Patent application title: USE OF VEGFR1 AS A BIOMARKER
Inventors:
Klaus Giese (Berlin, DE)
Klaus Giese (Berlin, DE)
Joerg Kaufmann (Berlin, DE)
IPC8 Class: AC12N15113FI
USPC Class:
514 44 A
Class name: Nitrogen containing hetero ring polynucleotide (e.g., rna, dna, etc.) antisense or rna interference
Publication date: 2015-12-24
Patent application number: 20150368650
Abstract:
The present invention is related to the use of VEGFR1 or of a nucleic
acid coding for VEGFR1 as a biomarker in a method for the treatment of a
subject, wherein the method for the treatment comprises administering to
the subject a PKN3 inhibitor.Claims:
1. A method for deciding whether a subject having undergone a first
method of treatment, wherein the first method of treatment comprises
administering to the subject a PKN3 inhibitor, shall be subject to a
second method of treatment, wherein the second method of treatment
comprises administering to the subject a PKN3 inhibitor, comprising:
determining the level of a VEGFR1 or of a nucleic acid coding for a
VEGFR1 in a sample from the subject prior to a first method of treatment,
determining the level of a VEGFR1 or of a nucleic acid coding for a
VEGFR1 in a sample from the subject after a first period of time after
the first method of treatment, and optionally determining the level of a
VEGFR1 or of a nucleic acid coding for a VEGFR1 in a sample from the
subject after a second period of time after the first method of
treatment, wherein if the level of a VEGFR1 or of a nucleic acid coding
for a VEGFR1 in a sample from the subject after the first or second
period of time after the first method of treatment is increased compared
to the level of a VEGFR1 or of a nucleic acid coding for a VEGFR1 in a
sample from the subject prior to a first method of treatment, the subject
is to be subjected to the second method of treatment.
2. The method according to claim 1, wherein the increase is 100% or more, 80% or more, 60% or more or 40% or more.
3. The method according to claim 1, wherein the subject is suffering from a disease or is at risk of suffering from a disease and wherein the method of treatment is a method of treatment for the disease.
4. The method according to claim 1, wherein first period of time after the first method of treatment is about 24 hours or 48 hours after the last administration of the PKN3 inhibitor being part of the first method of treatment.
5. The method according to claim 1, wherein the second period of time after the first method of treatment starts about 1 or two months after the first period of time after the first method of treatment.
6. The method according to claim 1, wherein the first method of treatment is the same as the second method of treatment, or wherein the first method of treatment is different from the second method of treatment.
7. The method according to claim 1, wherein the PKN3 inhibitor selected from the group consisting of an siRNA directed against an mRNA coding for PKN3, an antisense oligonucleotide directed against an mRNA coding for PKN3, a ribozyme directed against an mRNA coding for PKN3, an shRNA directed against an mRNA coding for PKN3, an miRNA or antagomir directed against an mRNA coding for PKN3, an aptamer directed against PKN3, a spiegelmer directed against PKN3, an antibody directed against PKN3, an anticalin directed against PKN3, and a small molecule.
8. The method according to claim 7, wherein the PKN3 inhibitor is an siRNA wherein the siRNA is as follows: 5' 5u8u7c6g8a6g5c7u7a6g5c5 3' 3' a6c6g8u7c5u7a8g6g5u7a8a 5' with unmodified ribonuclotides being as indicated and modified ribonucleotides being represented as follows: 5: 2'-O-Methyl-u, 6: 2'-O-Methyl-a, 7: 2'-O-Methyl-c, 8: 2'-O-Methyl-g.
9. The method according to claim 1, wherein the VEGFR1 is VEGFR1, VEGFR1 variant 2, VEGFR1 variant 1, VEGFR1 variant 3, VEGFR1 variant 4, soluble VEGFR1, or soluble VEGFR1 variant 2 or is a nucleic acid encoding VEGFR1, VEGFR1 variant 2, VEGFR1 variant 1, VEGFR1 variant 3, VEGFR1 variant 4, soluble VEGFR1, or soluble VEGFR1 variant 2.
10. A method for deciding whether a subject shall undergo a method of treatment, whereby the method of treatment comprises the administration of a PKN3 inhibitor, comprising determining the level of a VEGFR1 or of a nucleic acid coding for a VEGFR1, or determining the expression level of a VEGFR1 or of a nucleic acid coding for a VEGFR1, wherein if the expression level of a VEGFR1 or of a nucleic acid coding for a VEGFR1, expressed as titre of VEGF1 in blood or plasma of the subject, is equal to or greater than 200 pg/ml, the patient is amenable to the method of treatment comprising administration of a PKN3 inhibitor.
11. The method according to claim 10, wherein if the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, expressed as titre of VEGF1 in blood or plasma of the subject, is equal to or greater than 400 pg/ml, preferably equal to or greater than 600 pg/ml, more preferably equal to or greater than 800 pg/ml, the patient is amenable to the method of treatment comprising administration of a PKN3 inhibitor.
12. The method according to claim 10, wherein the disease is selected from the group consisting of tumor diseases, cancer diseases and pre-eclampsia.
13. The method according to claim 10, wherein the PKN3 inhibitor selected from the group consisting of an siRNA directed against an mRNA coding for PKN3, an antisense oligonucleotide directed against an mRNA coding for PKN3, a ribozyme directed against an mRNA coding for PKN3, an shRNA directed against an mRNA coding for PKN3, an miRNA or antagomir directed against an mRNA coding for PKN3, an aptamer directed against PKN3, a spiegelmer directed against PKN3, an antibody directed against PKN3, an anticalin directed against PKN3, and a small molecule.
14. The method according to claim 13, wherein the PKN3 inhibitor is an siRNA wherein the siRNA is as follows: 5' 5u8u7c6g8a6g5c7u7a6g5c5 3' 3' a6c6g8u7c5u7a8g6g5u7a8a 5' with unmodified ribonuclotides being as indicated and modified ribonucleotides being represented as follows: 5: 2'-O-Methyl-u, 6: 2'-O-Methyl-a, 7: 2'-O-Methyl-c, 8: 2'-O-Methyl-g.
15. The method according to claim 10, wherein the PKN3 inhibitor selected from the group consisting of an siRNA directed against an mRNA coding for PKN3, an antisense oligonucleotide directed against an mRNA coding for PKN3, a ribozyme directed against an mRNA coding for PKN3, an shRNA directed against an mRNA coding for PKN3, an miRNA or antagomir directed against an mRNA coding for PKN3, an aptamer directed against PKN3, a spiegelmer directed against PKN3, an antibody directed against PKN3, an anticalin directed against PKN3, and a small molecule.
16. The method according to claim 15, wherein the PKN3 inhibitor is an siRNA wherein the siRNA is as follows: 5' 5u8u7c6g8a6g5c7u7a6g5c5 3' 3' a6c6g8u7c5u7a8g6g5u7a8a 5' with unmodified ribonuclotides being as indicated and modified ribonucleotides being represented as follows: 5: 2'-O-Methyl-u, 6: 2'-O-Methyl-a, 7: 2'-O-Methyl-c, 8: 2'-O-Methyl-g.
17. The method according to claim 10, wherein the VEGFR1 is VEGFR1, VEGFR1 variant 2, VEGFR1 variant 1, VEGFR1 variant 3, VEGFR1 variant 4, soluble VEGFR1, or soluble VEGFR1 variant 2 or is a nucleic acid encoding VEGFR1, VEGFR1 variant 2, VEGFR1 variant 1, VEGFR1 variant 3, VEGFR1 variant 4, soluble VEGFR1, or soluble VEGFR1 variant 2.
18. A method for the treatment of a subject suffering from or being at risk of suffering from a disease associated with the expression level of a VEGFR1 or of a nucleic acid coding for a VEGFR1, said expression level being expressed as titre of a VEGF1 in blood or plasma of the subject that is equal to or greater than 200 pg/ml, comprising measuring the expression level of a VEGFR1 or of a nucleic acid coding for a VEGFR1 in said subject and administering a PKN3 inhibitor to said subject having an expression level that is equal to or greater than 200 pg/ml.
19. The method according to claim 18, wherein the expression level of a VEGFR1 or of a nucleic acid coding for a VEGFR1, expressed as titre of a VEGF1 in blood or plasma of the subject, is equal to or greater than 400 pg/ml, equal to or greater than 600 pg/ml or equal to or greater than 800 pg/ml.
20. The method according to claim 18, wherein the VEGFR1 is VEGFR1, VEGFR1 variant 2, VEGFR1 variant 1, VEGFR1 variant 3, VEGFR1 variant 4, soluble VEGFR1, or soluble VEGFR1 variant 2 or is a nucleic acid encoding VEGFR1, VEGFR1 variant 2, VEGFR1 variant 1, VEGFR1 variant 3, VEGFR1 variant 4, soluble VEGFR1, or soluble VEGFR1 variant 2.
Description:
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. Ser. No. 13/894,650, filed May 15, 2013, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.
[0002] The Sequence Listing for this application is labeled "29V9763.TXT" which was created on Jul. 19, 2013 and is 71 KB. The entire contents of the sequence listing is incorporated herein by reference in its entirety.
[0003] The present invention is related to the use of vascular endothelial growth factor receptor 1 (VEGFR1) or of a nucleic acid coding for VEGFR1 as a biomarker, a method for deciding whether a subject is a responder to a method of treatment, a method for deciding whether a subject having undergone a first method of treatment, wherein the first method of treatment comprises administering to the subject a PKN3 inhibitor, shall be subject to a second method of treatment, wherein the second method of treatment comprises administering to the subject a PKN3 inhibitor, a method for deciding whether a subject shall undergo a method of treatment, whereby the method of treatment comprises the administration of a PKN3 inhibitor, and a PKN3 inhibitor for use in a method for the treatment of a subject suffering from or being at risk of suffering from a disease.
[0004] Oncogenesis was described by Foulds (Foulds, L. (1958), J Chronic Dis, 8, 2-37) as a multistep biological process, which is presently known to occur by the accumulation of genetic damage. On a molecular level, the multistep process of tumorigenesis involves the disruption of both positive and negative regulatory effectors (Weinberg, R. A. (1989); Cancer Res, 49, 3713-3721). The molecular basis for human colon carcinomas has been postulated, by Vogelstein and coworkers (Fearon, E. R. and Vogelstein, B. (1990), Cell, 61, 759-767), to involve a number of oncogenes, tumor suppressor genes and repair genes. Similarly, defects leading to the development of retinoblastoma have been linked to another tumor suppressor gene (Lee, W. H., et al. (1987) Science, 235, 1394-1399). Still other oncogenes and tumor suppressors have been identified in a variety of other malignancies. Unfortunately, there remains an inadequate number of treatable cancers, and the effects of cancer are catastrophic--over half a million deaths per year in the United States alone.
[0005] Cancer is fundamentally a genetic disease in which damage to cellular DNA leads to disruption of the normal mechanisms that control cellular proliferation. Two of the mechanisms of action by which tumor suppressors maintain genomic integrity is by cell arrest, thereby allowing for repair of damaged DNA, or removal of the damaged DNA by apoptosis (Ellisen, L. W. and Haber, D. A. (1998), Annu Rev Med, 49, 425-436.). Apoptosis, otherwise called "programmed cell death," is a carefully regulated network of biochemical events which act as a cellular suicide program aimed at removing irreversibly damaged cells. Apoptosis can be triggered in a number of ways including binding of tumor necrosis factor, DNA damage, withdrawal of growth factors, and antibody cross-linking of Fas receptors. Although several genes have been identified that play a role in the apoptotic process, the pathways leading to apoptosis have not been fully elucidated. Many investigators have attempted to identify novel apoptosis-promoting genes with the objective that such genes would afford a means to induce apoptosis selectively in neoplastic cells to treat lancer in a patient.
[0006] An alternative approach to treating cancer involves the suppression of angiogenesis with an agent such as Endostatin® or anti-VEGF antibodies. In this approach, the objective is to prevent further vascularization of the primary tumor and potentially to constrain the size of metastatic lesions to that which can support neoplastic cell survival without substantial vascular growth.
[0007] A particular group of cancer diseases are those cancer diseases which are aggressive in terms of growth rate of the tumor, invasion into normal tissue, resistance to chemotherapy or other conventional treatments and the formation of metastasis throughout the body. In the case of more aggressive cancer, the cancer tissue is more different from the normal tissue and the tumor is more likely to spread. Therefore one objective in current cancer research is to develop agents which are inhibiting tumor growth and/or reducing the spreading of cancer cells throughout the body.
[0008] Definitions for what is an aggressive cancer disease may be taken from the homepage of the National Cancer Institute which is Worldwide Website: cancer.gov/Templates/db_alpha.aspx?CdrID=46053. Also, for the description of the aggressivity of a cancer disease, typically grading is used which is a system for classifying cancer cells in terms of how abnormal they appear when examined under a microscope. The objective of a grading system is to provide information about the probable growth rate of the tumor and its tendency to spread. The systems used to grade tumors vary with each type of cancer. Grading plays a role in treatment decisions.
[0009] Such grading systems are known to the ones skilled in the art. One of them is the Gleason score which is a system of grading prostate cancer tissue based on how it looks under a microscope. Gleason scores range from 2 to 10 and indicate how likely it is that a tumor will spread. A low Gleason score means the cancer tissue is similar to normal prostate tissue and the tumor is less likely to spread; a high Gleason score means the cancer tissue is very different from normal and the tumor is more likely to spread.
[0010] PKN3 which is also referred to as protein kinase N beta or PKN beta has been, among others, described by Oishi K, et al. (Oishi K. et al. Biochem. Biophys. Res. Commun. 261 (3): 808-14) or Shibata, H et al. (Shibata, H. et al.; J. Biochem. (Japan) 130 (1): 23-31). PKN3 has also been identified as a target molecule in tumors, in particular solid tumors, cancers, in particular metastatic cancers, and in pre-eclampsia. Also, as described in international patent application WO 2004/019973 protein kinase N beta is a downstream target of the PI-3 kinase/PTEN pathway which is linked to tumorigenesis and metastasis. Particularly the latter effect seems to be strongly related to the loss of suppressor function, more particularly PTEN tumour suppressor function.
[0011] The treatment of any disease is intended to provide relief or even cure to a patient while avoiding any undesired side effects or any adverse effects. In order to comply with this task there is a need for biomarkers which allow, in the broadest sense, to distinguish responders to a therapy from non-responders to such therapy. Furthermore, biomarkers are, for example, needed and useful as inclusion criterion for clinical trials, establishment or identification of optimum drug, dose and schedule of therapy and for identifying patients at high risk for adverse effects.
[0012] In the light of the above, there is a need for identifying biomarkers which can be used in the context of diseases and more specifically of diseases which can be treated by administering to a patient suffering from or being at risk of suffering from a disease which can be treated by a PKN3 inhibitor. Insofar, one problem underlying the present invention is the identification of such biomarkers.
[0013] A further problem underlying the present invention is the provision of a medicament for a particular group of patients suffering from or being at risk of suffering from a diseases which can be treated by administering to such patients a PKN3 inhibitor, whereby preferably such particular group of patients comprises a very high percentage of responders to such therapy comprising administering to the patients a PKN3 inhibitor.
[0014] These and further problems are solved by the subject matter of the attached independent claims. Particularly preferred embodiments may be taken from the attached dependent claims.
[0015] The problem underlying the present invention is also solved in a first aspect which is also the first embodiment of the first aspect by the use of VEGFR1 or of a nucleic acid coding for VEGFR1 as a biomarker in a method for the treatment of a subject, wherein the method for the treatment comprises administering to the subject a PKN3 inhibitor.
[0016] In a second embodiment of the first aspect which is also an embodiment of the first embodiment of the first aspect, the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1 is a or the biomarker.
[0017] The problem underlying the present invention is also solved in a second aspect which is also the first embodiment of the second aspect by the use of VEGFR1 or of a nucleic acid coding for VEGFR1 as a biomarker in designing a method for the treatment of a subject, wherein the method for the treatment comprises administering to the subject a PKN3 inhibitor.
[0018] In a second embodiment of the second aspect which is also an embodiment of the first embodiment of the second aspect, the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1 is a or the biomarker.
[0019] In a third embodiment of the first aspect which is also an embodiment of the first and the second embodiment of the first aspect, and in a third embodiment of the second aspect which is also an embodiment of the first and the second embodiment of the second aspect the VEGFR1 is selected from the group comprising VEGFR1 variant 2, VEGFR1 variant 1, VEGFR1 variant 3 and VEGFR1 variant 4.
[0020] In a fourth embodiment of the first aspect which is also an embodiment of the first, second and third embodiment of the first aspect, and in a fourth embodiment of the second aspect which is also an embodiment of the first, second and third embodiment of the second aspect the nucleic acid coding for VEGFR1 is selected from the group comprising VEGFR1 transcript variant 2, VEGFR1 transcript variant 1, VEGFR1 transcript variant 3 and VEGFR1 transcript variant 4.
[0021] In a fifth embodiment of the first aspect which is also an embodiment of the first, second, third and fourth embodiment of the first aspect, and in a fifth embodiment of the second aspect which is also an embodiment of the first, second, third and fourth embodiment of the second aspect the VEGFR1 is soluble VEGFR1 or VEGFR1 variant 2.
[0022] In a sixth embodiment of the first aspect which is also an embodiment of the fifth embodiment of the first aspect, and in a sixth embodiment of the second aspect which is also an embodiment of the fifth embodiment of the second aspect the soluble VEGFR1 or VEGFR1 variant 2 comprises an amino acid sequence according to SEQ ID NO:1 or is encoded by a nucleotide sequence according to SEQ ID NO: 2.
[0023] In a seventh embodiment of the first aspect which is also an embodiment of the first, second, third and fourth embodiment of the first aspect, and in a seventh embodiment of the second aspect which is also an embodiment of the first, second, third and fourth embodiment of the second aspect the nucleic acid coding for VEGFR1 is a nucleic acid coding for soluble VEGFR1 or VEGFR1 variant 2.
[0024] In an eighth embodiment of the first aspect which is also an embodiment of the seventh embodiment of the first aspect, and in an eighth embodiment of the second aspect which is also an embodiment of the seventh embodiment of the second aspect the nucleic acid coding for soluble VEGFR1 or VEGFR1 variant 2 comprises a nucleotide sequence according to SEQ ID NO:2 or a nucleotide sequence encoding an amino acid sequence according to SEQ ID NO: 1.
[0025] In a ninth embodiment of the first aspect which is also an embodiment of the first, second, third and fourth embodiment of the first aspect, and in a ninth embodiment of the second aspect which is also an embodiment of the first, second, third and fourth embodiment of the second aspect the VEGFR1 is VEGFR1 variant 1.
[0026] In a tenth embodiment of the first aspect which is also an embodiment of the ninth embodiment of the first aspect, and in a tenth embodiment of the second aspect which is also an embodiment of the ninth embodiment of the second aspect the VEGFR1 variant 1 comprises an amino acid sequence according to SEQ ID NO: 3 or is encoded by a nucleotide sequence according to SEQ ID NO: 4.
[0027] In an eleventh embodiment of the first aspect which is also an embodiment of the first, second, third and fourth embodiment of the first aspect, and in an eleventh embodiment of the second aspect which is also an embodiment of the first, second, third and fourth embodiment of the second aspect the nucleic acid coding for VEGFR1 is a nucleic acid coding for VEGFR1 variant 1.
[0028] In a twelfth embodiment of the first aspect which is also an embodiment of the eleventh embodiment of the first aspect, and in a twelfth embodiment of the second aspect which is also an embodiment of the eleventh embodiment of the second aspect the nucleic acid coding for VEGFR1 variant 1 comprises a nucleotide sequence according to SEQ ID NO: 4 or a nucleotide sequence encoding an amino acid sequence according to SEQ ID NO: 3.
[0029] In a 13th embodiment of the first aspect which is also an embodiment of the first, second, third and fourth embodiment of the first aspect, and in a 13th embodiment of the second aspect which is also an embodiment of the first, second, third and fourth embodiment of the second aspect the VEGFR1 is VEGFR1 variant 3.
[0030] In a 14th embodiment of the first aspect which is also an embodiment of the 13th embodiment of the first aspect, and in a 14th embodiment of the second aspect which is also an embodiment of the 13th embodiment of the second aspect the VEGFR1 variant 3 comprises an amino acid sequence according to SEQ ID NO: 5 or is encoded by a nucleotide sequence according to SEQ ID NO: 6.
[0031] In a 15th embodiment of the first aspect which is also an embodiment of the first, second, third and fourth embodiment of the first aspect, and in a 15th embodiment of the second aspect which is also an embodiment of the first, second, third and fourth embodiment of the second aspect the nucleic acid coding for VEGFR1 is a nucleic acid coding for VEGFR1 variant 3.
[0032] In a 16th embodiment of the first aspect which is also an embodiment of the 15th embodiment of the first aspect, and in a 16th embodiment of the second aspect which is also an embodiment of the 15th embodiment of the second aspect the nucleic acid coding for VEGFR1 variant 3 comprises a nucleotide sequence according to SEQ ID NO: 6 or a nucleotide sequence encoding an amino acid sequence according to SEQ ID NO: 5.
[0033] In a 17th embodiment of the first aspect which is also an embodiment of the first, second, third and fourth embodiment of the first aspect, and in a 17th embodiment of the second aspect which is also an embodiment of the first, second, third and fourth embodiment of the second aspect the VEGFR1 is VEGFR1 variant 4.
[0034] In an 18th embodiment of the first aspect which is also an embodiment of the 17th embodiment of the first aspect, and in an 18th embodiment of the second aspect which is also an embodiment of the 17th embodiment of the second aspect the VEGFR1 variant 4 comprises an amino acid sequence according to SEQ ID NO: 7 or is encoded by a nucleotide sequence according to SEQ ID NO: 8.
[0035] In a 19th embodiment of the first aspect which is also an embodiment of the first, second, third and fourth embodiment of the first aspect, and in a 19th embodiment of the second aspect which is also an embodiment of the first, second, third and fourth embodiment of the second aspect the nucleic acid coding for VEGFR1 is a nucleic acid coding for VEGFR1 variant 4.
[0036] In a 20th embodiment of the first aspect which is also an embodiment of the 19th embodiment of the first aspect, and in a 20th embodiment of the second aspect which is also an embodiment of the 19th embodiment of the second aspect the nucleic acid coding for VEGFR1 variant 4 comprises a nucleotide sequence according to SEQ ID NO: 8 or a nucleotide sequence encoding an amino acid sequence according to SEQ ID NO: 7.
[0037] In a 21st embodiment of the first aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th embodiment of the first aspect, and in a 21st embodiment of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th embodiment of the second aspect the biomarker is a pharmacodynamic biomarker.
[0038] In a 22nd embodiment of the first aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th embodiment of the first aspect, and in a 22nd embodiment of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th embodiment of the second aspect the biomarker is a predictive biomarker.
[0039] In a 23rd embodiment of the first aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th embodiment of the first aspect, and in a 23rd embodiment of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th embodiment of the second aspect the biomarker is a prognostic biomarker.
[0040] In a 24th embodiment of the first aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th embodiment of the first aspect, and in a 24th embodiment of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th and 20th embodiment of the second aspect the biomarker is a surrogate biomarker.
[0041] In a 25th embodiment of the first aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd and 24th embodiment of the first aspect, and in a 25th embodiment of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd and 24th embodiment of the second aspect the subject is suffering from or at risk of suffering from a disease, wherein the disease is a disease which can be treated, ameliorated and/or cured by a PKN3 inhibitor.
[0042] In a 26th embodiment of the first aspect which is also an embodiment of the 25th embodiment of the first aspect, and in a 26th embodiment of the second aspect which is also an embodiment of the 25th embodiment of the second aspect the disease is selected from the group comprising tumor diseases, cancer diseases and pre-eclampsia.
[0043] In a 27th embodiment of the first aspect which is also an embodiment of the 25th and 26th embodiment of the first aspect, and in a 27th embodiment of the second aspect which is also an embodiment of the 25th and 26th embodiment of the second aspect the disease is selected from the group comprising lung cancer, pancreas cancer, liver cancer, endometrial cancer, colorectal carcinomas, gliomas, adenocarcinomas, endometrial hyperplasias, hereditary non-polyposis colorectal carcinoma, breast-ovarian cancer, prostate cancer, gastrointestinal harmatomas, lipomas, thyroid adenomas, fibrocystic disease of the breast, cerebellar dysplastic gangliocytoma, breast and thyroid malignancies, large cell carcinoma, small cell carcinoma and squamous cell carcinoma.
[0044] In a 28th embodiment of the first aspect which is also an embodiment of the 25th and 26th embodiment of the first aspect, and in a 28th embodiment of the second aspect which is also an embodiment of the 25th and 26th embodiment of the second aspect the disease is a disease is selected from the group comprising cancers, metastatic cancers and any pathological conditions involving the PI 3-kinase pathway.
[0045] In a 29th embodiment of the first aspect which is also an embodiment of the 25th and 26th embodiment of the first aspect, and in a 29th embodiment of the second aspect which is also an embodiment of the 25th and 26th embodiment of the second aspect the disease is characterized in that the cells being involved in said disease lack PTEN activity, show an increased aggressive behavior, or are cells of a late stage tumor.
[0046] In a 30th embodiment of the first aspect which is also an embodiment of the 25th and 26th embodiment of the first aspect, and in a 30th embodiment of the second aspect which is also an embodiment of the 25th and 26th embodiment of the second aspect the disease is selected from the group comprising metastatic cancers and any pathological conditions involving the PI 3-kinase pathway, whereby such pathological condition consists of endometrial cancer, colorectal carcinomas, gliomas, adenocarcinomas, endometrial hyperplasias, Cowden's syndrome, hereditary non-polyposis colorectal carcinoma, Li-Fraumene's syndrome, breast-ovarian cancer, prostate cancer, Bannayan-Zonana syndrome, LDD (Lhermitte-Duklos' syndrome) hamartoma-macrocephaly diseases including Cow disease (CD) and Bannayan-Ruvalcaba-Rily syndrome (BRR), mucocutaneous lesions such as trichilemmonmas, macrocephaly, mental retardation, gastrointestinal harmatomas, lipomas, thyroid adenomas, fibrocystic disease of the breast, cerebellar dysplastic gangliocytoma, and breast and thyroid malignancies and large cell carcinoma, small cell carcinoma and squamous cell carcinoma.
[0047] In a 31st embodiment of the first aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th and 30th embodiment of the first aspect, and in a 31st embodiment of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th and 30th embodiment of the second aspect the PKN3 inhibitor is selected from the group comprising an siRNA directed against an mRNA coding for PKN3, an antisense oligonucleotide directed against an mRNA coding for PKN3, a ribozyme directed against an mRNA coding for PKN3, an shRNA directed against an mRNA coding for PKN3, an miRNA or antagomir directed against an mRNA coding for PKN3, an aptamer directed against PKN3, a spiegelmer directed against PKN3, an antibody directed against PKN3, an anticalin directed against PKN3, and a small molecule.
[0048] In a 32nd embodiment of the first aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th and 31st embodiment of the first aspect, and in a 32nd embodiment of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th and 31st embodiment of the second aspect the PKN3 inhibitor is an siRNA wherein the siRNA is as follows:
5' 5u8u7c6g8a6g5c7u7a6g5c5 3' (SEQ ID NO: 11)
3' a6c6g8u7c5u7a8g6g5u7a8a 5' (SEQ ID NO: 12)
with unmodified ribonucleotides being as indicated and modified ribonucleotides being represented as follows:
[0049] 5: 2'-O-Methyl-u,
[0050] 6: 2'-O-Methyl-a,
[0051] 7: 2'-O-Methyl-c,
[0052] 8: 2'-O-Methyl-g.
[0053] In a 33rd embodiment of the first aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st and 32nd embodiment of the first aspect, and in a 33rd embodiment of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st and 32nd embodiment of the second aspect PKN3 comprises an amino acid sequence according to SEQ ID NO: 9.
[0054] In a 34th embodiment of the first aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st and 32nd embodiment of the first aspect, and in a 34th embodiment of the second aspect which is also an embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth tenth, eleventh, twelfth, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 31st and 32nd embodiment of the second aspect PKN3 is encoded by a nucleotide sequence according to SEQ ID NO: 10 or a nucleotide sequence coding for an amino acid sequence according to SEQ ID NO: 9.
[0055] Additional non-limiting embodiments provide:
[0056] 1. Use of VEGFR1 or of a nucleic acid coding for VEGFR1 as a biomarker in a method for the treatment of a subject, wherein the method for the treatment comprises administering to the subject a PKN3 inhibitor.
[0057] 2. Use according to embodiment 1, wherein the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1 is a or the biomarker.
[0058] 3. Use of VEGFR1 or of a nucleic acid coding for VEGFR1 as a biomarker in designing a method for the treatment of a subject, wherein the method for the treatment comprises administering to the subject a PKN3 inhibitor.
[0059] 4. Use according to embodiment 3, wherein the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1 is a or the biomarker.
[0060] 5. Use according to any one of embodiments 1 to 4, wherein the VEGFR1 is selected from the group comprising VEGFR1 variant 2, VEGFR1 variant 1, VEGFR1 variant 3 and VEGFR1 variant 4.
[0061] 6. Use according to any one of embodiments 1 to 4, wherein the nucleic acid coding for VEGFR1 is selected from the group comprising VEGFR1 transcript variant 2, VEGFR1 transcript variant 1, VEGFR1 transcript variant 3 and VEGFR1 transcript variant 4.
[0062] 7. Use according to any one of embodiments 1 to 6, wherein the VEGFR1 is soluble VEGFR1 or VEGFR1 variant 2.
[0063] 8. Use according to embodiment 7, wherein the soluble VEGFR1 or VEGFR1 variant 2 comprises an amino acid sequence according to SEQ ID NO:1 or is encoded by a nucleotide sequence according to SEQ ID NO: 2.
[0064] 9. Use according to any one of embodiments 1 to 6, wherein the nucleic acid coding for VEGFR1 is a nucleic acid coding for soluble VEGFR1 or VEGFR1 variant 2.
[0065] 10. Use according to embodiment 9, wherein the nucleic acid coding for soluble VEGFR1 or VEGFR1 variant 2 comprises a nucleotide sequence according to SEQ ID NO:2 or a nucleotide sequence encoding an amino acid sequence according to SEQ ID NO: 1.
[0066] 11. Use according to any one of embodiments 1 to 6, wherein the VEGFR1 is VEGFR1 variant 1.
[0067] 12. Use according to embodiment 11, wherein the VEGFR1 variant 1 comprises an amino acid sequence according to SEQ ID NO: 3 or is encoded by a nucleotide sequence according to SEQ ID NO: 4.
[0068] 13. Use according to any one of embodiments 1 to 6, wherein the nucleic acid coding for VEGFR1 is a nucleic acid coding for VEGFR1 variant 1.
[0069] 14. Use according to embodiment 13, wherein the nucleic acid coding for VEGFR1 variant 1 comprises a nucleotide sequence according to SEQ ID NO: 4 or a nucleotide sequence encoding an amino acid sequence according to SEQ ID NO: 3.
[0070] 15. Use according to any one of embodiments 1 to 6, wherein the VEGFR1 is VEGFR1 variant 3.
[0071] 16. Use according to embodiment 15, wherein the VEGFR1 variant 3 comprises an amino acid sequence according to SEQ ID NO: 5 or is encoded by a nucleotide sequence according to SEQ ID NO: 6.
[0072] 17. Use according to any one of embodiments 1 to 6, wherein the nucleic acid coding for VEGFR1 is a nucleic acid coding for VEGFR1 variant 3.
[0073] 18. Use according to embodiment 17, wherein the nucleic acid coding for VEGFR1 variant 3 comprises a nucleotide sequence according to SEQ ID NO: 6 or a nucleotide sequence encoding an amino acid sequence according to SEQ ID NO: 5.
[0074] 19. Use according to any one of embodiments 1 to 6, wherein the VEGFR1 is VEGFR1 variant 4.
[0075] 20. Use according to embodiment 19, wherein the VEGFR1 variant 4 comprises an amino acid sequence according to SEQ ID NO: 7 or is encoded by a nucleotide sequence according to SEQ ID NO: 8.
[0076] 21. Use according to any one of embodiments 1 to 6, wherein the nucleic acid coding for VEGFR1 is a nucleic acid coding for VEGFR1 variant 4.
[0077] 22. Use according to embodiment 21, wherein the nucleic acid coding for VEGFR1 variant 4 comprises a nucleotide sequence according to SEQ ID NO: 8 or a nucleotide sequence encoding an amino acid sequence according to SEQ ID NO: 7.
[0078] 23. Use according to any one of embodiments 1 to 22, wherein the biomarker is a pharmacodynamic biomarker.
[0079] 24. Use according to any one of embodiments 1 to 22, wherein the biomarker is a predictive biomarker.
[0080] 25. Use according to any one of embodiments 1 to 22, wherein the biomarker is a prognostic biomarker.
[0081] 26. Use according to any one of embodiments 1 to 22, wherein the biomarker is a surrogate biomarker.
[0082] 27. Use according to any one of embodiments 1 to 26, wherein the subject is suffering from or at risk of suffering from a disease, wherein the disease is a disease which can be treated, ameliorated and/or cured by a PKN3 inhibitor.
[0083] 28. Use according to embodiment 27, wherein the disease is selected from the group comprising tumor diseases, cancer diseases and pre-eclampsia.
[0084] 29. Use according to any one of embodiments 27 to 28, wherein the disease is selected from the group comprising lung cancer, pancreas cancer, liver cancer, endometrial cancer, colorectal carcinomas, gliomas, adenocarcinomas, endometrial hyperplasias, hereditary non-polyposis colorectal carcinoma, breast-ovarian cancer, prostate cancer, gastrointestinal harmatomas, lipomas, thyroid adenomas, fibrocystic disease of the breast, cerebellar dysplastic gangliocytoma, breast and thyroid malignancies, large cell carcinoma, small cell carcinoma and squamous cell carcinoma.
[0085] 30. Use according to any one of embodiments 27 to 28, wherein the disease is a disease is selected from the group comprising cancers, metastatic cancers and any pathological conditions involving the PI 3-kinase pathway.
[0086] 31. Use according to any one of embodiments 27 to 28, wherein the disease is characterized in that the cells being involved in said disease lack PTEN activity, show an increased aggressive behavior, or are cells of a late stage tumor.
[0087] 32. Use according to any one of embodiments 27 to 28, wherein the disease is selected from the group comprising metastatic cancers and any pathological conditions involving the PI 3-kinase pathway, whereby such pathological condition consists of endometrial cancer, colorectal carcinomas, gliomas, adenocarcinomas, endometrial hyperplasias, Cowden's syndrome, hereditary non-polyposis colorectal carcinoma, Li-Fraumene's syndrome, breast-ovarian cancer, prostate cancer, Bannayan-Zonana syndrome, LDD (Lhermitte-Duklos' syndrome) hamartoma-macrocephaly diseases including Cow disease (CD) and Bannayan-Ruvalcaba-Rily syndrome (BRR), mucocutaneous lesions such as trichilemmonmas, macrocephaly, mental retardation, gastrointestinal harmatomas, lipomas, thyroid adenomas, fibrocystic disease of the breast, cerebellar dysplastic gangliocytoma, and breast and thyroid malignancies and large cell carcinoma, small cell carcinoma and squamous cell carcinoma.
[0088] 33. Use according to any one of embodiments 1 to 32, wherein the PKN3 inhibitor selected from the group comprising an siRNA directed against an mRNA coding for PKN3, an antisense oligonucleotide directed against an mRNA coding for PKN3, a ribozyme directed against an mRNA coding for PKN3, an shRNA directed against an mRNA coding for PKN3, an miRNA or antagomir directed against an mRNA coding for PKN3, an aptamer directed against PKN3, a spiegelmer directed against PKN3, an antibody directed against PKN3, an anticalin directed against PKN3, and a small molecule.
[0089] 34. Use according to any one of embodiments 1 to 33, wherein the PKN3 inhibitor is an siRNA wherein the siRNA is as follows:
5' 5u8u7c6g8a6g5c7u7a6g5c5 3' (SEQ ID NO: 11)
3' a6c6g8u7c5u7a8g6g5u7a8a 5' (SEQ ID NO: 12)
with unmodified ribonucleotides being as indicated and modified ribonucleotides being represented as follows:
[0090] 5: 2'-O-Methyl-u,
[0091] 6: 2'-O-Methyl-a,
[0092] 7: 2'-O-Methyl-c,
[0093] 8: 2'-O-Methyl-g.
[0094] 35. Use according to any one of embodiments 1 to 34, wherein PKN3 comprises an amino acid sequence according to SEQ ID NO: 9.
[0095] 36. Use according to any one of embodiments 1 to 34, wherein PKN3 is encoded by a nucleotide sequence according to SEQ ID NO: 10 or a nucleotide sequence coding for an amino acid sequence according to SEQ ID NO: 9.
[0096] 37. A method for deciding whether a subject is a responder to a method of treatment, wherein the method of treatment comprises administering to the subject a PKN3 inhibitor, comprising:
[0097] determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject prior to the subject being subjected to the method of treatment,
[0098] determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject after the subject having been subjected to the method of treatment; wherein if the level of VEGFR1 or a nucleic acid coding for VEGFR1 in a sample from the subject after the subject having been subjected to the method of treatment is decreased compared to the level of VEGFR1 or a nucleic acid coding for VEGFR1 in a sample from the subject prior to the subject having being subjected to the method of treatment, the subject is a responder to the method of treatment.
[0099] 38. The method according to embodiment 37, wherein the decrease is a decrease of 10% or more, of 20% or more, 40% or more, 60% or more or 80% or more.
[0100] 39. The method according to any one of embodiments 37 to 38, wherein the subject is suffering from a disease or is at risk of suffering from a disease and wherein the method of treatment is a method of treatment for the disease.
[0101] 40. A method for deciding whether a subject having undergone a first method of treatment, wherein the first method of treatment comprises administering to the subject a PKN3 inhibitor, shall be subject to a second method of treatment, wherein the second method of treatment comprises administering to the subject a PKN3 inhibitor, comprising:
[0102] determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject prior to a first method of treatment,
[0103] determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject after a first period of time after the first method of treatment, and
[0104] optionally determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject after a second period of time after the first method of treatment, wherein if the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject after the first or second period of time after the first method of treatment is increased compared to the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject prior to a first method of treatment, the subject is to be subjected to the second method of treatment.
[0105] 41. The method according to embodiment 40, wherein the increase is 100% or more, 80% or more, 60% or more or 40% or more.
[0106] 42. The method according to any one of embodiments 40 to 41, wherein the subject is suffering from a disease or is at risk of suffering from a disease and wherein the method of treatment is a method of treatment for the disease.
[0107] 43. The method according to any one of embodiments 40 to 41, wherein first period of time after the first method of treatment is about 24 hours or 48 hours after the last administration of the PKN3 inhibitor being part of the first method of treatment.
[0108] 44. The method according to any of embodiments 40 to 41, wherein the second period of time after the first method of treatment starts about 1 or two months after the first period of time after the first method of treatment.
[0109] 45. The method according to any one of embodiments 40 to 44, wherein the first method of treatment is the same as the second method of treatment, or wherein the first method of treatment is different from the second method of treatment.
[0110] 46. A method for deciding whether a subject shall undergo a method of treatment, whereby the method of treatment comprises the administration of a PKN3 inhibitor, comprising determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1, or determining the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, wherein if the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, expressed as titre of VEGF1 in blood or plasma of the subject, is equal to or greater than 200 pg/ml, the patient is amenable to the method of treatment comprising administration of a PKN3 inhibitor.
[0111] 47. The method according to embodiment 46, wherein if the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, expressed as titre of VEGF1 in blood or plasma of the subject, is equal to or greater than 400 pg/ml, preferably equal to or greater than 600 pg/ml, more preferably equal to or greater than 800 pg/ml, the patient is amenable to the method of treatment comprising administration of a PKN3 inhibitor.
[0112] 48. The method according to any one of embodiments 37 to 47, wherein the disease is any disease as defined in any of the preceding embodiments.
[0113] 49. The method according to any one of embodiments 37 to 48, wherein the PKN3 inhibitor is any PKN3 inhibitor as defined in any of the preceding embodiments.
[0114] 50. The method according to any one of embodiments 37 to 49, wherein the VEGFR1 or a nucleic acid coding thereof is any VEGFR1 or a nucleic acid coding for the VEGFR1 as defined in any of the preceding embodiments.
[0115] 51. A PKN3 inhibitor for use in a method for the treatment of a subject suffering from or being at risk of suffering from a disease, wherein the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, expressed as titre of VEGF1 in blood or plasma of the subject, is equal to or greater than 200 pg/ml.
[0116] 52. The PKN3 inhibitor according to embodiment 51, wherein the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, expressed as titre of VEGF1 in blood or plasma of the subject, is equal to or greater than 400 pg/ml, preferably equal to or greater than 600 pg/ml and more preferably equal to or greater than 800 pg/ml.
[0117] The problem underlying the present invention is also solved in a third aspect which is also the first embodiment of the third aspect by a method for deciding whether a subject is a responder to a method of treatment, wherein the method of treatment comprises administering to the subject a PKN3 inhibitor, comprising:
[0118] determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject prior to the subject being subjected to the method of treatment,
[0119] determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject after the subject having been subjected to the method of treatment; wherein if the level of VEGFR1 or a nucleic acid coding for VEGFR1 in a sample from the subject after the subject having been subjected to the method of treatment is decreased compared to the level of VEGFR1 or a nucleic acid coding for VEGFR1 in a sample from the subject prior to the subject having being subjected to the method of treatment, the subject is a responder to the method of treatment.
[0120] In a second embodiment of the third aspect which is also an embodiment of the first embodiment of the third aspect, the decrease is a decrease of 10% or more, of 20% or more, 40% or more, 60% or more or 80% or more.
[0121] In a third embodiment of the third aspect which is also an embodiment of the first and the second embodiment of the third aspect, the subject is suffering from a disease or is at risk of suffering from a disease and wherein the method of treatment is a method of treatment for the disease.
[0122] The problem underlying the present invention is also solved in a fourth aspect which is also the first embodiment of the fourth aspect by a method for deciding whether a subject having undergone a first method of treatment, wherein the first method of treatment comprises administering to the subject a PKN3 inhibitor, shall be subject to a second method of treatment, wherein the second method of treatment comprises administering to the subject a PKN3 inhibitor, comprising:
[0123] determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject prior to a first method of treatment,
[0124] determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject after a first period of time after the first method of treatment, and
[0125] optionally determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject after a second period of time after the first method of treatment,
[0126] wherein if the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject after the first or second period of time after the first method of treatment is increased compared to the level of VEGFR1 or of a nucleic acid coding for VEGFR1 in a sample from the subject prior to a first method of treatment, the subject is to be subjected to the second method of treatment.
[0127] In a second embodiment of the fourth aspect which is also an embodiment of the first embodiment of the fourth aspect, the increase is 100% or more, 80% or more, 60% or more or 40% or more.
[0128] In a third embodiment of the fourth aspect which is also an embodiment of the first and the second embodiment of the fourth aspect, the subject is suffering from a disease or is at risk of suffering from a disease and wherein the method of treatment is a method of treatment for the disease.
[0129] In a fourth embodiment of the fourth aspect which is also an embodiment of the first, second and third embodiment of the fourth aspect, the first period of time after the first method of treatment is about 24 hours or 48 hours after the last administration of the PKN3 inhibitor being part of the first method of treatment.
[0130] In a fifth embodiment of the fourth aspect which is also an embodiment of the first, second and third embodiment of the fourth aspect, the second period of time after the first method of treatment starts about 1 or two months after the first period of time after the first method of treatment.
[0131] In a sixth embodiment of the fourth aspect which is also an embodiment of the first, second, third, fourth and fifth embodiment of the fourth aspect, the first method of treatment is the same as the second method of treatment, or wherein the first method of treatment is different from the second method of treatment.
[0132] The problem underlying the present invention is also solved in a fifth aspect which is also the first embodiment of the fifth aspect by a method for deciding whether a subject shall undergo a method of treatment, whereby the method of treatment comprises the administration of a PKN3 inhibitor, comprising determining the level of VEGFR1 or of a nucleic acid coding for VEGFR1, or determining the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, wherein if the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, expressed as titre of VEGF1 in blood or plasma of the subject, is equal to or greater than 200 pg/ml, the patient is amenable to the method of treatment comprising administration of a PKN3 inhibitor.
[0133] In a second embodiment of the fifth aspect which is also an embodiment of the first embodiment of the fifth aspect, if the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, expressed as titre of VEGF1 in blood or plasma of the subject, is equal to or greater than 400 pg/ml, preferably equal to or greater than 600 pg/ml, more preferably equal to or greater than 800 pg/ml, the patient is amenable to the method of treatment comprising administration of a PKN3 inhibitor.
[0134] In an embodiment of any embodiment of the third, fourth and fifth aspect the disease is any disease as defined in any of the embodiments of any aspect of the present invention.
[0135] In an embodiment of any embodiment of the third, fourth and fifth aspect the PKN3 inhibitor is any PKN3 inhibitor as defined in any of the embodiments of any aspect of the present invention.
[0136] In an embodiment of any embodiment of the third, fourth and fifth aspect the VEGFR1 or a nucleic acid coding thereof is any VEGFR1 or a nucleic acid coding for the VEGFR1 as defined in any of the embodiments of any aspect of the present invention.
[0137] The problem underlying the present invention is also solved in a sixth aspect which is also the first embodiment of the sixth aspect by a PKN3 inhibitor for use in a method for the treatment of a subject suffering from or being at risk of suffering from a disease, wherein the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, expressed as titre of VEGF1 in blood or plasma of the subject, is equal to or greater than 200 pg/ml.
[0138] In a second embodiment of the sixth aspect which is also an embodiment of the sixth embodiment of the second aspect, the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1, expressed as titre of VEGF1 in blood or plasma of the subject, is equal to or greater than 400 pg/ml, preferably equal to or greater than 600 pg/ml and more preferably equal to or greater than 800 pg/ml.
[0139] The present invention, in its various aspects, is based on the surprising finding that VEGFR1 or a nucleic acid coding for VEGFR1, i.e. VEGF receptor 1 which is also referred to as Flt-1, is a biomarker for therapies which are different from antiangiogenic therapy. More specifically, the present invention is based on the surprising finding that VEGFR1 is a biomarker in connection with methods for the treatment of a subject, wherein the method for the treatment comprises administering to the subject a PKN3 inhibitor. Based on this finding it is also within the present invention that VEGFR1 or a nucleic acid coding therefor are used as a biomarker in a method for the treatment of a subject or in connection with designing a method for the treatment of a subject, wherein the method for the treatment comprises administering to the subject a PKN3 inhibitor. Such designing may comprise, among other, at least one of the following: Identifying responders from non-responders to the method for the treatment comprising administering a PKN3 inhibitor, defining the establishment or identification of optimum drug, dose and schedule of such method for the treatment and for identifying patients at high risk for adverse effects in connection with such method for the treatment.
[0140] As preferably used herein, a method for the treatment means a therapy and a method for the treatment comprising administration to a subject a PKN3 inhibitor means an anti-PKN3 therapy.
[0141] As preferably used herein, a PKN3 inhibitor is a compound which addresses or targets PKN3 or a fragment thereof, whereby the targeting of PKN3 or a fragment thereof results in a therapeutic effect in a subject to which the PKN3 inhibitor is administered.
[0142] A subject as preferably used is a vertebrate, more preferably a mammal. A particular preferred subject is man.
[0143] It will be understood by a person skilled in the art that the use of VEGFR1 or of a nucleic acid coding for VEGFR1 as a biomarker can be realized or put into practice by using or referring to the expression level of VEGFR1 or the expression level of a nucleic acid coding for VEGFR1. Insofar, the present invention in its various aspects also relates to the use of the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1 as a biomarker in a method for the treatment of a subject, wherein the method for the treatment comprises administering to the subject a PKN3 inhibitor, and to the use of the expression level of VEGFR1 or of a nucleic acid coding for VEGFR1 as a biomarker in a method for designing or in designing a method for the treatment of a subject, wherein the method for the treatment comprises administering to the subject a PKN3 inhibitor.
[0144] In accordance with the present invention, in its various aspects, either VEGFR1, i.e. the protein, or a nucleic acid which codes for VEGFR1 is used as a biomarker or is used in the methods and products of the present invention. It will be acknowledged by a person skilled in the art that, at the level of the nucleic acid, there are a total of four different transcript variants for VEGFR1, namely VEGFR1 transcript variant 2, VEGFR1 transcript variant 1, VEGFR1 1 transcript variant 3 and VEGFR1 transcript variant 4. It is understood that VEGFR1 transcript 2 codes for soluble VEGFR1 and VEGFR1 transcript 1 codes for the membrane-bound VEGFR1. VEGFR1 transcripts 3 and 4 code for fragments of VEGFR1. It is within the present invention that any of the VEGFR1 transcript variants 1 to 4 is, in principle, suitable for use in connection with the present invention. It is also within the present invention that any of the VEGFR1 variants 1 to 4 is, in principle suitable for use in connection with the various aspects of the present invention. As preferably used herein, a VEGFR1 variant 1 is a protein encoded by VEGFR1 splice variant 1, a VEGFR1 variant 2 is a protein encoded by VEGFR1 splice variant 2, a VEGFR1 variant 3 is a protein encoded by VEGFR1 splice variant 3, and a VEGFR1 variant 4 is a protein encoded by VEGFR1 splice variant 4.
[0145] As preferably used herein in connection with any aspect of the present invention a pharmacodynamics biomarker is a biomarker whose changes after treatment are associated with target modulation by a specific agent.
[0146] As preferably used herein in connection with any aspect of the present invention a predictive biomarker is a biomarker which can be used in advance of therapy to estimate response or survival of a specific patient on a specific treatment compared with another treatment.
[0147] As preferably used herein in connection with any aspect of the present invention a prognostic biomarker is a biomarker which provides information about the patient's overall disease outcome, regardless of therapy.
[0148] As preferably used herein in connection with any aspect of the present invention a surrogate biomarker is a biomarker intended to substitute for a clinical end point.
[0149] Whether a disease is a disease which can be treated, ameliorated and/or cured by a PKN3 inhibitor, can be determined by a person skilled in the art using routine methods and routine tests. For example, a person skilled in the art will use appropriate animal models, or any surrogate test system, and apply to such animal model or surrogate test system a PKN3 inhibitor and check whether the required read-out can be observed which goes along with the effect(s) of a PKN3 inhibitor. For example, such test may be the matrigel test described in international patent application WO 2008/009477, or any other tumor or cancer growth system involving PKN3 as a target molecule.
[0150] Preferably, a disease as used in the various aspects of the present invention is one which is selected from the group comprising:
A
Acute Lymphoblastic Leukemia (ALL)
Acute Myeloid Leukemia (AML)
Adolescents, Cancer in
Adrenocortical Carcinoma
[0151] Childhood
AIDS-Related Cancers
[0152] Kaposi Sarcoma
[0153] Lymphoma
Anal Cancer
Appendix Cancer
Astrocytomas, Childhood
[0154] Atypical Teratoid/Rhabdoid Tumor, Childhood, Central Nervous System
B
Basal Cell Carcinoma--see Skin Cancer (Nonmelanoma)
Bile Duct Cancer, Extrahepatic
Bladder Cancer
[0155] Childhood
Bone Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma
Brain Stem Glioma, Childhood
Brain Tumor
[0156] Astrocytomas, Childhood
[0157] Brain and Spinal Cord Tumors, Childhood
[0158] Brain Stem Glioma, Childhood
[0159] Central Nervous System Atypical Teratoid/Rhabdoid Tumor, Childhood
[0160] Central Nervous System Embryonal Tumors, Childhood
[0161] Central Nervous System Germ Cell Tumors, Childhood
[0162] Craniopharyngioma, Childhood
[0163] Ependymoblastoma, Childhood
[0164] Ependymoma, Childhood
[0165] Medulloblastoma, Childhood
[0166] Medulloepithelioma, Childhood
[0167] Pineal Parenchymal Tumors of Intermediate Differentiation, Childhood
[0168] Supratentorial Primitive Neuroectodermal Tumors and Pineoblastoma, Childhood Breast Cancer
[0169] Childhood
[0170] Male
[0171] Pregnancy, Breast Cancer and
Bronchial Tumors, Childhood
Burkitt Lymphoma--see Non-Hodgkin Lymphoma
C
Carcinoid Tumor
[0172] Childhood
[0173] Gastrointestinal
Carcinoma of Unknown Primary
[0174] Childhood
Central Nervous System
[0175] Atypical Teratoid/Rhabdoid Tumor, Childhood
[0176] Embryonal Tumors, Childhood
[0177] Germ Cell Tumor, Childhood
[0178] Lymphoma, Primary
Cervical Cancer
[0179] Childhood
Childhood Cancers
Chordoma, Childhood
Chronic Lymphocytic Leukemia (CLL)
Chronic Myelogenous Leukemia (CML)
Chronic Myeloproliferative Disorders
Colon Cancer
Colorectal Cancer
[0180] Childhood
Craniopharyngioma, Childhood
[0181] Cutaneous T-Cell Lymphoma--see Mycosis Fungoides and Sezary Syndrome
D
Duct, Bile, Extrahepatic
Ductal Carcinoma In Situ (DCIS)
E
Embryonal Tumors, Central Nervous System, Childhood
Endometrial Cancer
Ependymoblastoma, Childhood
Ependymoma, Childhood
Esophageal Cancer
[0182] Childhood
Esthesioneuroblastoma, Childhood
Ewing Sarcoma Family of Tumors
Extracranial Germ Cell Tumor, Childhood
Extragonadal Germ Cell Tumor
Extrahepatic Bile Duct Cancer
Eye Cancer
[0183] Intraocular Melanoma
[0184] Retinoblastoma
F
Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma
G
Gallbladder Cancer
Gastric (Stomach) Cancer
[0185] Childhood
Gastrointestinal Carcinoid Tumor
Gastrointestinal Stromal Tumors (GIST)--see Soft Tissue Sarcoma
Germ Cell Tumor
[0186] Central Nervous System, Childhood
[0187] Extracranial, Childhood
[0188] Extragonadal
[0189] Ovarian
Gestational Trophoblastic Tumor
Glioma--see Brain Tumor
[0190] Childhood Brain Stem
H
Hairy Cell Leukemia
Head and Neck Cancer
[0191] Childhood
Heart Cancer, Childhood
Hepatocellular (Liver) Cancer
Histiocytosis, Langerhans Cell
Hodgkin Lymphoma
Hypopharyngeal Cancer
I
Intraocular Melanoma
Islet Cell Tumors, Pancreatic Neuroendocrine Tumors
K
Kaposi Sarcoma
Kidney
[0192] Renal Cell
L
Langerhans Cell Histiocytosis
Laryngeal Cancer
[0193] Childhood
Leukemia
[0194] Acute Lymphoblastic (ALL)
[0195] Acute Myeloid (AML)
[0196] Chronic Lymphocytic (CLL)
[0197] Chronic Myelogenous (CML)
[0198] Hairy Cell
Lip and Oral Cavity Cancer
Liver Cancer (Primary)
Lobular Carcinoma In Situ (LCIS)
Lung Cancer
[0199] Non-Small Cell
[0200] Small Cell
Lymphoma
[0201] AIDS-Related
[0202] Burkitt--see Non-Hodgkin Lymphoma
[0203] Cutaneous T-Cell--see Mycosis Fungoides and Sezary Syndrome
[0204] Hodgkin
[0205] Non-Hodgkin
[0206] Primary Central Nervous System (CNS)
M
[0207] Macroglobulinemia, Waldenstrom
Male Breast Cancer
Malignant Fibrous Histiocytoma of Bone and Osteosarcoma
Medulloblastoma, Childhood
Medulloepithelioma, Childhood
Melanoma
[0208] Childhood
[0209] Intraocular (Eye)
Merkel Cell Carcinoma
Mesothelioma, Malignant
[0210] Childhood
Metastatic Squamous Neck Cancer with Occult Primary
Midline Tract Carcinoma Involving NUT Gene
Mouth Cancer
Multiple Endocrine Neoplasia Syndromes, Childhood
Multiple Myeloma/Plasma Cell Neoplasm
Mycosis Fungoides
Myelodysplastic Syndromes
Myelodysplastic/Myeloproliferative Neoplasms
Myelogenous Leukemia, Chronic (CML)
Myeloid Leukemia, Acute (AML)
Myeloma, Multiple
Myeloproliferative Disorders, Chronic
N
Nasal Cavity and Paranasal Sinus Cancer
Nasopharyngeal Cancer
[0211] Childhood
Neuroblastoma
Non-Hodgkin Lymphoma
Non-Small Cell Lung Cancer
O
Oral Cancer
[0212] Childhood
Oral Cavity Cancer, Lip and
Oropharyngeal Cancer
Osteosarcoma and Malignant Fibrous Histiocytoma of Bone
Ovarian Cancer
[0213] Childhood
[0214] Epithelial
[0215] Germ Cell Tumor
[0216] Low Malignant Potential Tumor
P
Pancreatic Cancer
[0217] Childhood
[0218] Pancreatic Neuroendocrine Tumors (Islet Cell Tumors)
Papillomatosis, Childhood
Paraganglioma
Paranasal Sinus and Nasal Cavity Cancer
Parathyroid Cancer
Penile Cancer
Pharyngeal Cancer
Pheochromocytoma
Pineal Parenchymal Tumors of Intermediate Differentiation, Childhood
Pineoblastoma and Supratentorial Primitive Neuroectodermal Tumors, Childhood
Pituitary Tumor
Plasma Cell Neoplasm/Multiple Myeloma
Pleuropulmonary Blastoma, Childhood
Pregnancy and Breast Cancer
Primary Central Nervous System (CNS) Lymphoma
Prostate Cancer
R
Rectal Cancer
Renal Cell (Kidney) Cancer
Renal Pelvis and Ureter, Transitional Cell Cancer
Retinoblastoma
Rhabdomyosarcoma, Childhood
S
Salivary Gland Cancer
[0219] Childhood
Sarcoma
[0220] Ewing Sarcoma Family of Tumors
[0221] Kaposi
[0222] Soft Tissue
[0223] Uterine
Sezary Syndrome
Skin Cancer
[0224] Childhood
[0225] Melanoma
[0226] Merkel Cell Carcinoma
[0227] Nonmelanoma
Small Cell Lung Cancer
Small Intestine Cancer
Soft Tissue Sarcoma
Squamous Cell Carcinoma--see Skin Cancer (Nonmelanoma)
[0228] Squamous Neck Cancer with Occult Primary, Metastatic
Stomach (Gastric) Cancer
[0229] Childhood
Supratentorial Primitive Neuroectodermal Tumors, Childhood
T
[0230] T-Cell Lymphoma, Cutaneous--see Mycosis Fungoides and Sezary Syndrome
Testicular Cancer
[0231] Childhood
Throat Cancer
Thymoma and Thymic Carcinoma
[0232] Childhood
Thyroid Cancer
[0233] Childhood
Transitional Cell Cancer of the Renal Pelvis and Ureter
Trophoblastic Tumor, Gestational
U
Unknown Primary, Carcinoma of
[0234] Childhood
Unusual Cancers of Childhood
Ureter and Renal Pelvis, Transitional Cell Cancer
Urethral Cancer
Uterine Cancer, Endometrial
Uterine Sarcoma
V
Vaginal Cancer
[0235] Childhood
Vulvar Cancer
W
[0236] Waldenstrom Macroglobulinemia
Wilms Tumor
Women's Cancers
Y
Young Adults, Cancer in
[0237] as may be taken from the list of cancers provided by the National Cancer Institute at the National Institutes of Health.
[0238] In each and any aspect of the present invention the PKN3 inhibitor may be a compound which is selected from the group comprising an siRNA directed against an mRNA coding for PKN3, an antisense oligonucleotide directed against an mRNA coding for PKN3, a ribozyme directed against an mRNA coding for PKN3, an shRNA directed against an mRNA coding for PKN3, an miRNA or antagomir directed against an mRNA coding for PKN3, an aptamer directed against PKN3, a spiegelmer directed against PKN3, an antibody directed against PKN3, an anticalin directed against PKN3, a small molecule. Each and any of the above classes of compounds are known to a person skilled in the art and a PKN3 inhibitor of each any of said classes can be generated by a person skilled in the art by applying standard technology.
[0239] The manufacture of an antibody specific for VEGFR1 in its diverse forms is known to the one skilled in the art and, for example, described in Harlow, E., and Lane, D., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988). Preferably, monoclonal antibodies may be used in connection with the present invention which may be manufactured according to the protocol of Cesar and Milstein and further developments based thereon. Antibodies as used herein, include, but are not limited to, complete antibodies, antibody fragments or derivatives such as Fab fragments, Fc fragments and single-stranded antibodies, as long as they are suitable and capable of binding to VEGFR1 as disclosed herein. Apart from monoclonal antibodies also polyclonal antibodies may be used and/or generated. The generation of polyclonal antibodies is also known to the one skilled in the art and, for example, described in Harlow, E., and Lane, D., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988). Preferably, the antibodies used for therapeutical purposes are humanized or human antibodies as defined above.
[0240] The antibodies which may be used according to the present invention may have one or several markers or labels. Such markers or labels may be useful to detect the antibody either in its diagnostic application or its therapeutic application. Preferably the markers and labels are selected from the group comprising avidine, streptavidine, biotin, gold and fluorescein and used, e. g., in ELISA methods. These and further markers as well as methods are, e. g. described in Harlow, E., and Lane, D., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988).
[0241] "Anticalins" are, among others, described in German patent application DE 197 42 706.
[0242] Aptamers are D-nucleic acids which are either single stranded or double stranded and which specifically interact with a target molecule. The manufacture or selection of aptamers is, e. g., described in European patent EP 0 533 838. Basically the following steps are realized. First, a mixture of nucleic acids, i. e. potential aptamers, is provided whereby each nucleic acid typically comprises a segment of several, preferably at least eight subsequent randomised nucleotides. This mixture is subsequently contacted with the target molecule whereby the nucleic acid(s) bind to the target molecule, such as based on an increased affinity towards the target or with a bigger force thereto, compared to the candidate mixture. The binding nucleic acid(s) are/is subsequently separated from the remainder of the mixture. Optionally, the thus obtained nucleic acid(s) is amplified using, e. g. polymerase chain reaction. These steps may be repeated several times giving at the end a mixture having an increased ratio of nucleic acids specifically binding to the target from which the final binding nucleic acid is then optionally selected. These specifically binding nucleic acid(s) are referred to aptamers. It is obvious that at any stage of the method for the generation or identification of the aptamers samples of the mixture of individual nucleic acids may be taken to determine the sequence thereof using standard techniques. It is within the present invention that the aptamers may be stabilized such as, e. g., by introducing defined chemical groups which are known to the one skilled in the art of generating aptamers. Such modification may for example reside in the introduction of an amino group at the 2'-position of the sugar moiety of the nucleotides. Aptamers are currently used as therapeutical agents. However, it is also within the present invention that the thus selected or generated aptamers may be used for target validation and/or as lead substance for the development of medicaments, preferably of medicaments based on small molecules. This is actually done by a competition assay whereby the specific interaction between the target molecule and the aptamer is inhibited by a candidate drug whereby upon replacement of the aptamer from the complex of target and aptamer it may be assumed that the respective drug candidate allows a specific inhibition of the interaction between target and aptamer, and if the interaction is specific, said candidate drug will, at least in principle, be suitable to block the target and thus decrease its biological availability or activity in a respective system comprising such target. The thus obtained small molecule may then be subject to further derivatisation and modification to optimise its physical, chemical, biological and/or medical characteristics such as toxicity, specificity, biodegradability and bioavailability.
[0243] The generation or manufacture of spiegelmers which may be used or generated according to the present invention using VEGFR1 as disclosed herein, is based on a similar principle. The manufacture of spiegelmers is described in the international patent application WO 98/08856. Spiegelmers are L-nucleic acids, which means that they are composed of L-nucleotides rather than aptamers which are composed of D-nucleotides as aptamers are. Spiegelmers are characterized by the fact that they have a very high stability in biological system and, comparable to aptamers, specifically interact with the target molecule against which they are directed. In the purpose of generating spiegelmers, a heterogonous population of D-nucleic acids is created and this population is contacted with the optical antipode of the target molecule, in the present case for example with the D-enantiomer of the naturally occurring L-enantiomer of the protein kinase N beta. Subsequently, those D-nucleic acids are separated which do not interact with the optical antipode of the target molecule. However, those D-nucleic acids interacting with the optical antipode of the target molecule are separated, optionally determined and/or sequenced and subsequently the corresponding L-nucleic acids are synthesized based on the nucleic acid sequence information obtained from the D-nucleic acids. These L-nucleic acids which are identical in terms of sequence with the aforementioned D-nucleic acids interacting with the optical antipode of the target molecule, will specifically interact with the naturally occurring target molecule rather than with the optical antipode thereof. Similar to the method for the generation of aptamers it is also possible to repeat the various steps several times and thus to enrich those nucleic acids specifically interacting with the optical antipode of the target molecule.
[0244] Ribozymes are catalytically active nucleic acids which preferably consist of RNA which basically comprises two moieties. The first moiety shows a catalytic activity whereas the second moiety is responsible for the specific interaction with the target nucleic acid, in the present case the nucleic acid coding for VEGFR1 as disclosed herein. Upon interaction between the target nucleic acid and the second moiety of the ribozyme, typically by hybridisation and Watson-Crick base pairing of essentially complementary stretches of bases on the two hybridising strands, the catalytically active moiety may become active which means that it catalyses, either intramolecularly or intermolecularly, the target nucleic acid in case the catalytic activity of the ribozyme is a phosphodiesterase activity. Subsequently, there may be a further degradation of the target nucleic acid which in the end results in the degradation of the target nucleic acid as well as the protein derived from the said target nucleic acid due to a lack of newly synthesized VEGFR1 as disclosed herein and a turn-over of prior existing VEGFR1 as disclosed herein. Ribozymes, their use and design principles are known to the one skilled in the art, and, for example described in Doherty and Doudna (Ribozym structures and mechanism. Annu ref. Biophys. Biomolstruct. 2001; 30:457-75) and Lewin and Hauswirth (Ribozyme Gene Therapy: Applications for molecular medicine. 2001 7: 221-8).
[0245] Antisense oligonucleotides are as such equally known in the art as is their use for the manufacture of a medicament and as a diagnostic agent, respectively. Basically, antisense oligonucleotides hybridise based on base complementarity, with a target RNA, preferably with a mRNA, thereby activate RNase H. RNase H is activated by both phosphodiester and phosphorothioate-coupled DNA. Phosphodiester-coupled DNA, however, is rapidly degraded by cellular nucleases with the exception of phosphorothioate-coupled DNA. These resistant, non-naturally occurring DNA derivatives do not inhibit RNase H upon hybridisation with RNA. In other words, antisense polynucleotides are only effective as DNA RNA hybride complexes. Examples for this kind of antisense oligonucleotides are described, among others, in U.S. Pat. No. 5,849,902 and U.S. Pat. No. 5,989,912. In other words, based on the nucleic acid sequence of the target molecule which in the present case is the nucleic acid coding for VEGFR1 as disclosed herein, either from the target protein from which a respective nucleic acid sequence may in principle be deduced, or by knowing the nucleic acid sequence as such, particularly the mRNA, suitable antisense oligonucleotides may be designed base on the principle of base complementarity.
[0246] siRNA is a double stranded RNA having typically a length of about 19 to about 23 nucleotides. The sequence of one of the two RNA strands corresponds to the sequence of the target nucleic acid such as the nucleic acid coding for target molecule, to be degraded. In other words, knowing the nucleic acid sequence of the target molecule, in the present case VEGFR1 in its various forms described herein, preferably the mRNA sequence, a double stranded RNA may be designed with one of the two strands being complementary to said, e. g. mRNA of the target molecule and, upon application of said siRNA to a system containing the gene, genomic DNA, hnRNA or mRNA coding for the target molecule, the respective target nucleic acid will be degraded and thus the level of the respective protein be reduced. The basic principles of designing, constructing and using said siRNA as medicament and diagnostic agent, respectively, is, among others, described in international patent applications WO 00/44895 and WO 01/75164.
[0247] Based on the aforementioned design principles, it is possible to generate such siRNA, antisense oligonucleotide and ribozyme, respectively, once the nucleic acid sequence coding for the target molecule is known. This is also true for precursor molecules of nucleic acid such as hnRNA, cDNA and the like, including genomic nucleic acid. Of course, also knowing the respective antisense strand may allow the design of such nucleic acid based compounds given the basic principle of base pair complementarity, preferably based on Watson-Crick base pairing. Accordingly, a further aspect of the present invention is related to specific siRNAs, ribozymes and antisense nucleotides which are directed against or specific for protein kinase N-beta. In the following, this is further illustrated by siRNA, however, this applies to antisense oligonucleotides and ribozymes as well, as will be acknowledged by the ones skilled in the art.
[0248] Such siRNA comprises preferably a length of from 15 to 25 nucleotides, whereby this means actually any length comprising 15, 16, 17, 18, 20, 21, 22, 23, 24 or 25 nucleotides. In further embodiments, the siRNA may even exhibit more nucleotides. According the design principles well known in the art, respective siRNA can be generated. Accordingly, the siRNA claimed herein comprises a stretch of preferably any nucleotide length from 15 to 25 consecutive nucleotides which is either at least partially complementary to the sense or to the antisense strand encoding the target molecule, and a second ribonucleotide strand which is at least partially complementary to the first one and thus to the antisense strand and sense strand respectively, encoding the target molecule. Any design principle known in the art of generation or manufacture of siRNA may be applied to this kind of duplex structure. The siRNA space disclosed herein comprises siRNA molecules the antisense strand of which starts with a nucleotides which corresponds to nucleotide no. 1 of a the target molecule as specified above. Further such siRNA molecules start with a nucleotide which corresponds to nucleotide no 2 of the target molecule encoding sequence as specified above, and so on. This kind of scanning over the target molecule encoding sequence is repeated so as to provide all possible siRNA molecules which can be directed against the target molecule. The length of any of the siRNA molecules thus generated may be any length suitable for siRNA, more particularly any length as specified above. Preferably, the various siRNA molecule of the siRNA molecule space disclosed herein, overlap except the most 5'terminal nucleotide of the antisense strand or sense strand. It is obvious that the thus obtained antisense sequences have to complemented through base pairing so as to form the at least partially double-stranded structure required for a functionally active siRNA.
[0249] MicroRNA for use in therapeutic applications are, for example, described in Soifer H S et al. (Soifer H S et al., Molecular therapy, vol. 15, no. 12, December 2007, pages 2070-2079).
[0250] Antagomirs for use in therapeutic applications are, for example, described in Stenvang J & Kauppinen S (Stenvang J & Kauppinen S, Expert Opin. Biol. Ther. (2008) 8(1), pages 59-81).
[0251] A preferred embodiment the PKN3 inhibitor is an siRNA which is double-stranded (ds 23mer) having the following composition and structure, respectively:
5' 5u8u7c6g8a6g5c7u7a6g5c5 3' (SEQ ID NO: 11)
3' a6c6g8u7c5u7a8g6g5u7a8a 5' (SEQ ID NO: 12)
with unmodified ribonucleotides being as indicated and modified ribonucleotides being represented as follows:
5: 2'-O-Methyl-u,
6: 2'-O-Methyl-a,
7: 2'-O-Methyl-c,
8: 2'-O-Methyl-g.
[0252] In a further preferred embodiment of the various aspects of the present invention the PKN3 inhibitor is a composition referred to in the prior art and herein as Atu027. The active pharmaceutical ingredient (API) of Atu027 is a PKN3-specific siRNA. The siRNA is a double stranded RNA molecule containing naturally occurring 2'-O-Methyl-modifications. The 2'-O-methyl modifications stabilize the RNA by protection from nuclease attack. The double strand (ds-23mer) is composed of the following sequences:
5' 5u8u7c6g8a6g5c7u7a6g5c5 3' (SEQ ID NO: 11)
3' a6c6g8u7c5u7a8g6g5u7a8a 5' (SEQ ID NO: 12)
with unmodified ribonucleotides being as indicated and modified ribonucleotides being represented as follows:
[0253] 5: 2'-O-Methyl-u,
[0254] 6: 2'-O-Methyl-a,
[0255] 7: 2'-O-Methyl-c,
[0256] 8: 2'-O-Methyl-g.
[0257] For production of Atu027 the PKN3-specific siRNA is formulated with positively charged liposomes composed of three lipids, i.e. the cationic AtuFect01, the neutral, fusogenic DPyPE helperlipid and the PEGylated lipid MPEG-2000-DSPE in a molar ratio of 50/49/1 to deliver the negatively charged siRNA in lipoplexed form. AtuFect01 and DPyPE helperlipid are novel excipients. The third lipid MPEG-2000-DSPE is a known excipient. The structure of the 3 lipids is presented below:
Cationic lipid AtuFect01: (β-(L-Arginyl)-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide tri-hydrochloride)
##STR00001##
Fusogenic/helperlipid DPyPE: 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine
##STR00002##
PEG-lipid MPEG-2000-DSPE: N-(Carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-p- hosphoethanolamine sodium salt
##STR00003##
[0258] It is within the present invention that any of the PKN3 inhibitors disclosed herein can be used in connection with each and any aspect of the present invention, including the method of the present invention and the PKN3 inhibitor for use in a method for the treatment of a subject suffering from or being at risk of suffering from a disease of the present invention.
[0259] It is within the present invention that any of the disease disclosed herein can be used in connection with each and any aspect of the present invention, including the method of the present invention and the PKN3 inhibitor for use in a method for the treatment of a subject suffering from or being at risk of suffering from a disease of the present invention.
[0260] It is also within the present invention that any of the VEGFR1 and nucleic acid coding therefor disclosed herein can be used in connection with each and any aspect of the present invention, including the method of the present invention and the PKN3 inhibitor for use in a method for the treatment of a subject suffering from or being at risk of suffering from a disease of the present invention.
[0261] Methods for the detection of VEGFR1 are known in the art and include, but are not limited to anti-VEGFR1 antibodies. In embodiments of the various aspects of the present invention VEGFR1 is detected by means of ELISA. However, other means are known to a person skilled in the art such as detection by mass spectrometry and the like.
[0262] In connection with the various aspects of the present invention a sample is a sample of a body fluid, whereby the body fluid is selected from the group comprising blood, plasma, liquor, urine and saliva. Preferred samples are blood samples and plasma samples.
[0263] Methods for the detection of a nucleic acid coding for VEGFR1 are also known in the art and include, but are not limited to PCR and RT-PCR.
[0264] VEGFR1 and soluble VEGFR1 (sVEGFR1) are known in the art and described, among others, in Caine G J et al. (Caine, G J, European Journal of Clinical Investigation (2003), 33, 883-890) or Barleon B et al. (Barleon B et al., Angionesis 4: 143-154 (2001)). Barleaon B et al. (supra) also disclose that the titre of soluble VEGFR1 is about 160 pg/ml in healthy volunteers.
[0265] The present invention is now further illustrated by the attached FIGS. and examples from which further feature, embodiments and advantages of the present invention may be taken.
[0266] The following list summarizes some of the SEQ ID NOs and provide an indication as to what they represent.
[0267] SEQ ID NO: 1 amino acid sequence of soluble VEGFR1 or VEGFR1 variant 2
[0268] SEQ ID NO: 2 nucleotide sequence coding for soluble VEGFR1 or VEGFR1 transcript variant 2
[0269] SEQ ID NO: 3 amino acid sequence of VEGFR1 variant 1
[0270] SEQ ID NO: 4 nucleotide sequence coding for VEGFR1 transcript variant 1
[0271] SEQ ID NO: 5 amino acid sequence of VEGFR1 variant 3
[0272] SEQ ID NO: 6 nucleotide sequence coding for VEGFR1 transcript variant 3
[0273] SEQ ID NO: 7 amino acid sequence of VEGFR1 variant 4
[0274] SEQ ID NO: 8 nucleotide sequence coding for VEGFR1 transcript variant 4
[0275] SEQ ID NO: 9 amino acid sequence of PKN3
[0276] SEQ ID NO: 10 nucleotide sequence coding for PKN3
BRIEF DESCRIPTION OF THE FIGURES
[0277] FIG. 1 is a diagram indicating PKN3 knock down, VEGFR1 expression and VEGFR2 expression in PC-3 cells. Human PC-3 cell line was obtained from American Type Culture Collection and cultivated according to the ATCC's recommendation. Cell line was transfected with Luciferase AtuPLEX and Atu027, respectively. Briefly, about 16 h after cell seeding siRNA-lipoplex solution diluted in 10% serum containing medium was added to the cells to achieve transfection concentration of 20 nM siRNA. After transfection (24 h, 48 h, 72 h, and 96 h), cells were lysed, total RNA was isolated and subjected to qRT-PCR. Graphs show respective mRNA level as mean of technical replicate relative to untreated @ 24 h. A decrease in VEGFR1 but not VEGFR2 expression in response to PKN3 knock down is observed.
[0278] FIG. 2 is a diagram indicating PKN3 knock down, VEGFR1 expression and VEGFR2 expression in MDA-MB-435 cells. Human MDA-MB-435 cell line was obtained from American Type Culture Collection and cultivated according to the ATCC's recommendation. Cell line was transfected with Luciferase AtuPLEX and Atu027, respectively. Briefly, about 16 h after cell seeding siRNA-lipoplex solution diluted in 10% serum containing medium was added to the cells to achieve transfection concentration of 20 nM siRNA. After transfection (24 h, 48 h, 72 h, and 96 h), cells were lysed, total RNA was isolated and subjected to qRT-PCR. Graphs show respective mRNA level as mean of technical replicate relative to untreated @ 24 h. A decrease in VEGFR1 but not VEGFR2 expression in response to PKN3 knock down is observed.
[0279] FIG. 3 is a diagram indicating PKN3 knock down, VEGFR1 expression and VEGFR2 expression in SKBR3 cells. Human SKBR3 cell line was obtained from American Type Culture Collection and cultivated according to the ATCC's recommendation. Cell line was transfected with Luciferase AtuPLEX and Atu027, respectively. Briefly, about 12 h after cell seeding siRNA-lipoplex solution diluted in 10% serum containing medium was added to the cells to achieve transfection concentration of 20 nM siRNA. After transfection (24 h, 48 h, 72 h, and 96 h), cells were lysed, total RNA was isolated and subjected to qRT-PCR. Graphs show respective mRNA level as mean of technical replicate relative to untreated @ 24 h. A decrease in VEGFR1 but not VEGFR2 expression in response to PKN3 knock down is observed.
[0280] FIG. 4 is a representation of the design of a clinical phase I study. Before (0 h) and 24 h after the last (24 h) repeated treatment with Atu027 blood was withdrawn for biomarker analysis.
[0281] FIG. 5 shows the result of a biomarker analysis in plasma from humans treated with Atu027. Before (0 h) and 24 h after the last (24 h) repeated treatment with Atu027 blood was withdrawn from 9 patients and plasma was prepared. Samples were analyzed for biomarker analyte concentration. In the graph the ratio of post/pre treatment plasma level is shown for the VEGF isoforms and their receptors.
[0282] FIG. 6 shows the result of a biomarker analysis in plasma from humans treated with Atu027: single analytes--VEGF. Before (0 h) and 24 h after the last (24 h) repeated treatment with Atu027 blood was withdrawn from 9 patients and plasma was prepared. Samples were analyzed for biomarker analyte concentration. In the graph the absolute values for VEGF are presented.
[0283] FIG. 7 shows the result of a biomarker analysis in plasma from humans treated with Atu027: single analytes--VEGF-B. Before (0 h) and 24 h after the last (24 h) repeated treatment with Atu027 blood was withdrawn from 9 patients and plasma was prepared. Samples were analyzed for biomarker analyte concentration. In the graph the absolute values for VEGF-B are presented.
[0284] FIG. 8 shows the result of a biomarker analysis in plasma from humans treated with Atu027: single analytes--VEGF-C. Before (0 h) and 24 h after the last (24 h) repeated treatment with Atu027 blood was withdrawn from 9 patients and plasma was prepared. Samples were analyzed for biomarker analyte concentration. In the graph the absolute values for VEGF-C are presented.
[0285] FIG. 9 shows the result of a biomarker analysis in plasma from humans treated with Atu027: single analytes--VEGF-D. Before (0 h) and 24 h after the last (24 h) repeated treatment with Atu027 blood was withdrawn from 9 patients and plasma was prepared. Samples were analyzed for biomarker analyte concentration. In the graph the absolute values for VEGF-D are presented.
[0286] FIG. 10 shows the result of a biomarker analysis in plasma from humans treated with Atu027: single analytes--VEGFR1 (which is also referred to as VEGFR-1). Before (0 h) and 24 h after the last (24 h) repeated treatment with Atu027 blood was withdrawn from 9 patients and plasma was prepared. Samples were analyzed for biomarker analyte concentration. In the graph the absolute values for VEGFR-1 are presented. In 7 out of 9 patient samples, VEGFR-1 was decreased after repeated Atu027 treatment.
[0287] FIG. 11 shows the result of a biomarker analysis in plasma from humans treated with Atu027: single analytes--VEGFR2 (which is also referred to as VEGFR-2). Before (0 h) and 24 h after the last (24 h) repeated treatment with Atu027 blood was withdrawn from 9 patients and plasma was prepared. Samples were analyzed for biomarker analyte concentration. In the graph the absolute values for VEGFR-2 is presented.
[0288] FIG. 12 shows the result of a biomarker analysis in plasma from humans treated with Atu027: single analytes--VEGFR3 (which is also referred to as VEGFR-3). Before (0 h) and 24 h after the last (24 h) repeated treatment with Atu027 blood was withdrawn from 9 patients and plasma was prepared. Samples were analyzed for biomarker analyte concentration. In the graph the absolute values for VEGFR-3 is presented.
[0289] FIG. 13 represents SEQ ID NO: 1.
[0290] FIG. 14 represents SEQ ID NO: 2.
[0291] FIG. 15 represents SEQ ID NO: 3.
[0292] FIG. 16 represents SEQ ID NO: 4.
[0293] FIG. 17 represents SEQ ID NO: 5.
[0294] FIG. 18 represents SEQ ID NO: 6.
[0295] FIG. 19 represents SEQ ID NO: 7.
[0296] FIG. 20 represents SEQ ID NO: 8.
[0297] FIG. 21 represents SEQ ID NO: 9.
[0298] FIG. 22 represents SEQ ID NO: 10.
EXAMPLES
Example 1
Synthesis of RNAi Molecule (AtuRNAi) for In Vitro Studies
[0299] The siRNA molecules (AtuRNAi) of this invention are described in Table 1. These molecules were synthesized by BioSpring GmbH (Frankfurt a. M., Germany).
TABLE-US-00001 TABLE 1 siRNA name sequence 5' to 3' SEQ ID NO PKN3 s agacuugaggacuuccuggacaa 13 PKN3 as uuguccaggaaguccucaagucu 14 Luciferase s aucacguacgcggaauacuucga 15 Luciferase as ucgaaguauuccgcguacgugau 16
[0300] Nucleotides with 2'-O-methyl modifications are underlined; "s" stands for the sense strand; and "as" stands for the antisense strand.
[0301] The duplexes formed by "PKN3 as" and "PKN3 s", and formed by "Luciferase as" and "Luciferase s" lack 3'-overhangs and are chemically stabilized by alternating 2'-O-methyl sugar modifications on both strands, whereby unmodified nucleotides face modified ones on the opposite strand (Table 1). These duplexes are also referred to herein as PKN3 and Luciferase and were resolved in water to obtain a stock concentration of 1 μM.
Example 2
Formulation of the AtuPLEX
[0302] The AtuPLEX formulation was prepared as essentially described in Santel et al. (Santel et al. Gene Therapy (2006) 13, 1222-1234). Cationic liposomes comprising the novel cationic lipid AtuFECT01 (β-L-arginyl-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride), the neutral phospholipid 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) and the PEGylated lipid N-(Carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-p- hospho-ethanolamine sodium salt (DSPE-PEG) in a molar ratio of 50/49/1 were prepared by lipid film re-hydration in 270 mM sterile RNase-free sucrose solution to a total lipid concentration of 4.335 mg/ml. Subsequently, the multilamellar dispersion was further processed by high-pressure homogenization using an EmulsiFlex C3 device (Avestin, Inc., Ottawa, Canada).
[0303] To generate siRNA-lipoplexes (AtuPLEX), the obtained liposomal dispersion was mixed with an equal volume of a 0.5625 mg/ml solution of siRNA in 270 mM sucrose, resulting in a calculated charge ratio of nucleic acid backbone phosphates to cationic lipid nitrogen atoms of approximately 1:4. The size of the liposome and the lipoplex dispersion and the zeta potential were measured using a Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK).
[0304] The active pharmaceutical ingredient (API) of Atu027 is the PKN3-siRNA. The siRNA is a double stranded RNA molecule containing naturally occurring 2'-O-Methyl-modifications. The 2'-O-methyl modifications stabilize the RNA by protection from nuclease attack.
[0305] The double strand (ds-23mer) is composed of the following sequence:
5' 5u8u7c6g8a6g5c7u7a6g5c5 3' (SEQ ID NO: 11)
3' a6c6g8u7c5u7a8g6g5u7a8a 5' (SEQ ID NO: 12)
Unmodified ribonucleotides: x;
Modifications: 5: 2'-O-Methyl-u, 6: 2'-O-Methyl-a, 7: 2'-O-Methyl-c, 8:2'-O-Methyl-g.
[0306] For Atu027 production PKN3-siRNA is formulated with positively charged liposomes composed of three lipids, i.e. the cationic AtuFect01, the neutral, fusogenic DPyPE helperlipid and the PEGylated lipid MPEG-2000-DSPE in a molar ratio of 50/49/1 to deliver the negatively charged siRNA in lipoplexed form. AtuFect01 and DPyPE helperlipid are novel excipients. The third lipid MPEG-2000-DSPE is a known excipient. The structure of the 3 lipids is presented below:
Cationic lipid AtuFect01: (β-(L-arginyl)-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide tri-hydrochloride)
##STR00004##
Fusogenic/helperlipid DPyPE: 1,2-Diphytanoyl-sn-Glycero-3-Phosphoethanolamine
##STR00005##
[0307] PEG-lipid MPEG-2000-DSPE: N-(Carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glycero-3-p- hosphoethanolamine sodium salt
##STR00006##
Example 3
Expression of VEGFR-1 in Response to In Vitro Gene Silencing of Human PKN3 in Human Prostate Cancer PC-3 Cells
[0308] 3×105 PC-3 cells were plated in 10-cm dishes, transfected 16 h later with 20 nM of the siRNA as described in Example 1 and 1 μg/ml liposome were prepared as described in Example 2, whereby the liposome consisted of the cationic lipid β-(L-arginyl)-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide tri-hydrochloride (AtuFECT01), the fusogenic lipid 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) and the PEGylated lipid MPEG-2000-DSPE in a molar ratio of 50/49/1. Cells were lysed for RNA extraction 24 h, 48 h, 72 h and 96 h after transfection, respectively.
[0309] Total RNA was prepared with the InviTrap® Spin Cell RNA Mini Kit (Invitek, Berlin, Germany). Inhibition of PKN3 mRNA expression, expression of VEGFR1 and VEGFR2 mRNA was detected by real time RT-PCR (TaqMan) analysis using 300 nM PKN3, VEGFR1 and VEGFR2 specific forward and reverse primer and 100 nM probe. The sequences of the primers in TaqMan are listed in the following table, whereby UPR means upper primer, LWR means lower primer, and PRB means probe.
TABLE-US-00002 TABLE 2 Primer/ SEQ Name Probe Sequence 5' to 3' ID NO Human UPR CACTTTGGGAAGGTCCTCCTG 17 PKN3 PRB FAM-TTCAAGGGGACAGGGAAATACTACGCC 18 A-BHQ1 LWR CCTCCTGCTTCTTCAGTGCTTT 19 Human UPR CCCCGATTATGTGAGAAAAGGA 20 VEGFR1 PRB FAM-CGACTTCCTCTGAAATGGATGGCTCCT 21 G-BHQ1 LWR CGCTCTTGGTGCTGTAGATTTTG 22 Human UPR TCTGCCTACCTCACCTGTTTCC 23 VEGFR2 PRB FAM-ATGGAGGAGGAGGAAGTATGTGACCCC 24 A-TAMRA LWR TGACTGATTCCTGCTGTGTTGTC 25
[0310] The reaction was carried out in 25 μl and assayed on the StepOnePlus® Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions under the following conditions: 48° C. for 30 min, 95° C. for 10 min, followed by 40 cycles of 15 s at 95° C. and 1 min at 60° C. mRNA amounts are shown relative to untreated control lysed 24 h after transfection.
[0311] The results are shown in FIG. 1 indicating in vitro a marked decrease in VEGFR1 but not VEGFR2 mRNA expression in response to PKN3 knock down 72 h and 96 h post-transfection in human prostate cancer PC-3 cells.
Example 4
Expression of VEGFR-1 in Response to In Vitro Gene Silencing of Human PKN3 in Human Breast Carcinoma MDA-MB-435 Cells
[0312] 3×105 MDA-MB-435 cells were plated in 10-cm dishes, transfected 16 h later with 20 nM of the siRNA as described in Example 1 and 1 μg/ml liposome were prepared as described in Example 2, whereby the liposome consisted of the cationic lipid β-(L-arginyl)-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide tri-hydrochloride (AtuFECT01), the fusogenic lipid 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) and the PEGylated lipid MPEG-2000-DSPE in a molar ratio of 50/49/1. Cells were lysed for RNA extraction 24 h, 48 h, 72 h and 96 h after transfection, respectively.
[0313] Total RNA was prepared with the InviTrap® Spin Cell RNA Mini Kit (Invitek, Berlin, Germany). Inhibition of PKN3 mRNA expression, expression of VEGFR1 and VEGFR2 mRNA was detected by real time RT-PCR (TaqMan) analysis as described in example 3. mRNA amounts are shown relative to untreated control lysed 24 h after transfection.
[0314] The results are shown in FIG. 2 indicating in vitro a marked decrease in VEGFR1 but not VEGFR2 mRNA expression in response to PKN3 knock down at all time-points analyzed (24 h, 48 h, 72 h and 96 h post-transfection) in human breast carcinoma MDA-MB-435 cells.
Example 5
Expression of VEGFR-1 in Response to In Vitro Gene Silencing of Human PKN3 in Human Ovarian Cancer SKBR3 Cells
[0315] 3×105 SKBR3 cells were plated in 10-cm dishes, transfected 16 h later with 20 nM of the siRNA as described in Example 1 and 1 μg/ml liposome were prepared as described in Example 2, whereby the liposome consisted of the cationic lipid β-(L-arginyl)-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide tri-hydrochloride (AtuFECT01), the fusogenic lipid 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) and the PEGylated lipid MPEG-2000-DSPE in a molar ratio of 50/49/1. Cells were lysed for RNA extraction 24 h, 48 h and 72 h after transfection, respectively.
[0316] Total RNA was prepared with the InviTrap® Spin Cell RNA Mini Kit (Invitek, Berlin, Germany). Inhibition of PKN3 mRNA expression, expression of VEGFR1 and VEGFR2 mRNA was detected by real time RT-PCR (TaqMan) analysis as described in example 3. mRNA amounts are shown relative to untreated control lysed 24 h after transfection.
[0317] The results are shown in FIG. 3 indicating in vitro a marked decrease in VEGFR1 but not VEGFR2 mRNA expression in response to PKN3 knock down 72 h post-transfection in human ovarian cancer SKBR3 cells.
Example 6
Design of Clinical Phase I Study with Atu027
[0318] The Phase I study with Atu027 "A prospective, open label, single-centre, dose finding phase I study with Atu027 (an siRNA formulation) in subjects with advanced solid cancer--Atu027-I-01" (EudraCT No. 2008-005588-32/NCT00938574) was conducted. Study design is shown in FIG. 4. Before (0 h) and 24 h after the last (24 h) repeated 4-h intravenous infusion treatment with Atu027 blood was withdrawn for biomarker analysis. EDTA plasma was prepared by centrifugation according to standard protocols and stored at -20° C.
Example 7
Biomarker Analysis in Plasma of Humans Treated with Atu027
[0319] Before (0 h) and 24 h after the last (24 h) repeated treatment with Atu027 blood was withdrawn from nine patients and EDTA plasma was prepared. Samples were analyzed in a multiplexed immunoassay for concentration of respective analytes using the Luminex technology.
[0320] In FIG. 5 the ratio of post/pre treatment plasma level (24 h level divided by the 0 h value) in the individual patient is shown for the VEGF isoforms VEGF, VEGF-B, VEGF-C, VEGF-D and their receptors VEGFR1, VEGFR2 and VEGFR3. The Atu027 doses based on the siRNA content are given in the right table. In seven out of nine patients VEGFR1 level were markedly decreased after Atu027 treatment.
[0321] In addition to the Atu027 doses indicated in FIG. 5 doses of 0.036 mg/kg Atu027 (based on siRNA content) were administered to three patients whereby two patients showed a marked decrease in the ratio of soluble FLT-1 (sFLT-1) post/pretreatment.
[0322] The absolute plasma level of the single analytes VEGF, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2 and VEGFR3 are shown in graphs and tables in FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11 and FIG. 12. Again, a marked decrease of VEGFR1 concentration after treatment could be detected (FIG. 10) whereas the VEGF, VEGF-B, VEGF-C, VEGF-D, VEGFR2 and VEGFR3 level remained unchanged in most patients and slightly increased or decreased in single individuals.
[0323] The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.
Sequence CWU
1
1
251687PRTHomo sapiensMISC_FEATURE(1)..(687)Soluble VEGFR1 1Met Val Ser Tyr
Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5
10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser
Gly Ser Lys Leu Lys Asp Pro 20 25
30 Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly
Gln Thr 35 40 45
Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50
55 60 Glu Met Val Ser Lys
Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70
75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser
Thr Leu Thr Leu Asn Thr 85 90
95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala
Val 100 105 110 Pro
Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115
120 125 Ser Asp Thr Gly Arg Pro
Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135
140 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val
Ile Pro Cys Arg Val 145 150 155
160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
165 170 175 Leu Ile
Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180
185 190 Ile Ile Ser Asn Ala Thr Tyr
Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200
205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr
Leu Thr His Arg 210 215 220
Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val 225
230 235 240 Lys Leu Leu
Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245
250 255 Pro Leu Asn Thr Arg Val Gln Met
Thr Trp Ser Tyr Pro Asp Glu Lys 260 265
270 Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser
Asn Ser His 275 280 285
Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290
295 300 Asp Lys Gly Leu
Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310
315 320 Ser Val Asn Thr Ser Val His Ile Tyr
Asp Lys Ala Phe Ile Thr Val 325 330
335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys
Arg Ser 340 345 350
Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val
355 360 365 Trp Leu Lys Asp
Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370
375 380 Thr Arg Gly Tyr Ser Leu Ile Ile
Lys Asp Val Thr Glu Glu Asp Ala 385 390
395 400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser
Asn Val Phe Lys 405 410
415 Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu
420 425 430 Lys Ala Val
Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435
440 445 Arg Gln Ile Leu Thr Cys Thr Ala
Tyr Gly Ile Pro Gln Pro Thr Ile 450 455
460 Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu
Ala Arg Cys 465 470 475
480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser
485 490 495 Asn Met Gly Asn
Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500
505 510 Glu Gly Lys Asn Lys Met Ala Ser Thr
Leu Val Val Ala Asp Ser Arg 515 520
525 Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly
Thr Val 530 535 540
Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545
550 555 560 Val Asn Leu Glu Lys
Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565
570 575 Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp
Val Thr Trp Ile Leu Leu 580 585
590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln
Lys 595 600 605 Met
Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610
615 620 Asn Val Ser Leu Gln Asp
Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn 625 630
635 640 Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys
Glu Ile Thr Ile Arg 645 650
655 Gly Glu His Cys Asn Lys Lys Ala Val Phe Ser Arg Ile Ser Lys Phe
660 665 670 Lys Ser
Thr Arg Asn Asp Cys Thr Thr Gln Ser Asn Val Lys His 675
680 685 26499DNAHomo
sapiensmisc_feature(1)..(6499)Soluble VEGFR1 2atcgaggtcc gcgggaggct
cggagcgcgc caggcggaca ctcctctcgg ctcctccccg 60gcagcggcgg cggctcggag
cgggctccgg ggctcgggtg cagcggccag cgggcgcctg 120gcggcgagga ttacccgggg
aagtggttgt ctcctggctg gagccgcgag acgggcgctc 180agggcgcggg gccggcggcg
gcgaacgaga ggacggactc tggcggccgg gtcgttggcc 240gcggggagcg cgggcaccgg
gcgagcaggc cgcgtcgcgc tcaccatggt cagctactgg 300gacaccgggg tcctgctgtg
cgcgctgctc agctgtctgc ttctcacagg atctagttca 360ggttcaaaat taaaagatcc
tgaactgagt ttaaaaggca cccagcacat catgcaagca 420ggccagacac tgcatctcca
atgcaggggg gaagcagccc ataaatggtc tttgcctgaa 480atggtgagta aggaaagcga
aaggctgagc ataactaaat ctgcctgtgg aagaaatggc 540aaacaattct gcagtacttt
aaccttgaac acagctcaag caaaccacac tggcttctac 600agctgcaaat atctagctgt
acctacttca aagaagaagg aaacagaatc tgcaatctat 660atatttatta gtgatacagg
tagacctttc gtagagatgt acagtgaaat ccccgaaatt 720atacacatga ctgaaggaag
ggagctcgtc attccctgcc gggttacgtc acctaacatc 780actgttactt taaaaaagtt
tccacttgac actttgatcc ctgatggaaa acgcataatc 840tgggacagta gaaagggctt
catcatatca aatgcaacgt acaaagaaat agggcttctg 900acctgtgaag caacagtcaa
tgggcatttg tataagacaa actatctcac acatcgacaa 960accaatacaa tcatagatgt
ccaaataagc acaccacgcc cagtcaaatt acttagaggc 1020catactcttg tcctcaattg
tactgctacc actcccttga acacgagagt tcaaatgacc 1080tggagttacc ctgatgaaaa
aaataagaga gcttccgtaa ggcgacgaat tgaccaaagc 1140aattcccatg ccaacatatt
ctacagtgtt cttactattg acaaaatgca gaacaaagac 1200aaaggacttt atacttgtcg
tgtaaggagt ggaccatcat tcaaatctgt taacacctca 1260gtgcatatat atgataaagc
attcatcact gtgaaacatc gaaaacagca ggtgcttgaa 1320accgtagctg gcaagcggtc
ttaccggctc tctatgaaag tgaaggcatt tccctcgccg 1380gaagttgtat ggttaaaaga
tgggttacct gcgactgaga aatctgctcg ctatttgact 1440cgtggctact cgttaattat
caaggacgta actgaagagg atgcagggaa ttatacaatc 1500ttgctgagca taaaacagtc
aaatgtgttt aaaaacctca ctgccactct aattgtcaat 1560gtgaaacccc agatttacga
aaaggccgtg tcatcgtttc cagacccggc tctctaccca 1620ctgggcagca gacaaatcct
gacttgtacc gcatatggta tccctcaacc tacaatcaag 1680tggttctggc acccctgtaa
ccataatcat tccgaagcaa ggtgtgactt ttgttccaat 1740aatgaagagt cctttatcct
ggatgctgac agcaacatgg gaaacagaat tgagagcatc 1800actcagcgca tggcaataat
agaaggaaag aataagatgg ctagcacctt ggttgtggct 1860gactctagaa tttctggaat
ctacatttgc atagcttcca ataaagttgg gactgtggga 1920agaaacataa gcttttatat
cacagatgtg ccaaatgggt ttcatgttaa cttggaaaaa 1980atgccgacgg aaggagagga
cctgaaactg tcttgcacag ttaacaagtt cttatacaga 2040gacgttactt ggattttact
gcggacagtt aataacagaa caatgcacta cagtattagc 2100aagcaaaaaa tggccatcac
taaggagcac tccatcactc ttaatcttac catcatgaat 2160gtttccctgc aagattcagg
cacctatgcc tgcagagcca ggaatgtata cacaggggaa 2220gaaatcctcc agaagaaaga
aattacaatc agaggtgagc actgcaacaa aaaggctgtt 2280ttctctcgga tctccaaatt
taaaagcaca aggaatgatt gtaccacaca aagtaatgta 2340aaacattaaa ggactcatta
aaaagtaaca gttgtctcat atcatcttga tttattgtca 2400ctgttgctaa ctttcaggct
cggaggagat gctcctccca aaatgagttc ggagatgata 2460gcagtaataa tgagaccccc
gggccccagc tctgggcccc ccattcaggc cgagggggct 2520gctccggggg gccgacttgg
tgcacgtttg gatttggagg atccctgcac tgccttctct 2580gtgtttgttg ctcttgctgt
tttctcctgc ctgataaaca acaacttggg atgatccttt 2640ccttccattt tgatgccaac
ctctttttat ttttaagtgt tgaagctgca caaactgaat 2700aatttaaaca aatgctggtt
tctgccaaag atggacacga ataagttaat tttccagctc 2760agaatgagta cagttgaatt
tgagactctg tcggacttct gcctggtttt atttgggact 2820atttcatctg ctcttgattt
gtaaatagca cctggatagc aagttataat gcttatttat 2880ttgaaaatgc tttttttttt
tttacgttaa gcacatttat cttgaactgg agcttctaaa 2940atgggcccca ggggtgcaag
atgttggtgt aattcagaga tagtaaaggt ttatcgcagt 3000gtgaattata agagtccatc
caaatcaacg tcccctccct cctctcatgc gatccaggta 3060attatgcagt tagtgccaca
gtagactagc ctagcaaagg gtttgctcct tgctgtctct 3120gactgcacca cacagctatt
gatggcagct gaaagaaagt ggatcatgcc ttaattttaa 3180atattcctgt cctctggtta
ttattttaag gaacttcatc atgttaaaat gacagcattc 3240aaaggtgtac cacaatcaat
ttatcaagga aataaaggct attgtaacca gagatttaat 3300gcattcttct aaatgtaaat
ttaaaatttg ccctttaaaa aagtccactt tccccatatg 3360caaatgttaa taggattttt
atggggatta agaagcggca aaactacaga agcagaattc 3420aaagtaattt aaaaaataca
caccagtttt aaatcaagag aagttgtaat ctcttgtttt 3480aagcttgcgt ttgagggaaa
atgacttttt caccaattta atatgcattg ttctgttgtt 3540tttatttatg attgatcatt
atatgtgact tgcataaact atttaaaaaa aaaaactata 3600atgaccaaaa tagccatggc
tgagaaacac agtggctggg cagttcaata ggaggtgaca 3660atatgacaac ttctcaagct
tgggaactca ccagactgtt tcctccttta ggtaacagat 3720tctgtcccac ggctaaactt
gtctttcacg tgggaattgc ttttgtcaaa cgtgaaagag 3780taaacaatag catttcccca
gaatgccagt tttatggagc cccaaatgct ctgaaaacaa 3840ttagtaacct ggaagttgtc
agcccaaagg aaagaaaaat caattgtatc ttgaaatttt 3900acctatggct ctttggcctg
gcttctttgt tcattataag ttagtgtgtt ccttcaggaa 3960acaatgcctt aataccatag
aacatggggg ccttaatagt tgctaacatt aaaaaagcaa 4020acagaatgat tgagggatcc
ttatgaaaac aaaatggtga attggacatg cagaacctac 4080catttccttc ccctgtttgc
aatttttgtg gggaggggag gatgttagta tttacaaaag 4140atgattttaa gaacttccaa
gagatgagtt taagaattcc atagagtatt agttgttcac 4200tgtgtaatta atccttccgg
agagtctttt tttttttttt taaagaaact tttgggtggg 4260ttttgttttt tattagttac
cctaggggta tgttaccctg gggtatgaag ggaggtgaag 4320ataacggagg ggggagaaaa
aaaaaaggag aaaaaaggag cctaaaatgg ggaataattg 4380aaatggaaca gggggtgtga
ggctggttcc tcagtcccca ttccaaacgg aggatagaag 4440ctgtgtattt atgtgacctg
gcagatctct ggggccataa cactgaaaag tgaaagaacc 4500tggtgggcag ctatctttgg
ctactgataa ccagcagaaa tgtctgttaa ttctgatttt 4560ctcaatttga agggatcagc
tacactgtta aattttggaa agccactacc tacttccatc 4620aagtaactta ggtttcgaaa
tatgggttca acgcacctcc cttattcaaa atgtcaaaat 4680agattattat aatgtataaa
gtaagaattg acaaaatatg attcttgggt tgattggtca 4740tttagaaact agccaaaagt
gagactttta atgtagaaca tttttcagaa atgggtacaa 4800agaaaaatgc atattactgt
atatttcaga gtgtttatgt gaaccttgta tttaattgag 4860agtcccatgt acgttctgca
gcctttttgc tgcttctatc atctgaagtt tgtgtagtac 4920aaataaggcc tttgggattc
ttaatgacat ttatgttaaa atgttctctt ctctttaaac 4980accgttttcc aatccacctg
tcagggagtc caaatcgtgt ctgtgttgat gatgctatac 5040tttgtagcta gaaaaacaat
tttagtgttg tgggctctgt attcagactt cctttttaca 5100agaccgatgg gcagtgatag
attattttat catatttaat gcatgggaaa tagtgtgctg 5160aggaagctat taaaagtata
actcagtgaa ttgggtctga gttttaaatg agatatttca 5220aaattggctt gccactgtaa
aagcgactaa ataataatat gatactgttc tttatgatct 5280tgtcatgttt cactgatatg
tttggggtct tcactatgta aaaaatgtca aaattgtaat 5340gagcaagcat gtacaagtag
tcgtaaatca aaggttttaa acaggactgc attttcaatt 5400aggaaaagct gtttggcaga
tagcatccaa tgcaaaaaca gaaatatcgt aacgttctgc 5460ttagtgggca agataagata
ggaaagacat gctcaaagag gcaaaagaat cattgctatc 5520attcattcta cactagtttg
aagaagtttt tgtacatcag agcacttcct tcagcacact 5580tttttgcctt cagatttcat
tttttataaa atgagaagac taatgataaa ctgtagaaat 5640caaaatttat tgagaaatct
gtttctccta acagatagta accctgccat gatatactac 5700ttcaacaatg ttataaaatt
tatgtgataa tatacatttt aacctgggat ttctaaattg 5760ctttaacaaa tgctaatcct
gagagttgcc ctgcaggact caaaagggaa aggttttggg 5820acgtggcaga accctgcagg
gacatggaat taaggccatt gcaatgtatc atctttgtag 5880cattgtcatc actcctaagc
tgccttcaca gttttagtac actaagatga ggaaatcgaa 5940aatgggcaga gaaagctcat
actgtataat tgaagacagt gacagagaac gtgtcagtta 6000tgccaaaact cttttgattt
ctgttccagg atttccaaca agaggggaaa ggaatgactt 6060gggagggtgg gaaagacatt
aggagttgtt tttatttttt accttggaag ctttagctac 6120caatccagta ccctcctaac
tagaatgtat acacatcagc aggactgact gactacttca 6180ttagagatat actgtactca
ttgggggcct tgggggtact gctgttctta tgtgggattt 6240taatgttgta atgtattgca
tcttaatgta ttgaattcat tttgttgtac tatattggtt 6300ggcattttat taaaataaat
tgtattgtat catatttgta tgttttaaga gaaaataata 6360taaaatacaa tatttgtact
attatatagt gcaaaaacta caaatctgtg cctctgcctc 6420ttgaattaat tctttggttg
cttgcatttg ggaagggaat ggagaaagga aagaaccaat 6480aaagctttca aagttcaag
649931338PRTHomo
sapiensMISC_FEATURE(1)..(1338)VEGFR1 variant 3 3Met Val Ser Tyr Trp Asp
Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5
10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser
Lys Leu Lys Asp Pro 20 25
30 Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln
Thr 35 40 45 Leu
His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50
55 60 Glu Met Val Ser Lys Glu
Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70
75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr
Leu Thr Leu Asn Thr 85 90
95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val
100 105 110 Pro Thr
Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115
120 125 Ser Asp Thr Gly Arg Pro Phe
Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135
140 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile
Pro Cys Arg Val 145 150 155
160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
165 170 175 Leu Ile Pro
Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180
185 190 Ile Ile Ser Asn Ala Thr Tyr Lys
Glu Ile Gly Leu Leu Thr Cys Glu 195 200
205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu
Thr His Arg 210 215 220
Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val 225
230 235 240 Lys Leu Leu Arg
Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245
250 255 Pro Leu Asn Thr Arg Val Gln Met Thr
Trp Ser Tyr Pro Asp Glu Lys 260 265
270 Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn
Ser His 275 280 285
Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290
295 300 Asp Lys Gly Leu Tyr
Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310
315 320 Ser Val Asn Thr Ser Val His Ile Tyr Asp
Lys Ala Phe Ile Thr Val 325 330
335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg
Ser 340 345 350 Tyr
Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355
360 365 Trp Leu Lys Asp Gly Leu
Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375
380 Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val
Thr Glu Glu Asp Ala 385 390 395
400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys
405 410 415 Asn Leu
Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420
425 430 Lys Ala Val Ser Ser Phe Pro
Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440
445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro
Gln Pro Thr Ile 450 455 460
Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys 465
470 475 480 Asp Phe Cys
Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485
490 495 Asn Met Gly Asn Arg Ile Glu Ser
Ile Thr Gln Arg Met Ala Ile Ile 500 505
510 Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala
Asp Ser Arg 515 520 525
Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530
535 540 Gly Arg Asn Ile
Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545 550
555 560 Val Asn Leu Glu Lys Met Pro Thr Glu
Gly Glu Asp Leu Lys Leu Ser 565 570
575 Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile
Leu Leu 580 585 590
Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys
595 600 605 Met Ala Ile Thr
Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610
615 620 Asn Val Ser Leu Gln Asp Ser Gly
Thr Tyr Ala Cys Arg Ala Arg Asn 625 630
635 640 Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu
Ile Thr Ile Arg 645 650
655 Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val
660 665 670 Ala Ile Ser
Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675
680 685 Glu Pro Gln Ile Thr Trp Phe Lys
Asn Asn His Lys Ile Gln Gln Glu 690 695
700 Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser Thr Leu Phe
Ile Glu Arg 705 710 715
720 Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys Lys Ala Thr Asn Gln
725 730 735 Lys Gly Ser Val
Glu Ser Ser Ala Tyr Leu Thr Val Gln Gly Thr Ser 740
745 750 Asp Lys Ser Asn Leu Glu Leu Ile Thr
Leu Thr Cys Thr Cys Val Ala 755 760
765 Ala Thr Leu Phe Trp Leu Leu Leu Thr Leu Phe Ile Arg Lys
Met Lys 770 775 780
Arg Ser Ser Ser Glu Ile Lys Thr Asp Tyr Leu Ser Ile Ile Met Asp 785
790 795 800 Pro Asp Glu Val Pro
Leu Asp Glu Gln Cys Glu Arg Leu Pro Tyr Asp 805
810 815 Ala Ser Lys Trp Glu Phe Ala Arg Glu Arg
Leu Lys Leu Gly Lys Ser 820 825
830 Leu Gly Arg Gly Ala Phe Gly Lys Val Val Gln Ala Ser Ala Phe
Gly 835 840 845 Ile
Lys Lys Ser Pro Thr Cys Arg Thr Val Ala Val Lys Met Leu Lys 850
855 860 Glu Gly Ala Thr Ala Ser
Glu Tyr Lys Ala Leu Met Thr Glu Leu Lys 865 870
875 880 Ile Leu Thr His Ile Gly His His Leu Asn Val
Val Asn Leu Leu Gly 885 890
895 Ala Cys Thr Lys Gln Gly Gly Pro Leu Met Val Ile Val Glu Tyr Cys
900 905 910 Lys Tyr
Gly Asn Leu Ser Asn Tyr Leu Lys Ser Lys Arg Asp Leu Phe 915
920 925 Phe Leu Asn Lys Asp Ala Ala
Leu His Met Glu Pro Lys Lys Glu Lys 930 935
940 Met Glu Pro Gly Leu Glu Gln Gly Lys Lys Pro Arg
Leu Asp Ser Val 945 950 955
960 Thr Ser Ser Glu Ser Phe Ala Ser Ser Gly Phe Gln Glu Asp Lys Ser
965 970 975 Leu Ser Asp
Val Glu Glu Glu Glu Asp Ser Asp Gly Phe Tyr Lys Glu 980
985 990 Pro Ile Thr Met Glu Asp Leu Ile
Ser Tyr Ser Phe Gln Val Ala Arg 995 1000
1005 Gly Met Glu Phe Leu Ser Ser Arg Lys Cys Ile
His Arg Asp Leu 1010 1015 1020
Ala Ala Arg Asn Ile Leu Leu Ser Glu Asn Asn Val Val Lys Ile
1025 1030 1035 Cys Asp Phe
Gly Leu Ala Arg Asp Ile Tyr Lys Asn Pro Asp Tyr 1040
1045 1050 Val Arg Lys Gly Asp Thr Arg Leu
Pro Leu Lys Trp Met Ala Pro 1055 1060
1065 Glu Ser Ile Phe Asp Lys Ile Tyr Ser Thr Lys Ser Asp
Val Trp 1070 1075 1080
Ser Tyr Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Gly Ser 1085
1090 1095 Pro Tyr Pro Gly Val
Gln Met Asp Glu Asp Phe Cys Ser Arg Leu 1100 1105
1110 Arg Glu Gly Met Arg Met Arg Ala Pro Glu
Tyr Ser Thr Pro Glu 1115 1120 1125
Ile Tyr Gln Ile Met Leu Asp Cys Trp His Arg Asp Pro Lys Glu
1130 1135 1140 Arg Pro
Arg Phe Ala Glu Leu Val Glu Lys Leu Gly Asp Leu Leu 1145
1150 1155 Gln Ala Asn Val Gln Gln Asp
Gly Lys Asp Tyr Ile Pro Ile Asn 1160 1165
1170 Ala Ile Leu Thr Gly Asn Ser Gly Phe Thr Tyr Ser
Thr Pro Ala 1175 1180 1185
Phe Ser Glu Asp Phe Phe Lys Glu Ser Ile Ser Ala Pro Lys Phe 1190
1195 1200 Asn Ser Gly Ser Ser
Asp Asp Val Arg Tyr Val Asn Ala Phe Lys 1205 1210
1215 Phe Met Ser Leu Glu Arg Ile Lys Thr Phe
Glu Glu Leu Leu Pro 1220 1225 1230
Asn Ala Thr Ser Met Phe Asp Asp Tyr Gln Gly Asp Ser Ser Thr
1235 1240 1245 Leu Leu
Ala Ser Pro Met Leu Lys Arg Phe Thr Trp Thr Asp Ser 1250
1255 1260 Lys Pro Lys Ala Ser Leu Lys
Ile Asp Leu Arg Val Thr Ser Lys 1265 1270
1275 Ser Lys Glu Ser Gly Leu Ser Asp Val Ser Arg Pro
Ser Phe Cys 1280 1285 1290
His Ser Ser Cys Gly His Val Ser Glu Gly Lys Arg Arg Phe Thr 1295
1300 1305 Tyr Asp His Ala Glu
Leu Glu Arg Lys Ile Ala Cys Cys Ser Pro 1310 1315
1320 Pro Pro Asp Tyr Asn Ser Val Val Leu Tyr
Ser Thr Pro Pro Ile 1325 1330 1335
47123DNAHomo sapiensmisc_feature(1)..(7123)VEGFR1 transcript
variat 1 4atcgaggtcc gcgggaggct cggagcgcgc caggcggaca ctcctctcgg
ctcctccccg 60gcagcggcgg cggctcggag cgggctccgg ggctcgggtg cagcggccag
cgggcgcctg 120gcggcgagga ttacccgggg aagtggttgt ctcctggctg gagccgcgag
acgggcgctc 180agggcgcggg gccggcggcg gcgaacgaga ggacggactc tggcggccgg
gtcgttggcc 240gcggggagcg cgggcaccgg gcgagcaggc cgcgtcgcgc tcaccatggt
cagctactgg 300gacaccgggg tcctgctgtg cgcgctgctc agctgtctgc ttctcacagg
atctagttca 360ggttcaaaat taaaagatcc tgaactgagt ttaaaaggca cccagcacat
catgcaagca 420ggccagacac tgcatctcca atgcaggggg gaagcagccc ataaatggtc
tttgcctgaa 480atggtgagta aggaaagcga aaggctgagc ataactaaat ctgcctgtgg
aagaaatggc 540aaacaattct gcagtacttt aaccttgaac acagctcaag caaaccacac
tggcttctac 600agctgcaaat atctagctgt acctacttca aagaagaagg aaacagaatc
tgcaatctat 660atatttatta gtgatacagg tagacctttc gtagagatgt acagtgaaat
ccccgaaatt 720atacacatga ctgaaggaag ggagctcgtc attccctgcc gggttacgtc
acctaacatc 780actgttactt taaaaaagtt tccacttgac actttgatcc ctgatggaaa
acgcataatc 840tgggacagta gaaagggctt catcatatca aatgcaacgt acaaagaaat
agggcttctg 900acctgtgaag caacagtcaa tgggcatttg tataagacaa actatctcac
acatcgacaa 960accaatacaa tcatagatgt ccaaataagc acaccacgcc cagtcaaatt
acttagaggc 1020catactcttg tcctcaattg tactgctacc actcccttga acacgagagt
tcaaatgacc 1080tggagttacc ctgatgaaaa aaataagaga gcttccgtaa ggcgacgaat
tgaccaaagc 1140aattcccatg ccaacatatt ctacagtgtt cttactattg acaaaatgca
gaacaaagac 1200aaaggacttt atacttgtcg tgtaaggagt ggaccatcat tcaaatctgt
taacacctca 1260gtgcatatat atgataaagc attcatcact gtgaaacatc gaaaacagca
ggtgcttgaa 1320accgtagctg gcaagcggtc ttaccggctc tctatgaaag tgaaggcatt
tccctcgccg 1380gaagttgtat ggttaaaaga tgggttacct gcgactgaga aatctgctcg
ctatttgact 1440cgtggctact cgttaattat caaggacgta actgaagagg atgcagggaa
ttatacaatc 1500ttgctgagca taaaacagtc aaatgtgttt aaaaacctca ctgccactct
aattgtcaat 1560gtgaaacccc agatttacga aaaggccgtg tcatcgtttc cagacccggc
tctctaccca 1620ctgggcagca gacaaatcct gacttgtacc gcatatggta tccctcaacc
tacaatcaag 1680tggttctggc acccctgtaa ccataatcat tccgaagcaa ggtgtgactt
ttgttccaat 1740aatgaagagt cctttatcct ggatgctgac agcaacatgg gaaacagaat
tgagagcatc 1800actcagcgca tggcaataat agaaggaaag aataagatgg ctagcacctt
ggttgtggct 1860gactctagaa tttctggaat ctacatttgc atagcttcca ataaagttgg
gactgtggga 1920agaaacataa gcttttatat cacagatgtg ccaaatgggt ttcatgttaa
cttggaaaaa 1980atgccgacgg aaggagagga cctgaaactg tcttgcacag ttaacaagtt
cttatacaga 2040gacgttactt ggattttact gcggacagtt aataacagaa caatgcacta
cagtattagc 2100aagcaaaaaa tggccatcac taaggagcac tccatcactc ttaatcttac
catcatgaat 2160gtttccctgc aagattcagg cacctatgcc tgcagagcca ggaatgtata
cacaggggaa 2220gaaatcctcc agaagaaaga aattacaatc agagatcagg aagcaccata
cctcctgcga 2280aacctcagtg atcacacagt ggccatcagc agttccacca ctttagactg
tcatgctaat 2340ggtgtccccg agcctcagat cacttggttt aaaaacaacc acaaaataca
acaagagcct 2400ggaattattt taggaccagg aagcagcacg ctgtttattg aaagagtcac
agaagaggat 2460gaaggtgtct atcactgcaa agccaccaac cagaagggct ctgtggaaag
ttcagcatac 2520ctcactgttc aaggaacctc ggacaagtct aatctggagc tgatcactct
aacatgcacc 2580tgtgtggctg cgactctctt ctggctccta ttaaccctct ttatccgaaa
aatgaaaagg 2640tcttcttctg aaataaagac tgactaccta tcaattataa tggacccaga
tgaagttcct 2700ttggatgagc agtgtgagcg gctcccttat gatgccagca agtgggagtt
tgcccgggag 2760agacttaaac tgggcaaatc acttggaaga ggggcttttg gaaaagtggt
tcaagcatca 2820gcatttggca ttaagaaatc acctacgtgc cggactgtgg ctgtgaaaat
gctgaaagag 2880ggggccacgg ccagcgagta caaagctctg atgactgagc taaaaatctt
gacccacatt 2940ggccaccatc tgaacgtggt taacctgctg ggagcctgca ccaagcaagg
agggcctctg 3000atggtgattg ttgaatactg caaatatgga aatctctcca actacctcaa
gagcaaacgt 3060gacttatttt ttctcaacaa ggatgcagca ctacacatgg agcctaagaa
agaaaaaatg 3120gagccaggcc tggaacaagg caagaaacca agactagata gcgtcaccag
cagcgaaagc 3180tttgcgagct ccggctttca ggaagataaa agtctgagtg atgttgagga
agaggaggat 3240tctgacggtt tctacaagga gcccatcact atggaagatc tgatttctta
cagttttcaa 3300gtggccagag gcatggagtt cctgtcttcc agaaagtgca ttcatcggga
cctggcagcg 3360agaaacattc ttttatctga gaacaacgtg gtgaagattt gtgattttgg
ccttgcccgg 3420gatatttata agaaccccga ttatgtgaga aaaggagata ctcgacttcc
tctgaaatgg 3480atggctcctg aatctatctt tgacaaaatc tacagcacca agagcgacgt
gtggtcttac 3540ggagtattgc tgtgggaaat cttctcctta ggtgggtctc catacccagg
agtacaaatg 3600gatgaggact tttgcagtcg cctgagggaa ggcatgagga tgagagctcc
tgagtactct 3660actcctgaaa tctatcagat catgctggac tgctggcaca gagacccaaa
agaaaggcca 3720agatttgcag aacttgtgga aaaactaggt gatttgcttc aagcaaatgt
acaacaggat 3780ggtaaagact acatcccaat caatgccata ctgacaggaa atagtgggtt
tacatactca 3840actcctgcct tctctgagga cttcttcaag gaaagtattt cagctccgaa
gtttaattca 3900ggaagctctg atgatgtcag atacgtaaat gctttcaagt tcatgagcct
ggaaagaatc 3960aaaacctttg aagaactttt accgaatgcc acctccatgt ttgatgacta
ccagggcgac 4020agcagcactc tgttggcctc tcccatgctg aagcgcttca cctggactga
cagcaaaccc 4080aaggcctcgc tcaagattga cttgagagta accagtaaaa gtaaggagtc
ggggctgtct 4140gatgtcagca ggcccagttt ctgccattcc agctgtgggc acgtcagcga
aggcaagcgc 4200aggttcacct acgaccacgc tgagctggaa aggaaaatcg cgtgctgctc
cccgccccca 4260gactacaact cggtggtcct gtactccacc ccacccatct agagtttgac
acgaagcctt 4320atttctagaa gcacatgtgt atttataccc ccaggaaact agcttttgcc
agtattatgc 4380atatataagt ttacaccttt atctttccat gggagccagc tgctttttgt
gattttttta 4440atagtgcttt tttttttttg actaacaaga atgtaactcc agatagagaa
atagtgacaa 4500gtgaagaaca ctactgctaa atcctcatgt tactcagtgt tagagaaatc
cttcctaaac 4560ccaatgactt ccctgctcca acccccgcca cctcagggca cgcaggacca
gtttgattga 4620ggagctgcac tgatcaccca atgcatcacg taccccactg ggccagccct
gcagcccaaa 4680acccagggca acaagcccgt tagccccagg gatcactggc tggcctgagc
aacatctcgg 4740gagtcctcta gcaggcctaa gacatgtgag gaggaaaagg aaaaaaagca
aaaagcaagg 4800gagaaaagag aaaccgggag aaggcatgag aaagaatttg agacgcacca
tgtgggcacg 4860gagggggacg gggctcagca atgccatttc agtggcttcc cagctctgac
ccttctacat 4920ttgagggccc agccaggagc agatggacag cgatgagggg acattttctg
gattctggga 4980ggcaagaaaa ggacaaatat cttttttgga actaaagcaa attttagaac
tttacctatg 5040gaagtggttc tatgtccatt ctcattcgtg gcatgttttg atttgtagca
ctgagggtgg 5100cactcaactc tgagcccata cttttggctc ctctagtaag atgcactgaa
aacttagcca 5160gagttaggtt gtctccaggc catgatggcc ttacactgaa aatgtcacat
tctattttgg 5220gtattaatat atagtccaga cacttaactc aatttcttgg tattattctg
ttttgcacag 5280ttagttgtga aagaaagctg agaagaatga aaatgcagtc ctgaggagag
gagttttctc 5340catatcaaaa cgagggctga tggaggaaaa aggtcaataa ggtcaaggga
aaaccccgtc 5400tctataccaa ccaaaccaat tcaccaacac agttgggacc caaaacacag
gaagtcagtc 5460acgtttcctt ttcatttaat ggggattcca ctatctcaca ctaatctgaa
aggatgtgga 5520agagcattag ctggcgcata ttaagcactt taagctcctt gagtaaaaag
gtggtatgta 5580atttatgcaa ggtatttctc cagttgggac tcaggatatt agttaatgag
ccatcactag 5640aagaaaagcc cattttcaac tgctttgaaa cttgcctggg gtctgagcat
gatgggaata 5700gggagacagg gtaggaaagg gcgcctactc ttcagggtct aaagatcaag
tgggccttgg 5760atcgctaagc tggctctgtt tgatgctatt tatgcaagtt agggtctatg
tatttatgat 5820gtctgcacct tctgcagcca gtcagaagct ggagaggcaa cagtggattg
ctgcttcttg 5880gggagaagag tatgcttcct tttatccatg taatttaact gtagaacctg
agctctaagt 5940aaccgaagaa tgtatgcctc tgttcttatg tgccacatcc ttgtttaaag
gctctctgta 6000tgaagagatg ggaccgtcat cagcacattc cctagtgagc ctactggctc
ctggcagcgg 6060cttttgtgga agactcacta gccagaagag aggagtggga cagtcctctc
caccaagatc 6120taaatccaaa caaaagcagg ctagagccag aagagaggac aaatctttgt
tcttcctctt 6180ctttacatac gcaaaccacc tgtgacagct ggcaatttta taaatcaggt
aactggaagg 6240aggttaaaca cagaaaaaag aagacctcag tcaattctct actttttttt
ttttttccaa 6300atcagataat agcccagcaa atagtgataa caaataaaac cttagctatt
catgtcttga 6360tttcaataat taattcttaa tcattaagag accataataa atactccttt
tcaagagaaa 6420agcaaaacca ttagaattgt tactcagctc cttcaaactc aggtttgtag
catacatgag 6480tccatccatc agtcaaagaa tggttccatc tggagtctta atgtagaaag
aaaaatggag 6540acttgtaata atgagctagt tacaaagtgc ttgttcatta aaatagcact
gaaaattgaa 6600acatgaatta actgataata ttccaatcat ttgccattta tgacaaaaat
ggttggcact 6660aacaaagaac gagcacttcc tttcagagtt tctgagataa tgtacgtgga
acagtctggg 6720tggaatgggg ctgaaaccat gtgcaagtct gtgtcttgtc agtccaagaa
gtgacaccga 6780gatgttaatt ttagggaccc gtgccttgtt tcctagccca caagaatgca
aacatcaaac 6840agatactcgc tagcctcatt taaattgatt aaaggaggag tgcatctttg
gccgacagtg 6900gtgtaactgt atgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgggt
gtatgtgtgt 6960tttgtgcata actatttaag gaaactggaa ttttaaagtt acttttatac
aaaccaagaa 7020tatatgctac agatataaga cagacatggt ttggtcctat atttctagtc
atgatgaatg 7080tattttgtat accatcttca tataataaac ttccaaaaac aca
71235733PRTHomo sapiensMISC_FEATURE(1)..(733)VEGFR1 variant 3
5Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1
5 10 15 Cys Leu Leu Leu
Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20
25 30 Glu Leu Ser Leu Lys Gly Thr Gln His
Ile Met Gln Ala Gly Gln Thr 35 40
45 Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser
Leu Pro 50 55 60
Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65
70 75 80 Cys Gly Arg Asn Gly
Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85
90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser
Cys Lys Tyr Leu Ala Val 100 105
110 Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe
Ile 115 120 125 Ser
Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130
135 140 Ile Ile His Met Thr Glu
Gly Arg Glu Leu Val Ile Pro Cys Arg Val 145 150
155 160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys
Phe Pro Leu Asp Thr 165 170
175 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe
180 185 190 Ile Ile
Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195
200 205 Ala Thr Val Asn Gly His Leu
Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215
220 Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr
Pro Arg Pro Val 225 230 235
240 Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr
245 250 255 Pro Leu Asn
Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260
265 270 Asn Lys Arg Ala Ser Val Arg Arg
Arg Ile Asp Gln Ser Asn Ser His 275 280
285 Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met
Gln Asn Lys 290 295 300
Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305
310 315 320 Ser Val Asn Thr
Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325
330 335 Lys His Arg Lys Gln Gln Val Leu Glu
Thr Val Ala Gly Lys Arg Ser 340 345
350 Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu
Val Val 355 360 365
Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370
375 380 Thr Arg Gly Tyr Ser
Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala 385 390
395 400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys
Gln Ser Asn Val Phe Lys 405 410
415 Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr
Glu 420 425 430 Lys
Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435
440 445 Arg Gln Ile Leu Thr Cys
Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455
460 Lys Trp Phe Trp His Pro Cys Asn His Asn His
Ser Glu Ala Arg Cys 465 470 475
480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser
485 490 495 Asn Met
Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500
505 510 Glu Gly Lys Asn Lys Met Ala
Ser Thr Leu Val Val Ala Asp Ser Arg 515 520
525 Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys
Val Gly Thr Val 530 535 540
Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545
550 555 560 Val Asn Leu
Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565
570 575 Cys Thr Val Asn Lys Phe Leu Tyr
Arg Asp Val Thr Trp Ile Leu Leu 580 585
590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser
Lys Gln Lys 595 600 605
Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610
615 620 Asn Val Ser Leu
Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn 625 630
635 640 Val Tyr Thr Gly Glu Glu Ile Leu Gln
Lys Lys Glu Ile Thr Ile Arg 645 650
655 Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His
Thr Val 660 665 670
Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro
675 680 685 Glu Pro Gln Ile
Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu 690
695 700 Pro Glu Leu Tyr Thr Ser Thr Ser
Pro Ser Ser Ser Ser Ser Ser Pro 705 710
715 720 Leu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
Ser 725 730 62968DNAHomo
sapiensmisc_feature(1)..(2968)VEGFR1 transcript variant 3 6atcgaggtcc
gcgggaggct cggagcgcgc caggcggaca ctcctctcgg ctcctccccg 60gcagcggcgg
cggctcggag cgggctccgg ggctcgggtg cagcggccag cgggcgcctg 120gcggcgagga
ttacccgggg aagtggttgt ctcctggctg gagccgcgag acgggcgctc 180agggcgcggg
gccggcggcg gcgaacgaga ggacggactc tggcggccgg gtcgttggcc 240gcggggagcg
cgggcaccgg gcgagcaggc cgcgtcgcgc tcaccatggt cagctactgg 300gacaccgggg
tcctgctgtg cgcgctgctc agctgtctgc ttctcacagg atctagttca 360ggttcaaaat
taaaagatcc tgaactgagt ttaaaaggca cccagcacat catgcaagca 420ggccagacac
tgcatctcca atgcaggggg gaagcagccc ataaatggtc tttgcctgaa 480atggtgagta
aggaaagcga aaggctgagc ataactaaat ctgcctgtgg aagaaatggc 540aaacaattct
gcagtacttt aaccttgaac acagctcaag caaaccacac tggcttctac 600agctgcaaat
atctagctgt acctacttca aagaagaagg aaacagaatc tgcaatctat 660atatttatta
gtgatacagg tagacctttc gtagagatgt acagtgaaat ccccgaaatt 720atacacatga
ctgaaggaag ggagctcgtc attccctgcc gggttacgtc acctaacatc 780actgttactt
taaaaaagtt tccacttgac actttgatcc ctgatggaaa acgcataatc 840tgggacagta
gaaagggctt catcatatca aatgcaacgt acaaagaaat agggcttctg 900acctgtgaag
caacagtcaa tgggcatttg tataagacaa actatctcac acatcgacaa 960accaatacaa
tcatagatgt ccaaataagc acaccacgcc cagtcaaatt acttagaggc 1020catactcttg
tcctcaattg tactgctacc actcccttga acacgagagt tcaaatgacc 1080tggagttacc
ctgatgaaaa aaataagaga gcttccgtaa ggcgacgaat tgaccaaagc 1140aattcccatg
ccaacatatt ctacagtgtt cttactattg acaaaatgca gaacaaagac 1200aaaggacttt
atacttgtcg tgtaaggagt ggaccatcat tcaaatctgt taacacctca 1260gtgcatatat
atgataaagc attcatcact gtgaaacatc gaaaacagca ggtgcttgaa 1320accgtagctg
gcaagcggtc ttaccggctc tctatgaaag tgaaggcatt tccctcgccg 1380gaagttgtat
ggttaaaaga tgggttacct gcgactgaga aatctgctcg ctatttgact 1440cgtggctact
cgttaattat caaggacgta actgaagagg atgcagggaa ttatacaatc 1500ttgctgagca
taaaacagtc aaatgtgttt aaaaacctca ctgccactct aattgtcaat 1560gtgaaacccc
agatttacga aaaggccgtg tcatcgtttc cagacccggc tctctaccca 1620ctgggcagca
gacaaatcct gacttgtacc gcatatggta tccctcaacc tacaatcaag 1680tggttctggc
acccctgtaa ccataatcat tccgaagcaa ggtgtgactt ttgttccaat 1740aatgaagagt
cctttatcct ggatgctgac agcaacatgg gaaacagaat tgagagcatc 1800actcagcgca
tggcaataat agaaggaaag aataagatgg ctagcacctt ggttgtggct 1860gactctagaa
tttctggaat ctacatttgc atagcttcca ataaagttgg gactgtggga 1920agaaacataa
gcttttatat cacagatgtg ccaaatgggt ttcatgttaa cttggaaaaa 1980atgccgacgg
aaggagagga cctgaaactg tcttgcacag ttaacaagtt cttatacaga 2040gacgttactt
ggattttact gcggacagtt aataacagaa caatgcacta cagtattagc 2100aagcaaaaaa
tggccatcac taaggagcac tccatcactc ttaatcttac catcatgaat 2160gtttccctgc
aagattcagg cacctatgcc tgcagagcca ggaatgtata cacaggggaa 2220gaaatcctcc
agaagaaaga aattacaatc agagatcagg aagcaccata cctcctgcga 2280aacctcagtg
atcacacagt ggccatcagc agttccacca ctttagactg tcatgctaat 2340ggtgtccccg
agcctcagat cacttggttt aaaaacaacc acaaaataca acaagagcct 2400gaactgtata
catcaacgtc accatcgtca tcgtcatcat caccattgtc atcatcatca 2460tcatcgtcat
catcatcatc atcatagcta tcatcattat catcatcatc atcatcatca 2520tcatagctac
catttattga aaactattat gtgtcaactt caaagaactt atcctttagt 2580tggagagcca
agacaatcat aacaataaca aatggccggg catggtggct cacgcctgta 2640atcccagcac
tttgggaggc caaggcaggt ggatcatttg aggtcaggag ttcaagacca 2700gcctgaccaa
gatggtgaaa tgctgtctct attaaaaata caaaattagc caggcatggt 2760ggctcatgcc
tgtaatgcca gctactcggg aggctgagac aggagaatca cttgaaccca 2820ggaggcagag
gttgcaggga gccgagatcg tgtactgcac tccagcctgg gcaacaagag 2880cgaaactccg
tctcaaaaaa caaataaata aataaataaa taaacagaca aaattcactt 2940tttattctat
taaacttaac atacatgc 29687541PRTHomo
sapiensMISC_FEATURE(1)..(541)VEGFR1 variant 4 7Met Val Ser Tyr Trp Asp
Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5
10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser
Lys Leu Lys Asp Pro 20 25
30 Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln
Thr 35 40 45 Leu
His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50
55 60 Glu Met Val Ser Lys Glu
Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70
75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr
Leu Thr Leu Asn Thr 85 90
95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val
100 105 110 Pro Thr
Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115
120 125 Ser Asp Thr Gly Arg Pro Phe
Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135
140 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile
Pro Cys Arg Val 145 150 155
160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr
165 170 175 Leu Ile Pro
Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180
185 190 Ile Ile Ser Asn Ala Thr Tyr Lys
Glu Ile Gly Leu Leu Thr Cys Glu 195 200
205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu
Thr His Arg 210 215 220
Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val 225
230 235 240 Lys Leu Leu Arg
Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245
250 255 Pro Leu Asn Thr Arg Val Gln Met Thr
Trp Ser Tyr Pro Asp Glu Lys 260 265
270 Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn
Ser His 275 280 285
Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290
295 300 Asp Lys Gly Leu Tyr
Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310
315 320 Ser Val Asn Thr Ser Val His Ile Tyr Asp
Lys Ala Phe Ile Thr Val 325 330
335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg
Ser 340 345 350 Tyr
Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355
360 365 Trp Leu Lys Asp Gly Leu
Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375
380 Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val
Thr Glu Glu Asp Ala 385 390 395
400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys
405 410 415 Asn Leu
Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420
425 430 Lys Ala Val Ser Ser Phe Pro
Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440
445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro
Gln Pro Thr Ile 450 455 460
Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys 465
470 475 480 Asp Phe Cys
Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485
490 495 Asn Met Gly Asn Arg Ile Glu Ser
Ile Thr Gln Arg Met Ala Ile Ile 500 505
510 Glu Gly Lys Asn Lys Leu Pro Pro Ala Asn Ser Ser Phe
Met Leu Pro 515 520 525
Pro Thr Ser Phe Ser Ser Asn Tyr Phe His Phe Leu Pro 530
535 540 81911DNAHomo
sapiensmisc_feature(1)..(1911)VEGFRa transcript variant 4 8atcgaggtcc
gcgggaggct cggagcgcgc caggcggaca ctcctctcgg ctcctccccg 60gcagcggcgg
cggctcggag cgggctccgg ggctcgggtg cagcggccag cgggcgcctg 120gcggcgagga
ttacccgggg aagtggttgt ctcctggctg gagccgcgag acgggcgctc 180agggcgcggg
gccggcggcg gcgaacgaga ggacggactc tggcggccgg gtcgttggcc 240gcggggagcg
cgggcaccgg gcgagcaggc cgcgtcgcgc tcaccatggt cagctactgg 300gacaccgggg
tcctgctgtg cgcgctgctc agctgtctgc ttctcacagg atctagttca 360ggttcaaaat
taaaagatcc tgaactgagt ttaaaaggca cccagcacat catgcaagca 420ggccagacac
tgcatctcca atgcaggggg gaagcagccc ataaatggtc tttgcctgaa 480atggtgagta
aggaaagcga aaggctgagc ataactaaat ctgcctgtgg aagaaatggc 540aaacaattct
gcagtacttt aaccttgaac acagctcaag caaaccacac tggcttctac 600agctgcaaat
atctagctgt acctacttca aagaagaagg aaacagaatc tgcaatctat 660atatttatta
gtgatacagg tagacctttc gtagagatgt acagtgaaat ccccgaaatt 720atacacatga
ctgaaggaag ggagctcgtc attccctgcc gggttacgtc acctaacatc 780actgttactt
taaaaaagtt tccacttgac actttgatcc ctgatggaaa acgcataatc 840tgggacagta
gaaagggctt catcatatca aatgcaacgt acaaagaaat agggcttctg 900acctgtgaag
caacagtcaa tgggcatttg tataagacaa actatctcac acatcgacaa 960accaatacaa
tcatagatgt ccaaataagc acaccacgcc cagtcaaatt acttagaggc 1020catactcttg
tcctcaattg tactgctacc actcccttga acacgagagt tcaaatgacc 1080tggagttacc
ctgatgaaaa aaataagaga gcttccgtaa ggcgacgaat tgaccaaagc 1140aattcccatg
ccaacatatt ctacagtgtt cttactattg acaaaatgca gaacaaagac 1200aaaggacttt
atacttgtcg tgtaaggagt ggaccatcat tcaaatctgt taacacctca 1260gtgcatatat
atgataaagc attcatcact gtgaaacatc gaaaacagca ggtgcttgaa 1320accgtagctg
gcaagcggtc ttaccggctc tctatgaaag tgaaggcatt tccctcgccg 1380gaagttgtat
ggttaaaaga tgggttacct gcgactgaga aatctgctcg ctatttgact 1440cgtggctact
cgttaattat caaggacgta actgaagagg atgcagggaa ttatacaatc 1500ttgctgagca
taaaacagtc aaatgtgttt aaaaacctca ctgccactct aattgtcaat 1560gtgaaacccc
agatttacga aaaggccgtg tcatcgtttc cagacccggc tctctaccca 1620ctgggcagca
gacaaatcct gacttgtacc gcatatggta tccctcaacc tacaatcaag 1680tggttctggc
acccctgtaa ccataatcat tccgaagcaa ggtgtgactt ttgttccaat 1740aatgaagagt
cctttatcct ggatgctgac agcaacatgg gaaacagaat tgagagcatc 1800actcagcgca
tggcaataat agaaggaaag aataagcttc caccagctaa cagttctttc 1860atgttgccac
ctacaagctt ctcttccaac tacttccatt tccttccgtg a 19119889PRTHomo
sapiensMISC_FEATURE(1)..(880)PKN3 9Met Glu Glu Gly Ala Pro Arg Gln Pro
Gly Pro Ser Gln Trp Pro Pro 1 5 10
15 Glu Asp Glu Lys Glu Val Ile Arg Arg Ala Ile Gln Lys Glu
Leu Lys 20 25 30
Ile Lys Glu Gly Val Glu Asn Leu Arg Arg Val Ala Thr Asp Arg Arg
35 40 45 His Leu Gly His
Val Gln Gln Leu Leu Arg Ser Ser Asn Arg Arg Leu 50
55 60 Glu Gln Leu His Gly Glu Leu Arg
Glu Leu His Ala Arg Ile Leu Leu 65 70
75 80 Pro Gly Pro Gly Pro Gly Pro Ala Glu Pro Val Ala
Ser Gly Pro Arg 85 90
95 Pro Trp Ala Glu Gln Leu Arg Ala Arg His Leu Glu Ala Leu Arg Arg
100 105 110 Gln Leu His
Val Glu Leu Lys Val Lys Gln Gly Ala Glu Asn Met Thr 115
120 125 His Thr Cys Ala Ser Gly Thr Pro
Lys Glu Arg Lys Leu Leu Ala Ala 130 135
140 Ala Gln Gln Met Leu Arg Asp Ser Gln Leu Lys Val Ala
Leu Leu Arg 145 150 155
160 Met Lys Ile Ser Ser Leu Glu Ala Ser Gly Ser Pro Glu Pro Gly Pro
165 170 175 Glu Leu Leu Ala
Glu Glu Leu Gln His Arg Leu His Val Glu Ala Ala 180
185 190 Val Ala Glu Gly Ala Lys Asn Val Val
Lys Leu Leu Ser Ser Arg Arg 195 200
205 Thr Gln Asp Arg Lys Ala Leu Ala Glu Ala Gln Ala Gln Leu
Gln Glu 210 215 220
Ser Ser Gln Lys Leu Asp Leu Leu Arg Leu Ala Leu Glu Gln Leu Leu 225
230 235 240 Glu Gln Leu Pro Pro
Ala His Pro Leu Arg Ser Arg Val Thr Arg Glu 245
250 255 Leu Arg Ala Ala Val Pro Gly Tyr Pro Gln
Pro Ser Gly Thr Pro Val 260 265
270 Lys Pro Thr Ala Leu Thr Gly Thr Leu Gln Val Arg Leu Leu Gly
Cys 275 280 285 Glu
Gln Leu Leu Thr Ala Val Pro Gly Arg Ser Pro Ala Ala Ala Leu 290
295 300 Ala Ser Ser Pro Ser Glu
Gly Trp Leu Arg Thr Lys Ala Lys His Gln 305 310
315 320 Arg Gly Arg Gly Glu Leu Ala Ser Glu Val Leu
Ala Val Leu Lys Val 325 330
335 Asp Asn Arg Val Val Gly Gln Thr Gly Trp Gly Gln Val Ala Glu Gln
340 345 350 Ser Trp
Asp Gln Thr Phe Val Ile Pro Leu Glu Arg Ala Arg Glu Leu 355
360 365 Glu Ile Gly Val His Trp Arg
Asp Trp Arg Gln Leu Cys Gly Val Ala 370 375
380 Phe Leu Arg Leu Glu Asp Phe Leu Asp Asn Ala Cys
His Gln Leu Ser 385 390 395
400 Leu Ser Leu Val Pro Gln Gly Leu Leu Phe Ala Gln Val Thr Phe Cys
405 410 415 Asp Pro Val
Ile Glu Arg Arg Pro Arg Leu Gln Arg Gln Glu Arg Ile 420
425 430 Phe Ser Lys Arg Arg Gly Gln Asp
Phe Leu Arg Arg Ser Gln Met Asn 435 440
445 Leu Gly Met Ala Ala Trp Gly Arg Leu Val Met Asn Leu
Leu Pro Pro 450 455 460
Cys Ser Ser Pro Ser Thr Ile Ser Pro Pro Lys Gly Cys Pro Arg Thr 465
470 475 480 Pro Thr Thr Leu
Arg Glu Ala Ser Asp Pro Ala Thr Pro Ser Asn Phe 485
490 495 Leu Pro Lys Lys Thr Pro Leu Gly Glu
Glu Met Thr Pro Pro Pro Lys 500 505
510 Pro Pro Arg Leu Tyr Leu Pro Gln Glu Pro Thr Ser Glu Glu
Thr Pro 515 520 525
Arg Thr Lys Arg Pro His Met Glu Pro Arg Thr Arg Arg Gly Pro Ser 530
535 540 Pro Pro Ala Ser Pro
Thr Arg Lys Pro Pro Arg Leu Gln Asp Phe Arg 545 550
555 560 Cys Leu Ala Val Leu Gly Arg Gly His Phe
Gly Lys Val Leu Leu Val 565 570
575 Gln Phe Lys Gly Thr Gly Lys Tyr Tyr Ala Ile Lys Ala Leu Lys
Lys 580 585 590 Gln
Glu Val Leu Ser Arg Asp Glu Ile Glu Ser Leu Tyr Cys Glu Lys 595
600 605 Arg Ile Leu Glu Ala Val
Gly Cys Thr Gly His Pro Phe Leu Leu Ser 610 615
620 Leu Leu Val Cys Phe Gln Thr Ser Ser His Ala
Arg Phe Val Thr Glu 625 630 635
640 Phe Val Pro Gly Gly Asp Leu Met Met Gln Ile His Glu Asp Val Phe
645 650 655 Pro Glu
Pro Gln Ala Arg Phe Tyr Val Ala Cys Val Val Leu Gly Leu 660
665 670 Gln Phe Leu His Glu Lys Lys
Ile Ile Tyr Arg Asp Leu Lys Leu Asp 675 680
685 Asn Leu Leu Leu Asp Ala Gln Gly Phe Leu Lys Ile
Ala Asp Phe Gly 690 695 700
Leu Cys Lys Glu Gly Ile Gly Phe Gly Asp Arg Thr Ser Thr Phe Cys 705
710 715 720 Gly Thr Pro
Glu Phe Leu Ala Pro Glu Val Leu Thr Gln Glu Ala Tyr 725
730 735 Thr Gln Ala Val Asp Trp Trp Ala
Leu Gly Val Leu Leu Tyr Glu Met 740 745
750 Leu Val Gly Glu Cys Pro Phe Pro Gly Asp Thr Glu Glu
Glu Val Phe 755 760 765
Asp Cys Ile Val Asn Met Asp Ala Pro Tyr Pro Gly Phe Leu Ser Val 770
775 780 Gln Gly Leu Glu
Phe Ile Gln Lys Leu Leu Gln Lys Cys Pro Glu Lys 785 790
795 800 Arg Leu Gly Ala Gly Glu Gln Asp Ala
Glu Glu Ile Lys Val Gln Pro 805 810
815 Phe Phe Arg Thr Thr Asn Trp Gln Ala Leu Leu Ala Arg Thr
Ile Gln 820 825 830
Pro Pro Phe Val Pro Thr Leu Cys Gly Pro Ala Asp Leu Arg Tyr Phe
835 840 845 Glu Gly Glu Phe
Thr Gly Leu Pro Pro Ala Leu Thr Pro Pro Ala Pro 850
855 860 His Ser Leu Leu Thr Ala Arg Gln
Gln Ala Ala Phe Arg Asp Phe Asp 865 870
875 880 Phe Val Ser Glu Arg Phe Leu Glu Pro
885 102670DNAHomo sapiensmisc_feature(1)..(2670)PKN3
10atggaggagg gggcgccgcg gcagcctggg ccgagccagt ggcccccaga ggatgagaag
60gaggtgatcc gccgggccat ccagaaagag ctgaagatca aggagggggt ggagaacctg
120cggcgcgtgg ccacagaccg ccgccacttg ggccatgtgc agcagctgct gcggtcctcc
180aaccgccgcc tggagcagct gcatggcgag ctgcgggagc tgcacgcccg aatcctgctg
240cccggccctg ggcctggccc agctgagcct gtggcctcag gaccccggcc gtgggcagag
300cagctcaggg ctcggcacct agaggctctc cggaggcagc tgcatgtgga gctgaaggtg
360aaacaggggg ctgagaacat gacccacacg tgcgccagtg gcacccccaa ggagaggaag
420ctccttgcag ctgcccagca gatgctgcgg gacagccagc tgaaggtggc cctgctgcgg
480atgaagatca gcagcctgga ggccagtggg tccccggagc cagggcctga gctactggcg
540gaggagctac agcatcgact gcacgttgag gcagcggtgg ctgagggcgc caagaacgtg
600gtgaaactgc ttagtagccg gagaacacag gaccgcaagg cactggctga ggcccaggcc
660cagctacagg agtcctctca gaaactggac ctcctgcgcc tggccttgga gcagctgctg
720gagcaactgc ctcctgccca ccctttgcgc agcagagtga cccgagagtt gcgggctgcg
780gtgcctggat acccccagcc ttcagggaca cctgtgaagc ccaccgccct aacagggaca
840ctgcaggtcc gcctcctggg ctgtgaacag ttgctgacag ccgtgcctgg gcgctcccca
900gcggccgcac tggccagcag cccctccgag ggctggcttc ggaccaaggc caagcaccag
960cgtggccgag gcgagcttgc cagtgaggtg ctggctgtgc taaaggtgga caaccgtgtt
1020gtggggcaga cgggctgggg gcaggtggcc gaacagtcct gggaccagac ctttgtcatc
1080ccactggagc gagcccgtga gctggagatt ggggtacact ggcgggactg gcggcagcta
1140tgtggcgtgg ccttcctgag acttgaagac ttcctggaca atgcctgtca ccaactgtcc
1200ctcagcctgg taccgcaggg actgcttttt gcccaggtga ccttctgcga tcctgtcatt
1260gagaggcggc cccggctgca gaggcaggaa cgcatcttct ctaaacgcag aggccaggac
1320ttcctgaggc gttcgcagat gaacctcggc atggcggcct gggggcgcct cgtcatgaac
1380ctgctgcccc cctgcagctc cccgagcaca atcagccccc ctaaaggatg ccctcggacc
1440ccaacaacac tgcgagaggc ctctgaccct gccactccca gtaatttcct gcccaagaag
1500acccccttgg gtgaagagat gacaccccca cccaagcccc cacgcctcta cctcccccag
1560gagccaacat ccgaggagac tccgcgcacc aaacgtcccc atatggagcc taggactcga
1620cgtgggccat ctccaccagc ctcccccacc aggaaacccc ctcggcttca ggacttccgc
1680tgcttagctg tgctgggccg gggacacttt gggaaggtcc tcctggtcca gttcaagggg
1740acagggaaat actacgccat caaagcactg aagaagcagg aggtgctcag ccgggacgag
1800atagagagcc tgtactgcga gaagcggatc ctggaggctg tgggctgcac agggcaccct
1860ttcctgctct ccctccttgt ctgcttccag acctccagcc atgcccgctt tgtgactgag
1920tttgtgcctg gtggtgacct catgatgcag atccacgagg atgtcttccc cgagccccag
1980gcccgcttct acgtggcttg tgttgtcctg gggctgcagt tcttacacga gaagaagatc
2040atttacaggg acctgaagtt ggataacctt ctgctggatg cccagggatt cctgaagatc
2100gcagactttg gactctgcaa ggaagggatc ggcttcgggg accggactag caccttctgt
2160ggcaccccgg agttcctggc tcccgaggtg ctgacccagg aggcatacac acaggccgtc
2220gactggtggg cgctgggtgt gctgctctac gagatgctgg tgggtgagtg cccgttccca
2280ggggacacag aggaagaggt gtttgactgc atcgtcaaca tggacgcccc ctaccccggc
2340tttctgtcgg tgcaagggct tgagttcatt cagaagctcc tccagaagtg cccggagaag
2400cgcctcgggg caggtgagca ggatgccgag gagatcaagg tccagccatt cttcaggacc
2460accaactggc aagccctgct cgcccgcacc atccagcccc ccttcgtgcc taccctgtgt
2520ggccctgcgg acctgcgcta ctttgagggc gagttcacag ggctgccgcc tgccctgacc
2580ccacctgcac cccacagcct cctcactgcc cgccaacagg ccgccttccg ggacttcgac
2640tttgtgtcag agcgattcct ggaaccctga
26701123RNAArtificialSynthetic 11uuguccagga aguccucaag ucu
231223RNAArtificialSynthetic 12agacuugagg
acuuccugga caa
231323RNAArtificialSynthetic 13agacuugagg acuuccugga caa
231423RNAArtificialSynthetic 14uuguccagga
aguccucaag ucu
231523RNAArtificialSynthetic 15aucacguacg cggaauacuu cga
231623RNAArtificialSynthetic 16ucgaaguauu
ccgcguacgu gau
231721DNAArtificialSynthetic 17cactttggga aggtcctcct g
211828DNAArtificialSynthetic 18ttcaagggga
cagggaaata ctacgcca
281922DNAArtificialSynthetic 19cctcctgctt cttcagtgct tt
222022DNAArtificialSynthetic 20ccccgattat
gtgagaaaag ga
222128DNAArtificialSynthetic 21cgacttcctc tgaaatggat ggctcctg
282223DNAArtificialSynthetic 22cgctcttggt
gctgtagatt ttg
232322DNAArtificialSynthetic 23tctgcctacc tcacctgttt cc
222428DNAArtificialSynthetic 24atggaggagg
aggaagtatg tgacccca
282523DNAArtificialSynthetic 25tgactgattc ctgctgtgtt gtc
23
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