 to about 70 %t of the active composition. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active composition with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is in association with it.

Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be as solid forms suitable for oral administration. . Multiple- unit-dosage granules can be prepared as well. Tablets and granules of the above cores can be coated with concentrated solutions of sugar, etc. The cores can also be coated with polymers which change the dissolution rate in the gastrointestinal tract, such as anionic polymers having a pka of above 5.5. Such polymers are hydroxypropylmethyl cellulose phthalate, cellulose acetate phthalate, and polymers sold under the trade mark Eudragit S100 andU OO. In preparation of gelatine capsules these can be soft or hard. In the former case the active compound is mixed with oil, and in the latter case the multiple-unit-dosage granules are filled therein.

Drops according to the present invention may comprise sterile or non-sterile aqueous or oil solutions or suspensions, and may be prepared by dissolving the active ingredient in a suitable aqueous solution, optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100 degree C for half an hour. Alternatively, the solution may be sterilized by filtration and transferred to the container aseptically. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01 %) and chlorhexidine acetate (0.01 %). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like.

Other forms suitable for oral administration include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpaste, gel dentrifrice, chewing gum, or solid form preparations which are intended to be converted shortly before use to liquid form preparations. Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents. Solid form preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like.

Parenteral administration

The compositions of the present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.

Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides; (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and

polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-. beta.-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations typically will contain from about 0.5 to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

Topical administration

The compositions of the invention can also be delivered topically. Regions for topical administration include the skin surface and also mucous membrane tissues of the vagina, rectum, nose, mouth, and throat. Compositions for topical administration via the skin and mucous membranes should not give rise to signs of irritation, such as swelling or redness.

The topical composition may include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example. Methods for preparing such compositions are well known in the pharmaceutical industry.

The compositions of the present invention may be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also containing one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Formulations suitable for topical administration in the mouth include lozenges comprising active agents in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives or a fatty acid such as steric or oleic acid together with an alcohol such as propylene glycol or a macrogel. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a

polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.

Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.

Transdermal Delivery

The pharmaceutical agent-chemical modifier complexes described herein can be administered transdermally. Transdermal administration typically involves the delivery of a pharmaceutical agent for percutaneous passage of the drug into the systemic circulation of the patient. The skin sites include anatomic regions for transdermally administering the drug and include the forearm, abdomen, chest, back, buttock, mastoidal area, and the like.

Transdermal delivery is accomplished by exposing a source of the complex to a patient's skin for an extended period of time. Transdermal patches have the added advantage of providing controlled delivery of a pharmaceutical agent-chemical modifier complex to the body. See Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987); and Transdermal Delivery of Drugs, Vols. 1 -3, Kydonieus and Berner (eds.), CRC Press, (1987). Such dosage forms can be made by dissolving, dispersing, or otherwise incorporating the pharmaceutical agent-chemical modifier complex in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

Passive Transdermal Drug Delivery

A variety of types of transdermal patches will find use in the methods described herein. For example, a simple adhesive patch can be prepared from a backing material and an acrylate adhesive. The pharmaceutical agent-chemical modifier complex and any enhancer are formulated into the adhesive casting solution and allowed to mix thoroughly. The solution is cast directly onto the backing material and the casting solvent is evaporated in an oven, leaving an adhesive film. The release liner can be attached to complete the system.

Alternatively, a polyurethane matrix patch can be employed to deliver the

pharmaceutical agent-chemical modifier complex. The layers of this patch comprise a backing, a polyurethane drug/enhancer matrix, a membrane, an adhesive, and a release liner. The polyurethane matrix is prepared using a room temperature curing polyurethane prepolymer. Addition of water, alcohol, and complex to the prepolymer results in the formation of a tacky firm elastomer that can be directly cast only the backing material.

A further embodiment of this invention will utilize a hydrogel matrix patch. Typically, the hydrogel matrix will comprise alcohol, water, drug, and several hydrophilic polymers. This hydrogel matrix can be incorporated into a transdermal patch between the backing and the adhesive layer.

The liquid reservoir patch will also find use in the methods described herein. This patch comprises an impermeable or semipermeable, heat sealable backing material, a heat sealable membrane, an acrylate based pressure sensitive skin adhesive, and a siliconized release liner. The backing is heat sealed to the membrane to form a reservoir which can then be filled with a solution of the complex, enhancers, gelling agent, and other excipients.

Foam matrix patches are similar in design and components to the liquid reservoir system, except that the gelled pharmaceutical agent-chemical modifier solution is constrained in a thin foam layer, typically a polyurethane. This foam layer is situated between the backing and the membrane which have been heat sealed at the periphery of the patch.

For passive delivery systems, the rate of release is typically controlled by a membrane placed between the reservoir and the skin, by diffusion from a monolithic device, or by the skin itself serving as a rate-controlling barrier in the delivery system. See U.S. Pat. Nos. 4,816,258; 4,927,408; 4,904,475; 4,588,580, 4,788,062; and the like. The rate of drug delivery will be dependent, in part, upon the nature of the membrane. For example, the rate of drug delivery across membranes within the body is generally higher than across dermal barriers. The rate at which the complex is delivered from the device to the membrane is most advantageously controlled by the use of rate-limiting membranes which are placed between the reservoir and the skin. Assuming that the skin is sufficiently permeable to the complex (i.e., absorption through the skin is greater than the rate of passage through the membrane), the membrane will serve to control the dosage rate experienced by the patient.

Suitable permeable membrane materials may be selected based on the desired degree of permeability, the nature of the complex, and the mechanical considerations related to constructing the device. Exemplary permeable membrane materials include a wide variety of natural and synthetic polymers, such as polydimethylsiloxanes (silicone rubbers), ethylenevinylacetate copolymer (EVA), polyurethanes, polyurethane- polyether copolymers, polyethylenes, polyamides, polyvinylchlorides (PVC), polypropylenes, polycarbonates, polytetrafluoroethylenes (PTFE), cellulosic materials, e.g., cellulose triacetate and cellulose nitrate/acetate, and hydrogels, e.g., 2- hydroxyethylmethacrylate (HEMA).

Other items may be contained in the device, such as other conventional components of therapeutic products, depending upon the desired device characteristics. For example, the compositions according to this invention may also include one or more

preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like. These pharmaceutical compositions also can contain other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents. Administration as suppositories

The compositions of the present invention may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.

The active composition may be formulated into a suppository comprising, for example, about 0.5% to about 50% of a composition of the invention, disposed in a polyethylene glycol (PEG) carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%].

The compositions of the present invention may be formulated for vaginal

administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Respiratory tract administration

The compositions of the present invention may be formulated for nasal administration. The solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in a single or multidose form. In the latter case of a dropper or pipette this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray this may be achieved for example by means of a metering atomizing spray pump.

The compositions of the present invention may be formulated for aerosol

administration, particularly to the respiratory tract and including intranasal

administration. The composition will generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by a metered valve. Alternatively the active ingredients may be provided in a form of a dry powder, for example a powder mix of the composition in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and

polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of e.g., gelatin or blister packs from which the powder may be administered by means of an inhaler. When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient. The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

Pharmaceutically acceptable salts

Pharmaceutically acceptable salts of the instant compounds, where they can be prepared, are also intended to be covered by this invention. These salts will be ones which are acceptable in their application to a pharmaceutical use. By that it is meant that the salt will retain the biological activity of the parent compound and the salt will not have untoward or deleterious effects in its application and use in treating diseases.

Pharmaceutically acceptable salts are prepared in a standard manner. If the parent compound is a base it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it is treated with an inorganic or organic base in a suitable solvent. The compounds of the invention may be

administered in the form of an alkali metal or earth alkali metal salt thereof, concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount. Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example.

Administration forms

The pharmaceutical composition may be prepared so it is suitable for one or more particular administration methods. Furthermore, the method of treatment described herein may involve different administration methods.

In general any administration method, wherein at least one binding member and/or HER4 encoding polynucleotide, transcriptional product and/or polypeptide thereof, functional equivalent thereof, variants or fragments thereof may be administered to an individual in a manner so that active binding member and/or polynucleotide, transcriptional product and/or polypeptide thereof, functional equivalent thereof, variants or fragments thereof may reach the site of disease may be employed with the present invention.

The main routes of drug delivery, in the treatment method are intravenous, oral, and topical, as will be described below. Other drug-administration methods, such as subcutaneous injection or via inhalation, which are effective to deliver the drug to a target site or to introduce the drug into the bloodstream, are also contemplated.

The mucosal membrane to which the pharmaceutical preparation of the invention is administered may be any mucosal membrane of the mammal to which the biologically active substance is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, preferably the mucosa of the nose, mouth or vagina.

Compositions of the invention may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred. Appropriate dosage forms for such administration may be prepared by conventional techniques. The compositions may also be administered by inhalation, that is, by intranasal and oral inhalation administration. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques. The compositions according to the invention may be administered with at least one other compound. The compounds may be administered simultaneously, either as separate formulations or combined in a unit dosage form, or administered sequentially.

Medical use and therapeutic methods

The compositions and splice-switching oligonucleotides of the invention are also in one aspect provided for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.

In one embodiment, the composition for treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder of the present invention is combined with another treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or is combined with another medicament for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. The compositions and/or medicaments of the combined treatment are provided for separate, successive or simultaneous administration.

The composition of the invention is in one embodiment claimed for use in a curative treatment, and in another embodiment for ameliorating treatment and in yet another embodiment for prophylactic treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.

The present invention also provides for the use of a composition comprising at least one binding member capable of binding to a HER4 polypeptide and/or a HER4 RNA, thereby a) specifically downregulating the level of HER4 CYT-1 polypeptide and/or

RNA encoding a HER4 CYT-1 polypeptide and/or b) specifically upregulating the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 polypeptide, for the manufacture of a medicament for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.

The present invention also provides a method of treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, said method comprising administering to a person in need thereof a therapeutically efficient amount of at least one binding member capable of binding to a HER4 polypeptide and/or a HER4 RNA, thereby a) specifically downregulating the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 polypeptide and/or b) specifically upregulating the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 polypeptide. For the uses and methods of treatments described above, examples of binding members are provided elsewhere herein, and specific cancer forms are also indicated herein above. In a preferred embodiment, the binding member is a splice-switching oligonucleotide as described herein above. The medicament produced according to the use of the invention is applicable for curative treatment as well as ameliorating or prophylactic treatment.

Disorders

The compositions, methods and medicaments are provided herein for medical use. Specifically, the compositions, methods and medicaments are provided for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder.

In one embodiment, the cancer is a growth factor dependent cancer, and in another embodiment, cells derived from cancer or tumour tissue expresses HER4 CYT-1 polypeptide or a functional homolog or fragment thereof and/or RNA encoding said HER4 CYT-1 polypeptide or functional homolog or fragment thereof. The cancer is in one embodiment selected from the group consisting of breast carcinoma, bladder carcinoma, anaplastic large cell lymphoma, B-cell lymphoma, chronic lymphoblastic leukemia, multiple myeloma, testicular tumor, astrocytoma, acute myelogenous leukemia, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, esophageal squamous cell carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, and urothelial carcinoma.

In a preferred embodiment, the cancer is breast carcinoma or bladder carcinoma. A cancer or a cancer cell can e.g. be neuronal, glial, lung, liver, brain, breast, bladder, blood, leukemic, colon, endometrial, stomach, skin, ovarian, fat, bone, cervical, esophageal, pancreatic, prostate, kidney, testicular or thyroid cell. The term cancer also includes includes, but is not limited to anaplastic large cell lymphoma, B-cell lymphoma, chronic lymphoblastic leukemia, multiple myeloma, testicular tumor, astrocytoma, acute myelogenous leukemia, breast carcinoma, bladder carcinoma, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, esophageal squamous cell carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, urothelial carcinoma.

Embodiments of the present invention are also directed to methods for treating a subject with a disease or pathological condition, wherein the method comprises one or more of the steps of (a) determining an expression profile of one or more genes expressed in said subject; (b) assessing the sensitivity of the subject to therapy based on the expression profile; (c) selecting a therapy based on the assessed sensitivity; and (d) treating the subject using the selected therapy. Samples

In the method for assisting in diagnosing cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or diagnosing cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or determining cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or determining a predisposition for cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder in an individual, the determination of the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide and/or the determination of the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is performed on a sample from said individual.

The determination may be performed on a sample removed from the individual.

In one embodiment, the determination is performed on samples selected from the group consisting of breast tissue, ovarian tissue, uterine tissue, colon tissue, prostate tissue, lung tissue, renal tissue, thymus tissue, testis tissue, hematopoietic tissue, bone marrow, urogenital tissue, expiration air, stem cells, including cancer stem cell, and body fluids, such as sputum, urine, blood and sweat. In one embodiments the sample is selected from the group consisting of skin tissue, including epidermal and dermal tissue, breast tissue, ovarian tissue, uterine tissue, colon tissue, prostate tissue, lung tissue, renal tissue, thymus tissue, testis tissue, hematopoietic tissue, bone marrow, urogenital tissue, expiration air, stem cells, including cancer stem cell, and body fluids, such as sputum, urine, blood and sweat.

In a preferred embodiment the sample is selected from the group consisting of breast tissue, ovarian tissue, uterine tissue, colon tissue, prostate tissue, lung tissue and urogenital tissue. In one embodiment the sample is lung tissue. In another especially preferred embodiment the sample is prostate tissue. In another especially preferred embodiment the sample is ovarian tissue. The sample is in yet another embodiment derived from cancer tissue, such as a tissue of a cancer as described herein.

In the method for identifying a compound for the treatment and/or diagnosis of cancer or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, according to the present invention, the provided sample is derived from any of the tissues as defined elsewhere herein. For example, the sample is selected from the group consisting of skin tissue, including epidermal and dermal tissue, breast tissue, ovarian tissue, uterine tissue, colon tissue, prostate tissue, lung tissue, renal tissue, thymus tissue, testis tissue, hematopoietic tissue, bone marrow, urogenital tissue, expiration air, stem cells, including cancer stem cell, and body fluids, such as sputum, urine, blood and/or sweat.

A cell, tissue, or subject may thus be a cancer cell, a cancerous tissue, harbor cancerous tissue, or it may be a subject or patient diagnosed or at risk of developing a disease or condition. A cell, tissue, or subject may in principle be or suffer from an abnormal or pathologic condition, or in the case of a cell or tissue, the component of a pathological condition. In certain aspects, a cell, tissue, or subject is a cancer cell, a cancerous tissue or harbor cancerous tissue, or a cancer patient. In a particular aspect the cancer is neuronal, glial, lung, liver, brain, breast, bladder, blood, leukemic, colon, endometrial, stomach, skin, ovarian, esophageal, pancreatic, prostate, kidney, or thyroid cancer.

In one embodiment, the determination is performed in the live animal. In another embodiment, evaluation of the transcriptional and/or translational products is performed without removing the tissue from the live animal. In another embodiment, however, evaluation of the transcriptional and/or translational products is performed on a sample removed from the animal.

Combination treatment

In one important embodiment, the composition and/or splice-switching oligonucleotides of the present invention for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder is combined with a second treatment, or pharmaceutical composition, compound or therapy, for example a combined with another treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or is combined with another medicament for the treatment of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. Also the methods or treatment may be combined with a second therapy for the treatment of cancer. A second therapy or therapeutic agent can include a second microRNA, siRNA, shRNA, antisense RNA, ribozymes and RNA decoys, or another nucleic acid therapy, or one or more standard treatment therapies, such as chemotherapy, drug therapy, radiation therapy, immunotherapy, thermal therapy, and the like. A second therapy can also include administration of a siRNA or antisense oligonucleotide. In a particular aspect, a second therapy is chemotherapy. A chemotherapy can include, but is not limited to paclitaxel, cisplatin, carboplatin, doxorubicin, oxaliplatin, larotaxel, taxol, lapatinib, docetaxel, methotrexate, capecitabine, vinorelbine, cyclophosphamide, gemcitabine, amrubicin, cytarabine, etoposide, camptothecin, dexamethasone, dasatinib, tipifarnib, bevacizumab, sirolimus, temsirolimus, everolimus, lonafarnib, cetuximab, erlotinib, gefitinib, imatinib mesylate, rituximab, trastuzumab, nocodazole, sorafenib, sunitinib, bortezomib, alemtuzumab, gemtuzumab, tositumomab or ibritumomab.

Chemotherapy

A wide variety of chemotherapeutic agents may be used in combination with the compositions and treatments in accordance with the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

Alkylating agents

Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific.

Alkylating agents can be implemented to treat chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, and particular cancers of the breast, lung, and ovary. They include: busulfan, chlorambucil, cisplatin, cyclophosphamide (Cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan. Troglitazaone can be used to treat cancer in combination with any one or more of these alkylating agents.

Antimetabolites

Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. They have been used to combat chronic leukemias in addition to tumors of breast, ovary and the gastrointestinal tract. Antimetabolites include 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.

5-Fluorouracil (5-FU) has the chemical name of 5-fluoro-2,4(IH,3H)- pyrimidinedione. Its mechanism of action is thought to be by blocking the methylation reaction of deoxyuridylic acid to thymidylic acid. Thus, 5-FU interferes with the synthesis of deoxyribonucleic acid (DNA) and to a lesser extent inhibits the formation of ribonucleic acid (RNA). Since DNA and RNA are essential for cell division and proliferation, it is thought that the effect of 5-FU is to create a thymidine deficiency leading to cell death. Thus, the effect of 5-FU is found in cells that rapidly divide, a characteristic of metastatic cancers.

Antitumor Antibiotics

Antitumor antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle. Thus, they are widely used for a variety of cancers. Examples of antitumor antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), and idarubicin, some of which are discussed in more detail below. Widely used in clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m<2> at 21 day intervals for adriamycin, to 35- 100 mg/m<2> for etoposide intravenously or orally, d. Mitotic Inhibitors Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors comprise docetaxel, etoposide (VP 16), paclitaxel, taxol, taxotere, vinblastine, vincristine, and vinorelbine. Nitrosureas

Nitrosureas, like alkylating agents, inhibit DNA repair proteins. They are used to treat non-Hodgkin's lymphomas, multiple myeloma, malignant melanoma, in addition to brain tumors. Examples include carmustine and lomustine. Radiotherapy

Radiotherapy, also called radiation therapy, is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly.

Radiotherapy may be used to treat localized solid tumors, such as cancers of the skin, tongue, larynx, brain, breast, or cervix. It can also be used to treat leukemia and lymphoma (cancers of the blood-forming cells and lymphatic system, respectively). Radiation therapy used according to the present invention may include, but is not limited to, the use of [gamma]-rays, X-rays, and/or the directed delivery of

radioisotopes to tumor cells. Other forms of DNA damaging factors are also

contemplated such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287) and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. Radiotherapy may comprise the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmunotherapy). Once injected into the body, the antibodies actively seek out the cancer cells, which are destroyed by the cell-killing (cytotoxic) action of the radiation. This approach can minimize the risk of radiation damage to healthy cells.

Stereotactic radio-surgery (gamma knife) for brain and other tumors does not use a knife, but very precisely targeted beams of gamma radiotherapy from hundreds of different angles. Only one session of radiotherapy, taking about four to five hours, is needed. For this treatment a specially made metal frame is attached to the head. Then, several scans and x- rays are carried out to find the precise area where the treatment is needed. During the radiotherapy for brain tumors, the patient lies with their head in a large helmet, which has hundreds of holes in it to allow the radiotherapy beams through. Related approaches permit positioning for the treatment of tumors in other areas of the body.

Immunotherapy

In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Trastuzumab (Herceptin(TM)) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers. In one aspect of immunotherapy, the tumor or disease cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma- IFN, and chemokines such as MIP-I, MCP-I, IL-8 and growth factors such as FLT3 ligand. Combining immune stimulating molecules, either as proteins or using gene delivery in combination with a tumor suppressor such as MDA-7 has been shown to enhance anti-tumor effects (Ju et al, 2000). Moreover, antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein. Examples of immunotherapies currently under investigation or in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Patents 5,801 ,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy e.g., interferons [alpha], [beta] and [gamma]; IL-I, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al, 1998;

Hellstrand et al, 1998) gene therapy e.g., TNF, IL-I, IL-2, p53 (Qin et al, 1998; Austin- Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945) and monoclonal antibodies e.g., anti-ganglioside GM2, anti-HER- 2, anti-pl85; Pietras et al, 1998;

Hanibuchi et al, 1998; U.S. Patent 5,824,31 1 ). Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It possesses anti-tumor activity and has been approved for use in the treatment of malignant tumors (Dillman, 1999). A non-limiting list of several known anti-cancer immunotherapeutic agents and their targets includes, but is not limted to (Generic Name (Target))

Cetuximab (EGFR), Panitumumab (EGFR), Trastuzumab (erbB2 receptor),

Bevacizumab (VEGF), Alemtuzumab (CD52), Gemtuzumab ozogamicin (CD33), Rituximab (CD20), Tositumomab (CD20), Matuzumab (EGFR), Ibritumomab tiuxetan (CD20), Tositumomab (CD20), HuP AM4 (MUCI), MORAb-009 (Mesothelin), G250 (carbonic anhydrase IX), mAb 8H9 (8H9 antigen), M195 (CD33), Ipilimumab (CTLA4), HuLuc63 (CSI), Alemtuzumab (CD53), Epratuzumab (CD22), BC8 (CD45), HuJ591 (Prostate specific membrane antigen), hA20 (CD20), Lexatumumab (TRAIL receptor- 2), Pertuzumab (HER-2 receptor), Mik-beta-1 (IL-2R), RAV12 (RAAG12), SGN-30 (CD30), AME-133v (CD20), HeFi-l (CD30), BMS-663513 (CD137), Volociximab (anti- [alpha]5[beta]l integrin), GC1008 (TGF[beta]), HCD 122 (CD40), Siplizumab (CD2), MORAb-003 (Folate receptor alpha), CNTO 328 (IL- 6), MDX-060 (CD30),

Ofatumumab (CD20), or SGN-33 (CD33). It is contemplated that one or more of these therapies may be employed with the miRNA or siRNA therapies described herein.

A number of different approaches for passive immunotherapy of cancer exist. They may be broadly categorized into the following: injection of antibodies alone; injection of antibodies coupled to toxins or chemotherapeutic agents; injection of antibodies coupled to radioactive isotopes; injection of anti-idiotype antibodies; and finally, purging of tumor cells in bone marrow.

Gene Therapy

In yet another embodiment, a combination treatment involves gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as one or more therapeutic miRNA, siRNA, shRNA, antisense, ribozymes or RNA decoys.

Delivery of a therapeutic polypeptide or encoding nucleic acid in conjunction with a miRNA, siRNA, shRNA, antisense, ribozymes or RNA decoys may have a combined therapeutic effect on target tissues. A variety of proteins are encompassed within the invention, some of which are described below. Various genes that may be targeted for gene therapy of some form in combination with the present invention include, but are not limited to inducers of cellular proliferation, inhibitors of cellular proliferation, regulators of programmed cell death, cytokines and other therapeutic nucleic acids or nucleic acid that encode therapeutic proteins.

The tumor suppressor oncogenes function to inhibit excessive cellular proliferation. The inactivation of these genes destroys their inhibitory activity, resulting in unregulated proliferation. The tumor suppressors (e.g., therapeutic polypeptides) p53, FHIT, pi 6 and C-CAM can be employed.

In addition to p53, another inhibitor of cellular proliferation is pi 6. The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, or CDK' s. One CDK, cyclin-dependent kinase 4 (CDK4), regulates progression through the Gl. The activity of this enzyme may be to phosphorylate Rb at late Gl. The activity of CDK4 is controlled by an activating subunit, D-type cyclin, and by an inhibitory subunit, the pl6INK4 has been biochemically characterized as a protein that specifically binds to and inhibits CDK4, and thus may regulate Rb phosphorylation (Serrano et al, 1993; Serrano et al, 1995). Since the pl6INK4 protein is a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the activity of CDK4, resulting in

hyperphosphorylation of the Rb protein, pi 6 also is known to regulate the function of CDK6. pl6INK4 belongs to a newly described class of CDK-inhibitory proteins that also includes pl6B, pi 9, p2 IWAFI, and p27KIPI . The pl6INK4 gene maps to 9p21 , a chromosome region frequently deleted in many tumor types. Homozygous deletions and mutations of the pl6INK4 gene are frequent in human tumor cell lines. This evidence suggests that the pl6INK4 gene is a tumor suppressor gene. This

interpretation has been challenged, however, by the observation that the frequency of the pl6INK4 gene alterations is much lower in primary uncultured tumors than in cultured cell lines (Caldas et al, 1994; Cheng et al, 1994; Hussussian et al, 1994; Kamb et al, 1994; Mori et al, 1994; Okamoto et al, 1994; Nobori et al, 1995; Orlow et al, 1994; Arap et al, 1995). Restoration of wild-type pl6INK4 function by transfection with a plasmid expression vector reduced colony formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

Other genes that may be employed according to the present invention include Rb, APC, DCC, NF-I, NF-2, WT-I, MEN-I, MEN-II, zacl, p73, VHL, MMACI / PTEN,

DBCCR-I, FCC, rsk-3, p27, p27/pl6 fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-I, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp, hst, abl, EIA, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-I, GDAIF, or their receptors) and MCC.

Surgery

Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1 , 2, 3, 4, 5, 6, or 7 days, or every 1 , 2, 3, 4, and 5 weeks or every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 12 months. These treatments may be of varying dosages as well. 6. Other Agents It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-I, MIP-I beta, MCP-I, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL (Apo-2 ligand) would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a

hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy. Apo2 ligand (Apo2L, also called TRAIL) is a member of the tumor necrosis factor (TNF) cytokine family. TRAIL activates rapid apoptosis in many types of cancer cells, yet is not toxic to normal cells. TRAIL mRNA occurs in a wide variety of tissues. Most normal cells appear to be resistant to TRAIL'S cytotoxic action, suggesting the existence of mechanisms that can protect against apoptosis induction by TRAIL. The first receptor described for TRAIL, called death receptor 4 (DR4), contains a cytoplasmic "death domain"; DR4 transmits the apoptosis signal carried by TRAIL. Additional receptors have been identified that bind to TRAIL. One receptor, called DR5, contains a cytoplasmic death domain and signals apoptosis much like DR4. The DR4 and DR5 mRNAs are expressed in many normal tissues and tumor cell lines. Recently, decoy receptors such as DcRI and DcR2 have been identified that prevent TRAIL from inducing apoptosis through DR4 and DR5. These decoy receptors thus represent a novel mechanism for regulating sensitivity to a pro-apoptotic cytokine directly at the cell's surface. The preferential expression of these inhibitory receptors in normal tissues suggests that TRAIL may be useful as an anticancer agent that induces apoptosis in cancer cells while sparing normal cells. (Marsters et ah, 1999).

There have been many advances in the therapy of cancer following the introduction of cytotoxic chemotherapeutic drugs. However, one of the consequences of

chemotherapy is the development/acquisition of drug-resistant phenotypes and the development of multiple drug resistance. The development of drug resistance remains a major obstacle in the treatment of such tumors and therefore, there is an obvious need for alternative approaches such as gene therapy.

Another form of therapy for use in conjunction with chemotherapy, radiation therapy or biological therapy includes hyperthermia, which is a procedure in which a patient's tissue is exposed to high temperatures (up to 106[deg.]F). External or internal heating devices may be involved in the application of local, regional, or whole-body

hyperthermia. Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body. Internal heat may involve a sterile probe , including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae, or radiofrequency electrodes. A patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets. Alternatively, some of the patient's blood may be removed and heated before being perfused into an area that will be internally heated. Whole-body heating may also be implemented in cases where cancer has spread throughout the body. Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose.

Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases. Expression vector

In one aspect, the present invention provides a composition comprising at least one binding member capable of binding to a HER4 polypeptide and/or a HER4 RNA, thereby a) specifically downregulating the level of HER4 CYT-1 polypeptide and/or RNA encoding a HER4 CYT-1 polypeptide and/or b) specifically upregulating the level or activity of HER4 CYT-2 polypeptide and/or RNA encoding a HER4 CYT-2 polypeptide for use as a medicament, wherein the composition further comprises a) HER4 CYT-2 polypeptide or a functional homolog or fragment thereof, and/or b) nucleic acid encoding HER4 CYT-2 polypeptide or a functional homolog or fragment thereof. Thus, in one embodiment the composition also comprises a HER4 CYT-2 polypeptide or a functional homolog or fragment thereof. In this way, a binding member may specifically downregulate HER4 CYT-1 , while HER4 CYT-2 is provided as a mature polypeptide. However, in another embodiment, the composition further comprises a nucleic acid encoding HER4 CYT-2 polypeptide or a functional homolog or fragment thereof. In this way, HER4 CYT-2 polypeptide is provided via a nucleic acid sequence encoding HER4 CYT-2 or part thereof, for example an RNA, DNA or cDNA sequence encoding CYT-2. The nucleic acid is for example comprised in an expression vector allowing the functional expression of HER4 CYT-2 in a human cell. By having the polynucleotide positioned in a vector the HER4 polynucleotide can also easily be stored, amplified, modified and expressed. The term "expression vector" refers to a DNA molecule used as a vehicle to transfer recombinant genetic material into a host cell. The four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artifical chromosomes. The vector itself is generally a DNA sequence that consists of an insert (a heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the

"backbone" of the vector. The purpose of a vector which transfers genetic information to the host is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (expression constructs) are specifically adapted for the expression of the heterologous sequences in the target cell, and generally have a promoter sequence that drives expression of the heterologous sequences. Simpler vectors called transcription vectors are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed, unlike expression vectors. Transcription vectors are used to amplify the inserted heterologous

sequences. The transcripts may subsequently be isolated and used in as templates suitable in vitro translations systems.

Construction of vectors for recombinant expression of HER4, in particular HER4 CYT-2 polypeptides for use in the invention may be accomplished using conventional techniques which do not require detailed explanation to one of ordinary skill in the art. For review, however, those of ordinary skill may wish to consult Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, (NY 1982). Expression vectors may be used for generating producer cells for recombinant production of NsG33 polypeptides for medical use, and for generating therapeutic cells secreting NsG33 polypeptides for naked or encapsulated therapy.

For generation of efficient expression vectors, these should contain regulatory sequences necessary for expression of the encoded gene in the correct reading frame. Expression of a gene is controlled at the transcription, translation or post-translation levels. Transcription initiation is an early and critical event in gene expression. This depends on the promoter and enhancer sequences and is influenced by specific cellular factors that interact with these sequences. The transcriptional unit of many genes consists of the promoter and in some cases enhancer or regulator elements (Banerji et al., Cell 27: 299 (1981 ); Corden et al., Science 209: 1406 (1980); and Breathnach and Chambon, Ann. Rev. Biochem. 50: 349 (1981 )). For retroviruses, control elements involved in the replication of the retroviral genome reside in the long terminal repeat (LTR) (Weiss et al., eds., The molecular biology of tumor viruses: RNA tumor viruses, Cold Spring Harbor Laboratory, (NY 1982)). Moloney murine leukemia virus (MLV) and Rous sarcoma virus (RSV) LTRs contain promoter and enhancer sequences (Jolly et al., Nucleic Acids Res. 1 1 : 1855 (1983); Capecchi et al., In :

Enhancer and eukaryotic gene expression, Gulzman and Shenk, eds., pp. 101 -102, Cold Spring Harbor Laboratories (NY 1991 ). Other potent promoters include those derived from cytomegalovirus (CMV) and other wild-type viral promoters. The invention provides medical use of genomic DNA and cDNA coding for HER4 CYT- 2, including for example the nucleotide sequence (SEQ ID No. 3). Variants of these sequences are also included within the scope of the present invention.

One aspect of the present invention pertains to an isolated polynucleotide for use as a medicament comprising or consisting of a nucleic acid or its complementary sequence, said polynucleotide being selected from the group consisting of SEQ ID NO: 1 -3, or shares at least 70%, such as at least 80%, for example at least 90%, for example at least 91 , for example at least 92, for example at least 93, for example at least 94, for example at least 95, for example at least 96, for example at least 97, for example at least 98, for example at least 99, for example at least 100% identity with any one of SEQ ID NO: 1 -3 or the complement thereof.

In a further, preferred embodiment of the invention the polynucleotide is

complementary to

i) a polynucleotide encoding an amino acid sequence consisting of SEQ ID No. 1 -3; or

ii) a polynucleotide encoding a functional fragment or homolog of the amino acid sequence, having at least 70% sequence identity to said SEQ ID NO. 1 -3, or

iii) a polynucleotide encoding a biologically active fragment of at least 50 contiguous amino acids of any of a) through b), wherein said fragment is a fragment of SEQ ID NO 1 -3.

The polynucleotide may comprise the nucleotide sequence of a naturally occurring allelic nucleic acid variant of HER4. The polynucleotide of the invention may encode a variant polypeptide, wherein the variant polypeptide has the polypeptide sequence of a naturally occurring polypeptide variant.

In one embodiment, the nucleic acid sequence of the polynucleotide has at least 80% sequence identity, such as preferably at least 81 % sequence identity, more preferably e.g. at least 82% sequence identity, such as more preferably at least 83% sequence identity, e.g. more preferably at least 84% sequence identity, more preferably such as at least 85% sequence identity, more preferably e.g. at least 86% sequence identity, more preferably such as at least 87% sequence identity, more preferably e.g. at least 88% sequence identity, more preferably such as at least 89% sequence identity, more preferably e.g. at least 90% sequence identity, more preferably such as at least 91 % sequence identity, more preferably e.g. at least 92% sequence identity, such as at least 93% sequence identity, more preferably e.g. at least 94% sequence identity, more preferably such as at least 95% sequence identity, more preferably e.g. at least 96% sequence identity, more preferably such as at least 97% sequence identity, more preferably e.g. at least 98% sequence identity, more preferably such as at least 99% sequence identity, more preferably e.g. at least 99.5% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID No: 1 ,2 and 3. The nucleic acid sequence of the polynucleotide may contain less than 99.5%, such as less than 98%, e.g. less than 97%, such as less than 96%, e.g. less than 95%, such as less than 94%, e.g. less than 93%, such as less than 92%, e.g. less than 91 %, such as less than 90%, e.g. less than 88%, such as less than 86%, e.g. less than 84%, e.g. less than 82%, such as less than 80%, e.g. less than 75%, such as less than 70%, e.g. less than 65%, such as less than 60%, e.g. less than 55%, such as less than 50%, e.g. less than 45%, such as less than 40%, e.g. less than 35%, such as less than 30%, e.g. less than 25%, such as less than 20%, such as less than 15%, e.g. less than 10% of nucleotide sequence selected from the group consisting of SEQ ID No: 1 ,2 and 3. In a preferred embodiment the encoded polypeptide has at least 70% sequence identity to SEQ ID No. 5, more preferably at least 75%, more preferably at least 80%, more preferably at least 95%, more preferably at least 98%, more preferably wherein said polypeptide has the sequence of SEQ ID No. 5. In yet another embodiment the polynucleotide is capable of hybridizing to the nucleic acid selected from the group consisting of SEQ ID NO: SEQ ID NO: 1 , 2, and 3, or a fragment hereof, under stringent conditions as described below. A portion of the polynucleotide may hybridize under stringent conditions to a nucleotide probe corresponding to at least 10 consecutive nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 1 , 2, and 3.

Dosage regimes

The dosage requirements of monomeric alpha-lactalbuminin complex, preferably LAC to be administered will vary with the particular drug composition employed, the route of administration and the particular subject being treated. Ideally, a patient to be treated by the present method will receive a pharmaceutically effective amount of the compound in the maximum tolerated dose, generally no higher than that required before drug resistance develops.

For all methods of use disclosed herein for the compounds, the daily oral dosage regimen will preferably be from about 0.01 to about 80 mg/kg of total body weight. The daily parenteral dosage regimen may be about 0.001 to about 80 mg/kg of total body weight. The daily topical dosage regimen will preferably be from 0.1 mg to 150 mg, administered one to four, preferably two or three times daily. The daily inhalation dosage regimen will preferably be from about 0.01 mg/kg to about 1 mg/kg per day. It will also be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound or a pharmaceutically acceptable salt thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound or a pharmaceutically acceptable salt thereof given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

The daily dose of the active compound varies and is dependant on the type of administrative route, but as a general rule it is 1 to 100 mg/dose of active compound at personal administration, and 2 to 200 mg/dose in topical administration. The number of applications per 24 hours depend of the administration route, but may vary, e. g. in the case of a topical application in the no. se from 3 to 8 times per 24 hours, i. e. , depending on the flow of phlegm produced by the body treated in therapeutic use. The compound according to the present invention is given in an effective amount to an individual in need there of. The amount of compound according to the present invention in one preferred embodiment is in the range of from about 0.01 milligram per kg body weight per dose to about 20 milligram per kg body weight per dose, such as from about 0.02 milligram per kg body weight per dose to about 18 milligram per kg body weight per dose, for example from about 0.04 milligram per kg body weight per dose to about 16 milligram per kg body weight per dose, such as from about 0.06 milligram per kg body weight per dose to about 14 milligram per kg body weight per dose, for example from about 0.08 milligram per kg body weight per dose to about 12 milligram per kg body weight per dose, such as from about 0.1 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 0.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 0.3 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 0.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 0.5 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 0.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 0.7 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 0.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 0.9 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 1 .0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 1 .2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 1 .4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 1 .6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 1 .8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 2.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 2.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 2.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 2.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 2.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 3.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 3.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 3.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 3.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 3.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 4.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 4.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 4.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 4.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 4.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 5.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 5.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 5.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 5.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 5.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 6.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 6.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 6.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 6.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 6.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 7.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 7.2 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 7.4 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 7.6 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 7.8 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, for example from about 8.0 milligram per kg body weight per dose to about 10 milligram per kg body weight per dose, such as from about 0.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 0.3 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 0.4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 0.5 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 0.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 0.7 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 0.8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 0.9 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 1 .0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 1 .2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 1 .4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 1 .6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 1 .8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 2.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 2.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 2.4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 2.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 2.8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 3.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 3.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 3.4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 3.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 3.8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 4.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 4.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 4.4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 4.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 4.8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 5.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 5.2 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 5.4 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 5.6 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 5.8 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, for example from about 6.0 milligram per kg body weight per dose to about 8 milligram per kg body weight per dose, such as from about 0.2 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 0.3 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 0.4 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 0.5 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 0.6 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 0.7 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 0.8 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 0.9 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 1 .0 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 1 .2 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 1 .4 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 1 .6 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 1 .8 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 2.0 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 2.2 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 2.4 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 2.6 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 2.8 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 3.0 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 3.2 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 3.4 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 3.6 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 3.8 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 4.0 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 4.2 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 4.4 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 4.6 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, for example from about 4.8 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose, such as from about 5.0 milligram per kg body weight per dose to about 6 milligram per kg body weight per dose.

The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a

predetermined quantity of a compound, alone or in combination with other agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound or compounds employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. The dose administered should be an "effective amount" or an amount necessary to achieve an "effective level" in the individual patient.

Since the "effective level" is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on individual differences in pharmacokinetics, drug distribution, and metabolism. The "effective level" can be defined, for example, as the blood or tissue level desired in the patient that corresponds to a concentration of one or more compounds according to the invention.

Diagnostic methods and kits

In a further aspect, the present invention relates to a method for assisting in diagnosing cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or diagnosing cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or determining cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or determining a predisposition for cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder in an individual, said method comprising the steps of

a. determining the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT- 1 polypeptide in a sample from said individual, and/or

b. determining in said sample the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide, and

c. comparing the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide with the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide.

In the preferred embodiment of the method, the presence of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide is indicative of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or predisposition of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder, and/or the presence of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is indicative of a reduced risk of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or a reduced predisposition for cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. In one

embodiment a higher level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide relative to HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is indicative of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder or predisposition of cancer and/or neuropsyciatric disorders, such as schizophrenia or bipolar disorder. In another embodiment, the level of HER4 CYT-1 polypeptide and/or RNA encoding HER4 CYT-1 polypeptide and/or the level of HER4 CYT-2 polypeptide and/or RNA encoding HER4 CYT-2 polypeptide is determined by using a binding member of the present invention. The cancer determined or diagnosed according to the method of the invention is in one embodiment a growth factor dependent cancer, and in another embodiment, cells derived from cancer or tumour tissue expresses HER4 CYT-1 polypeptide or a functional homolog or fragment thereof and/or RNA encoding said HER4 CYT-1 polypeptide or functional homolog or fragment thereof. The cancer is in one

embodiment selected from the group consisting of breast carcinoma, bladder carcinoma, anaplastic large cell lymphoma, B-cell lymphoma, chronic lymphoblastic leukemia, multiple myeloma, testicular tumor, astrocytoma, acute myelogenous leukemia, cervical carcinoma, colorectal carcinoma, endometrial carcinoma, esophageal squamous cell carcinoma, glioma, glioblastoma, gastric carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, leukemia, lipoma, melanoma, mantle cell lymphoma, myxofibrosarcoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma, esophageal carcinoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, squamous cell carcinoma of the head and neck, thyroid carcinoma, and urothelial carcinoma.

In a preferred embodiment, the cancer is breast carcinoma or bladder carcinoma. The sample wherein HER4 polypeptide or RNA is determined is preferably derived from cancer or tumor tissue. The present invention also provides a diagnostic kit comprising at least one binding member or detection member capable of binding to a HER4 polypeptide and/or a HER4 RNA. The kit comprises in one embodiment buffers and/or primers for nucleic acid amplification. In one embodiment, the detection member is an oligonucleotide primer comprising at least 10 consecutive nucleic acids, such as between 10 and 50 nucleotides selected from a HER4 gene, for example selected from any of SEQ ID NO: 1 -3. In a specifically preferred embodiment, the detection member is an oligonucleotide primer comprising or consisting of at least one of the following sequences:

CYT-1 :

Forward: 5'- GGATGAAGAGGATTTGGAAG -3';

Reverse: 5'- TCCTGACATGGGGGTGTA-3'.

CYT2:

Forward: 5'- GAATAGGAACCAGTTTGTATACCG-3';

Reverse: 5'- ACAGCAGGAGTCATCAAAAATC-3'. Diagnosticum The term "diagnosticum" refers in the present context to a compound or composition used in diagnosis of a disease or medical state. In the present text the diagnosticum is a binding member or a detection member of the present invention or active derivative thereof for use in the diagnosis of a disease or condition, as described herein above.

The binding members, polynucleotides (eg. PCR primers, such as SEQ ID NO: 9-12), polypeptides, methods, compositions and kits of the invention may also be used as a diagnosticum, Thus, in an aspect the invention relates to an isolated binding member or an active derivative thereof according to the invention for use as a diagnosticum.

The expression can be detected using a standard sandwich assay known to the person skilled in the art. If no secondary antibody able to bind to the polypeptides of the present invention (after binding to the receptor) a tag may be positioned, in frame, on the polypeptides, thereby allowing binding and detection of the secondary antibody. Thus in an embodiment the invention relates to the polypeptides for use in determining the expression level of a HER4 receptor in a sample. The amount of receptor may be used for determining the amount of a pharmaceutical composition, or polypeptide of the present invention is needed in a specific treatment of an individual. The binding members may also be used as a diagnosticum to provide precise measurements of HER4 CYT-1 or CYT-2 in a sample.

Detection

Certain embodiments of the invention can include a determination of the expression of one or more markers, genes, or nucleic acids representative thereof, by using e.g. an amplification assay, a hybridization assay, or protein assay, a variety of which are well known to one of ordinary skill in the art.

In certain aspects, an amplification assay can be a quantitative amplificationassay, such as quantitative RT-PCR or the like. In still further aspects, a hybridization assay can include array hybridization assays or solution hybridization assays. The nucleic acids from a sample may be labeled from the sample and/or hybridizing the labeled nucleic acid to one or more nucleic acid probes. Nucleic acids, mRNA, and/or nucleic acid probes may be coupled to a support. Such supports are well known to those of ordinary skill in the art and include, but are not limited to glass, plastic, metal, or latex. In particular aspects of the invention, the support can be planar or in the form of a bead or other geometric shapes or configurations known in the art. Proteins are typically assayed by immunoblotting, chromatography, mass spectrometry or other methods known to those of ordinary skill in the art. Examples

Example 1

The CYT-2 isoform was found to be associated with decreased cell growth as compared to CYT1 .

SSOs have shown their potential as therapeutics and therapies using SSOs are in clinical trial (12).

Here, we show that SSO-induced skipping of exon 26 in HER4 pre-mRNA results in a significant reduction of the CYT1 isoform of HER4 and concomitant reduced cell proliferation of HER4 expressing NCI358 lung cancer cells and MDA-MB-231 breast cancer cells.

Methods

Cell lines:

Human lung cancer cell line NCI358 and human breast cancer cell line MDA-MB-231 were obtained from the American Type Culture Collection. NCI358 cells were cultured in RPMI medium supplemented with 10 % fetal bovine serum, 1 % penicillin streptomycin 1 % hepes and 1 % sodium pyruvate. MDA-MB-231 cells were cultured in DMEM + GlutaMAX-1 medium containing 10 % fetal bovine serum and 1 % penicillin streptomycin.

Oligonucleotide treatment:

15-mer LNA modified oligoribonucleotide SSOe26 (5'-(l)T (m)U (l)A (m)C (m)U (l)C (m)C (m)U (l)G (m)A (m)C (l)A (m)U (l)G (l)G-3' (I = LNA, m = methylatet)) antisense to the 5' splice site of HER4 exon 26 was used (covering nucleotide no. 3271 -3285 according to NM 005235.2, NCBI). A scrambled oligonucleotide, SSOsc, containing the same nucleotides as SSOe26 at scrambled positions and modifications at the same positions as in SSOe26, was used as a negative control (5'-(l)G (m)T (l)T (m)C (m)A (l)G (m)T (m)C (l)G (m)C (m)A (l)T (m)A (l)C (Ι)Τ-3'). All oligonucleotides were synthesized and purified. Twenty-four hours prior to oligonucleotide treatment, the cells were plated in2 ml of medium in 6-well plates at a density of 2.5 x 105 cells/well. The cells were treated with oligonucleotides complexed with LipofectamineTM 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's directions at the concentrations indicated on the figures. Analyses were carried out 24 hr after transfection for Q-PCR or continuously for cell proliferation assays.

RNA isolation and quantitative reverse transcription polymerase chain reaction (Q- PCR):

RNA was purified according to the manufacturer's directions (Qiagen "Total RNA isolation kit"). The RNA was resuspended in RNase free water. The amount of RNA was quantified by UV spectroscopy (A260nm = 1 corresponds to 40 μg/ml RNA). 0.4 μg of RNA was used for cDNA synthesis.

2 μΙ of cDNA was used for Q-PCR on a Roche lightcycler 480 at: 95 °C, 10 min; 50 cycles of 95 °C for 10 sec, 56 °C for 10 sec, 72 °C for 5 sec; 99 °C for 1 sec; 59 °C for 15 sec; 95 °C for 1 sec; cooling to 40 °C. The forward and reverse primers were CYT1 :

Forward: 5'- GGATGAAGAGGATTTGGAAG -3';

Reverse: 5'- TCCTGACATGGGGGTGTA-3'.

CYT2:

Forward: 5'- GAATAGGAACCAGTTTGTATACCG-3';

Reverse: 5'- ACAGCAGGAGTCATCAAAAATC-3'.

Cell proliferation assay:

Cell proliferation was measured using Roche xCELLigence system from Roche.

The System measures electrical impedance across interdigitated micro-electrodes integrated on the bottom of tissue culture E-Plates.

The presence of the cells on top of the electrodes will affect the local ionic environment at the electrode/solution interface, leading to an increase in the electrode impedance. The more cells are attached on the electrodes, the larger the increases in electrode impedance. In addition, the impedance depends on the quality of the cell interaction with the electrodes. For example, increased cell adhesion or spreading will lead to a larger change in electrode impedance. Thus, electrode impedance, which is displayed as cell index (CI) values, can be used to monitor cell viability, number, morphology, and adhesion degree in a number of cell-based assays. A dimensionless parameter termed Cell Index (CI) is derived as a relative change in measured electrical impedance to represent cell status. Several features of CI are summarized below:

• When cells are not present or are not well-adhered on the electrodes, the CI is zero.

• Under the same physiological conditions, when more cells are attached on the electrodes, the CI values are larger. Thus, CI is a quantitative measure of cell number present in a well.

• Additionally, change in a cell status, such as cell morphology, cell adhesion, or cell viability will lead to a change in CI.

(From Roche homepage)

Cell index was measured using the software provided by the manufacturer. 50 μΙ_ of cell culture media was added into each well of an E-plate 96 and background impedance of the plate was determined. Subsequently 100 μΙ of the cell suspension was added (5000 cells/well). Cells were incubated for 30 min at room temperature and E-plates were placed into the Real-Time Cell Analyzer (RTCA) station. Cells were grown for 120 h, with impedance measured every 1 h. After 24 h 50 μΙ of fresh media/media containing lipofectamineTM 2000/oligonucleotides complexed with LipofectamineTM 2000 was added.

Results:

To be able to inhibit the inclusion of exon 26, we have designed a 15-mer LNA modified SSO antisense to the 5' splice site of HER4 exon 26 (SSOe26). A control oligo was designed by scrambling the nucleotides present in SSOe26 and keeping the positions of the modifications (SSOsc) (figurel ).

Figure 1 A) Inclusion of exon 26 in HER4 pre-mRNA generates the CYT1 isoform. Skipping of exon 26 generates the CYT2 isoform. B) Part of the HER4 mRNA sequence showing the 3'-end of intron 25, exon 26 (green) and the 5'-end of intron 26. The sequence of 15 nucleotides annealing with SSOe26 is underlined (red). The numbers indicate the nucleotide number of the first and the last nucleotide in exon 26 according to NM_005235.2, NCBI C) Sequence and modifications of SSOe26 and SSOsc. (I) = LNA, (m) = methylation. Skipping of HER4 exon 26 in SSO-treated NCI358 lung cancer cells

The ability of SSOe26 to inhibit inclusion of HER4 exon 26 was tested in human lung cancer cells NCI358. We transfected the cells with increasing amounts of SSOe26. Q- PCR analysis of total cellular mRNA 24-hr post-treatment showed that SSOe26 induced a dose-dependent skipping of exon 26, as indicated by a decrease in the concentration of CYT1 mRNA and an increase in CYT2 mRNA (figure 2). The exon- skipping effect was sequence- specific, as SSOsc did not induce any exon skipping (figure kommer snarest). Figure 2 mRNA expression levels of CYT1 and CYT2. Cells were treated with fresh media (Control), transfection reagent only (Lipo) or increasing amounts of SSOe26 as indicated. Expressions are the ratio of CYT1 or CYT2 expression to the β-actin expression.

Growth inhibition of NCI358 and MDA-MB-231 cells caused by SSOe26

Having confirmed that SSOe26 is able to inhibit the inclusion of exon26, we investigated the effect of the oligo on cell proliferation in NCI358 and MDA-MB-231 cells (figure 3). As can be seen, SSOe26 has an inhibitory effect on cell proliferation in both cell lines, whereas the control SSO with a scrambled sequence (SSOsc), showed only a minor (NCI358) or no effect (MDA-MB-231 ) on cell growth.

Figure 3 Growth inhibition of NCI358 and MDA-MB-231 cells by SSOe26 treatment. Cells were treated with fresh media containing lipofectamin (green), control oligo SSOsc (100 nM) (red) or SSOe26 (100 nM) (blue). Shown are the mean of triplicates. Proliferation was measured as the cell index using a Real-Time Cell Analyzer (RTCA) station.

To investigate the concentration of oligo needed for inhibition of cell proliferation, increasing amounts of SSOe26 was transfected into NCI358 cells (1 -100 nM) (figure 4). The results show, that almost the same inhibitory effect is achieved when treating only 50 nM of SSOe26 as is achieved with 100 nM.

Figure 4 Growth inhibition of NCI358 cells by increasing concentrations of SSOe26. Cells were treated with fresh media (light blue, top) fresh media containing lipofectamin (purple, 2nd line from top), 1 nM SSOe26 (red, 3rd line from top), 20 nM SSOe26 (green, 4th from top), 50 nM SSOe26 (blue, 5th from top) or 100 nM SSOe26 (pink, 6th from top). Shown are the mean of triplicates. Proliferation was measured as the cell index using a Real-Time Cell Analyzer (RTCA) station.

Our results show that alternative splicing of HER4 can be directed by the use of SSOs to inhibit expression of the CYT1 isoform while the expression of the CYT2 isoform is maintained. We also show that this switch in alternative splicing decreases cell proliferation of cancer cells. We anticipate this oligo to have potential as a specific drug which can be used to treat cancer patients with tumors that express the CYT1 isoform of HER4. Example 2

Effect of SSOe26 on growth inhibition of cancer cells

Growth inhibition of MCF7 and T47D cells by SSOe26 treatment were investigated, see figure 5.

Dose dependency of SSOe26 treatment of MCF7 and T47D breast cancer cell lines were also investigated. Growth inhibition of MCF7 and T47D cells by increasing concentrations of SSOe26 were studies, see figure 6. Method confirmation

To confirm, that the effect of SSOe26 on cell growth is not method dependent, the experiment was repeated using an MTT assay. This is a colorimetric assay in which increasing number of cells give an increase in transformation of one colorless compound to another colored compound. The amount of color formation is then measured, see figure 7 A). It is verified that SSOe26 inhibit eel growth.

Apoptosis assay

An apoptosis assay was also performed to see if protein expression of PARP in affected by HER4 splicing; see figure 5, which shows protein expression of PARP in NCI-358 lung cancer cells. PARP cleavage is indicative of apoptosis. It is seen that the SSOe26 increases apoptosis. Example 3

SEQ ID NO: 7 has been tested at the RNA level, and was found to be capable of regulating splicing in experiments corresponding to figure 2, although less efficient than SSOe26.

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ErbB/HER receptors. Int J Radiat Oncol Biol Phys 2004 Mar 1 ;58(3):903-13.

(2) Tovey SM, Witton CJ, Bartlett JM, Stanton PD, Reeves JR, Cooke TG. Outcome and human epidermal growth factor receptor (HER) 1 -4 status in invasive breast carcinomas with proliferation indices evaluated by bromodeoxyuridine labelling. Breast Cancer Res 2004;6(3):R246-R251 .

(3) Wiseman SM, Griffith OL, Melck A, Masoudi H, Gown A, Nabi IR, et al. Evaluation of type 1 growth factor receptor family expression in benign and malignant thyroid lesions. Am J Surg 2008 May;195(5):667-73.

(4) Witton CJ, Reeves JR, Going JJ, Cooke TG, Bartlett JM. Expression of the HER1 -4 family of receptor tyrosine kinases in breast cancer. J Pathol 2003 Jul;200(3):290-7.

(5) Suo Z, Risberg B, Kalsson MG, Willman K, Tierens A, Skovlund E, et al. EGFR family expression in breast carcinomas. c-erbB-2 and c-erbB-4 receptors have different effects on survival. J Pathol 2002 Jan;196(1 ):17-25.

(6) Tang CK, Concepcion XZ, Milan M, Gong X, Montgomery E, Lippman ME.

Ribozyme-mediated down-regulation of ErbB-4 in estrogen receptor-positive breast cancer cells inhibits proliferation both in vitro and in vivo. Cancer Res 1999 Oct 15;59(20):5315-22.

(7) Lodge AJ, Anderson JJ, Gullick WJ, Haugk B, Leonard RC, Angus B. Type 1 growth factor receptor expression in node positive breast cancer: adverse prognostic significance of c-erbB-4. J Clin Pathol 2003 Apr;56(4):300-4.

(8) Elenius K, Choi CJ, Paul S, Santiestevan E, Nishi E, Klagsbrun M. Characterization of a naturally occurring ErbB4 isoform that does not bind or activate phosphatidyl inositol 3-kinase. Oncogene 1999 Apr 22;18(16):2607-15.

(9) Browne BC, O'Brien N, Duffy MJ, Crown J, O'Donovan N. HER-2 signaling and inhibition in breast cancer. Curr Cancer Drug Targets 2009 May;9(3):419-38. (10) Okamoto I. Epidermal growth factor receptor in relation to tumor development: EGFR-targeted anticancer therapy. FEBS J 2010 Jan;277(2):309-15.

(1 1 ) Burgess AW. EGFR family: structure physiology signalling and therapeutic targets. Growth Factors 2008 Oct;26(5):263-74.

(12) Kinali M, rechavala-Gomeza V, Feng L, Cirak S, Hunt D, Adkin C, et al. Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in

Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol 2009 Oct;8(10):918-28. Sequences

SEQ ID NO: 1

HER4 genomic DNA

The complete HER4 genomic DNA sequence is retrievable from the NCBI database, cf. http://www.ncbi.nlm.nih.goV/nuccore/NC_000002.1 1 ?&from=212240441 &to=21340335 1 &report=genbank&strand=true

NCBI Reference Sequence: NC_000002.1 1 , Gl:22458981 1

The HER4 exons are located in the genomic sequence as indicated below:

1 ..179 (EXON 1 ),413724..413875 (EXON 2),59101 1 ..591 197 (EXON

3),750468..750602 (EXON 4), 787923.-787988 (EXON 5), 813433..813551 (EXON 6), 816093..816234 (EXON 7), 824979..825092 (EXON 8), 826451 ..826577 (EXON 9), 833236..833309 (EXON 10), 834433.-834523 (EXON 1 1 ), 836461 ..836660 (EXON 12), 859443.-859575 (EXON 13), 865370-865463 (EXON 14), 873150-873304 (EXON 15), 880799-880873 (EXON 16), 908033-908165 (EXON 17), 914583-914705 (EXON 18), 919352-919450 (EXON 19), 976539-976724 (EXON 20), 1 107527-1 107682 (EXON 21 ), 1 1 10144-1 1 10219 (EXON 22), 1 1 14326-1 1 14472 (EXON 23), 1 1 16523-1 1 16620 (EXON 24), 1 1 18016-1 1 18186 (EXON 25), 1 150635-1 150682 (EXON

26), 1 151477..1 151774, (EXON 27), 1 154567..1 162910 (EXON 28)

gene="ERBB47product="v-erb- aerythroblasticleukemiaviraloncogenehomolog4(avian),transcriptvariantJM-a/CVT-1 "

The genomic sequence comprising intron 25-exon 26-intron 26 is provided below; intron 25 (pos. 207-32654), exon 26 (pos. 32655-32702), intron 26 (pos. 32703-33496) SEQ ID NO: 2

HER4 CYT-1 cDNA

NCBI Reference Sequence: NM_005235.2

Nucleotides encoding exon 26 are 3233-3281

SEQ ID NO: 3

HER4 CYT-2 cDNA

NCBI Reference Sequence: NM_001042599.1 SEQ ID NO: 4

HER4 CYT-1 polypeptide

NCBI Reference Sequence: NM_005235.2

EXON26 (amino acids 1046-1061 ) is underlined

MKPATGLWVWVSLLVAAGTVQPSDSQSVCAGTENKLSSLSDLEQQYRALRKYYENC EVVMGNLEITSIEHNRDLSFLRSVREVTGYVLVALNQFRYLPLENLRIIRGTKLYEDRYA LAIFLNYRKDGNFGLQELGLKNLTEILNGGVYVDQNKFLCYADTIHWQDIVRNPWPSN LTLVSTNGSSGCGRCHKSCTGRCWGPTENHCQTLTRTVCAEQCDGRCYGPYVSDC CHRECAGGCSGPKDTDCFACMNFNDSGACVTQCPQTFVYNPTTFQLEHNFNAKYTY GAFCVKKCPHNFVVDSSSCVRACPSSKMEVEENGIKMCKPCTDICPKACDGIGTGSL MSAQTVDSSNIDKFINCTKINGNLIFLVTGIHGDPYNAIEAIDPEKLNVFRTVREITGFLNI QSWPPNMTDFSVFSNLVTIGGRVLYSGLSLLILKQQGITSLQFQSLKEISAGNIYITDNS NLCYYHTINWTTLFSTINQRIVIRDNRKAENCTAEGMVCNHLCSSDGCWGPGPDQCL SCRRFSRGRICIESCNLYDGEFREFENGSICVECDPQCEKMEDGLLTCHGPGPDNCT KCSHFKDGPNCVEKCPDGLQGANSFIFKYADPDRECHPCHPNCTQGCNGPTSHDCI YYPWTGHSTLPQHARTPLIAAGVIGGLFILVIVGLTFAVYVRRKSIKKKRALRRFLETEL VEPLTPSGTAPNQAQLRILKETELKRVKVLGSGAFGTVYKGIWVPEGETVKIPVAIKILN ETTGPKANVEFMDEALIMASMDHPHLVRLLGVCLSPTIQLVTQLMPHGCLLEYVHEHK DNIGSQLLLNWCVQIAKGMMYLEERRLVHRDLAARNVLVKSPNHVKITDFGLARLLEG DEKEYNADGGKMPIKWMALECIHYRKFTHQSDVWSYGVTIWELMTFGGKPYDGIPTR EIPDLLEKGERLPQPPICTIDVYMVMVKCWMIDADSRPKFKELAAEFSRMARDPQRYL VIQGDDRMKLPSPNDSKFFQNLLDEEDLEDMMDAEEYLVPQAFNIPPPIYTSRARIDS NRSEIGHSPPPAYTPMSGNQFVYRDGGFAAEQGVSVPYRAPTSTIPEAPVAQGATAE IFDDSCCNGTLRKPVAPHVQEDSSTQRYSADPTVFAPERSPRGELDEEGYMTPMRD KPKQEYLNPVEENPFVSRRKNGDLQALDNPEYHNASNGPPKAEDEYVNEPLYLNTFA NTLGKAEYLKNNILSMPEKAKKAFDNPDYWNHSLPPRSTLQHPDYLQEYSTKYFYKQ NGRIRPIVAENPEYLSEFSLKPGTVLPPPPYRHRNTVV

SEQ ID NO: 5

HER4 CYT-2 polypeptide

NCBI Reference Sequence: NM_001042599.1

MKPATGLWVWVSLLVAAGTVQPSDSQSVCAGTENKLSSLSDLEQQYRALRKYYENC

EVVMGNLEITSIEHNRDLSFLRSVREVTGYVLVALNQFRYLPLENLRIIRGTKLYEDRYA

LAIFLNYRKDGNFGLQELGLKNLTEILNGGVYVDQNKFLCYADTIHWQDIVRNPWPSN LTLVSTNGSSGCGRCHKSCTGRCWGPTENHCQTLTRTVCAEQCDGRCYGPYVSDC CHRECAGGCSGPKDTDCFACMNFNDSGACVTQCPQTFVYNPTTFQLEHNFNAKYTY GAFCVKKCPHNFVVDSSSCVRACPSSKMEVEENGIKMCKPCTDICPKACDGIGTGSL MSAQTVDSSNIDKFINCTKINGNLIFLVTGIHGDPYNAIEAIDPEKLNVFRTVREITGFLNI QSWPPNMTDFSVFSNLVTIGGRVLYSGLSLLILKQQGITSLQFQSLKEISAGNIYITDNS NLCYYHTINWTTLFSTINQRIVIRDNRKAENCTAEGMVCNHLCSSDGCWGPGPDQCL SCRRFSRGRICIESCNLYDGEFREFENGSICVECDPQCEKMEDGLLTCHGPGPDNCT KCSHFKDGPNCVEKCPDGLQGANSFIFKYADPDRECHPCHPNCTQGCNGPTSHDCI YYPWTGHSTLPQHARTPLIAAGVIGGLFILVIVGLTFAVYVRRKSIKKKRALRRFLETEL VEPLTPSGTAPNQAQLRILKETELKRVKVLGSGAFGTVYKGIWVPEGETVKIPVAIKILN ETTGPKANVEFMDEALIMASMDHPHLVRLLGVCLSPTIQLVTQLMPHGCLLEYVHEHK DNIGSQLLLNWCVQIAKGMMYLEERRLVHRDLAARNVLVKSPNHVKITDFGLARLLEG DEKEYNADGGKMPIKWMALECIHYRKFTHQSDVWSYGVTIWELMTFGGKPYDGIPTR EIPDLLEKGERLPQPPICTIDVYMVMVKCWMIDADSRPKFKELAAEFSRMARDPQRYL VIQGDDRMKLPSPNDSKFFQNLLDEEDLEDMMDAEEYLVPQAFNIPPPIYTSRARIDS NRNQFVYRDGGFAAEQGVSVPYRAPTSTIPEAPVAQGATAEIFDDSCCNGTLRKPVA PHVQEDSSTQRYSADPTVFAPERSPRGELDEEGYMTPMRDKPKQEYLNPVEENPFV SRRKNGDLQALDNPEYHNASNGPPKAEDEYVNEPLYLNTFANTLGKAEYLKNNILSM PEKAKKAFDNPDYWNHSLPPRSTLQHPDYLQEYSTKYFYKQNGRIRPIVAENPEYLSE FSLKPGTVLPPPPYRHRNTVV

SEQ ID NO: 6

15-mer LNA modified oligoribonucleotide SSOe26

(5'-(l)T (m)U (l)A (m)C (m)U (l)C (m)C (m)U (l)G (m)A (m)C (l)A (m)U (l)G (l)G-3' (I = LNA, m = methylatet)) antisense to the 5' splice site of HER4 exon 26 (covering nucleotide no. 3271 -3285 according to NM_005235.2, NCBI).

Same sequence without modification indicated:

5'-TUACUCCUGACAUGG-3'

SEQ ID NO: 7

LNA modified oligoribonucleotide SSO targeting exon 26.

5'-TUTCACUCTAATAGG-3' 5'-(l)T (m)U (l)T (m)C (m)A (l)C (m)U (m)C (l)T (m)A (m)A (l)T (m)A (l)G (l)G-3'

SEQ ID NO: 8

A scrambled oligonucleotide, SSOsc, containing the same nucleotides as SSOe26 at scrambled positions and modifications at the same positions as in SSOe26 (a negative control)

(5'-(l)G (m)T (l)T (m)C (m)A (l)G (m)T (m)C (l)G (m)C (m)A (l)T (m)A (l)C (Ι)Τ-3'). Same sequence without modification indicated:

5'-GTTCAGTCGCATACT-3'. SEQ ID NO: 9-12

Example (PCR primers)

CYT1 :

Forward: 5'- GGATGAAGAGGATTTGGAAG -3';

Reverse: 5'- TCCTGACATGGGGGTGTA-3'.

CYT2:

Forward: 5'- GAATAGGAACCAGTTTGTATACCG-3';

Reverse: 5'- ACAGCAGGAGTCATCAAAAATC-3'.

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Clasificaciones
Clasificación internacionalA61P35/00, A61P25/18, C12N15/113, A61K31/7088
Clasificación cooperativaC12N2320/33, C12N2310/3231, A61K31/7088, C12N15/1135, C12N15/1137, C12Y207/1102, C12N2310/14, C12N2310/11
Clasificación europeaA61K31/7088, C12N15/113D, C12N15/113B, C12Y207/11020
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