Inhibitor of metadherin expression

The present disclosure refers to an inhibitor of metadherin (MTDH) expression consisting of an antisense oligonucleotide hybridizing with a nucleic acid sequence of MTDH and to a pharmaceutical composition comprising such antisense oligonucleotide and a pharmaceutically acceptable carrier, excipient and/or dilutant. Further, the invention refers to the use of the inhibitor or the pharmaceutical composition comprising the inhibitor in a method of preventing and/or treating a benign or malign tumor.

TECHNICAL BACKGROUND

Metadherin (MTDH) also known as protein LYRIC or astrocyte elevated gene-1 protein (AEG-1) is for example involved in HIF-1 alpha mediated angiogenesis. MTDH also interacts with SND1 and is involved in the RNA-induced silencing complex (RISC) and plays very important role in RISC and miRNA functions. It induces an oncogene called Late SV40 factor (LSF/TFCP2) which is involved in thymidylate synthase (TS) induction and DNA biosynthesis synthesis. Late SV40 factor (LSF/TFCP2) enhances angiogenesis by transcriptionally up-regulating matrix metalloproteinase-9 (MMP9). MTDH also regulates multiple signaling pathways including PI3K/Akt, NF-κB, Wnt/6-catenin, and MAPK which cooperate to promote the tumorigenic and metastatic potential of transformed cells. Several microRNA have also been found to be associated with the increased MTDH expression in different cancers.

MTDH is a type-two transmembrane protein containing an extracellular lung homing domain which is for example implicated in breast cancer metastasis to the lung. MTDH encodes a single-pass transmembrane protein with the molecular mass of 64-kDa expressed mainly in the endoplasmic reticulum and perinuclear space. In polarized epithelial cells, it co-localizes with tight junction protein ZO-1 and occludin; however, MTDH is not a native component of tight junctions but becomes incorporated during tight junction complex maturation. The sub-cellular location of MTDH protein varies depending on the physiological state of the cell. In non-malignant tissue, MTDH was shown to be expressed in the nucleus, whereas in malignant cells it becomes translocated into the cytoplasm. It is believed that cytoplasmic translocation of MTDH promotes disease progression by mediating mechanisms that support pro-angiogenesic and metastatic pathways.

It appears that TNF-α is the key regulator of MTDH expression. TNF-α upregulates MTDH expression via NF-κB pathways. TNF-α causes NF-κB nuclear translocation and consequent interaction with MTDH, which is essential for activation of downstream genes. The N-terminal domain of MTDH interacts with NF-κB and triggers gene expression via several convergent mechanisms. NF-κB nuclear translocation coincides with a significant reduction of IκBα level, suggesting MTDH involvement in IκBα degradation. Studies have also revealed that MTDH interacts with Cyclic AMP-responsive element binding protein—binding protein (CBP) which is a NF-κB coactivator. Hence, MTDH may function as a bridging element among p50-p65, NF-κB CBP, and the basal transcription machinery and therefore consequent induction of NF-κB related gene expression enhances migration and invasion. MTDH promoted NF-κB gene expression results in anchorage independent cell growth, possibly mediated by direct activation of matrix metalloproteinase 1 (MMP1) expression. MTDH also serves as a link between NF-κB and matrix metalloprotease 9 (MMP9) expression (Dhiman G. et al., Front. Oncol., 3 May 2019, Vol. 9, p. 1-8).

MTDH has recently been identified as being overexpressed in different cancers and is correlated with worse prognosis (Wan L. et al., Cancer Res 74, 2014, p. 5336-5347; Hu G. et al., Cancer Cell 15, 2009, p. 9-20; Song et al., J Pathol 219, 2009, p. 317-326). It has been identified as a factor that is responsible for cancer cell growth, progression and metastasis as shown for example in models of breast cancer (Wan L et al., Cancer Cell 26, 2014, p. 92-105) and prostate cancer (Wan L. et al., Cancer Res 74, 2014, p. 5336-5347). The proliferative effects of MTDH are related to attenuation of the key cell cycle inhibitors p27Kip1 and p21Cip1. MTDH furthermore mediates chemoresistance of cancer cells by increasing cell survival, probably by inhibiting pro-apoptotic genes like BNIP3 and TRAIL.

As MTDH is an intracellular factor with no enzymatic function it cannot directly be inhibited by monoclonal antibodies and specific suppression of its diverse functions with small molecule inhibitors is nearly impossible. It therefore represents an ideal target for antisense oligonucleotides.

So far no antisense oligonucleotide exists which is highly efficient in reduction and inhibition, respectively, of MTDH expression and hybridizes with MTDH mRNA and/or pre-mRNA.

An oligonucleotide of the present invention is very successful in the inhibition of the expression of MTDH. The mode of action of an oligonucleotide differs from the mode of action of an antibody or small molecule, and oligonucleotides are highly advantageous regarding for example

- (i) the penetration of tumor tissue in solid tumors,
- (ii) the blocking of multiple functions and activities, respectively, of a target, (iii) the combination of oligonucleotides with each other or an antibody or a small molecule, and
- (iv) the inhibition of intracellular effects which are not accessible for an antibody or inhibitable via a small molecule.

SUMMARY

The present invention refers to a MTDH inhibitor consisting of an antisense oligonucleotide comprising 12 to 25 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide hybridizes with a nucleic acid sequence of MTDH of SEQ ID NO.1, of SEQ ID NO.2 or a combination thereof, wherein the oligonucleotide inhibits at least 50% of the MTDH expression compared to an untreated control.

The modified nucleotide is for example selected from the group consisting of a bridged nucleic acid such as LNA, cET, ENA, 2′Fluoro modified nucleotide, 2′O-Methyl modified nucleotide, a 2′O-Methoxy modified nucleotide, a FANA and a combination thereof.

The inhibitor and antisense oligonucleotide, respectively, of the present invention hybridizes for example with a hybridizing active region selected from the group consisting of position 52000 to 52499, position 32000 to 32499, position 87500 to 87999, position 90500 to 90999, position 65500 to 65999, position 8500 to 8999, position 9000 to 9499, position 9500 to 9999, position 1000 to 10499, position 10500 to 10999, position 11000 to 11499, position 13000 to 13499, position 14000 to 14499, position 15500 to 15999, position 16500 to 16999, position 17500 to 17999, position 18000 to 18499, position 20500 to 20999, position 21000 to 21499, position 22500 to 22999, position 24000 to 24499, position 25000 to 25499, position 25500 to 25999, position 27000 to 27499, position 29000 to 29499, position 29500 to 29999, position 37000 to 37499, position 37500 to 37999, position 43500 to 43999, position 44500 to 44999, position 45500 to 45999, position 46500 to 46999, position 47000 to 47499, position 49000 to 49499, position 50500 to 50999, position 52000 to 52499, position 54000 to 54499, position 55500 to 55999, position 61500 to 61999, position 64000 to 64499, position 64500 to 64999, position 65000 to 65499, position 68000 to 68499, position 68500 to 68999, position 71500 to 71999, position 72000 to 72499, position 74500 to 74999, position 76000 to 76499, position 77000 to 77499, position 77500 to 77999, position 78000 to 78499, position 80500 to 80999, position 81000 to 81499, position 81500 to 81999, position 82000 to 82499, position 83500 to 83999, position 85000 to 85499, position 86000 to 86499, position 88500 to 88999, position 89000 to 89499, position 89500 to 89999, position 90000 to 90499, position 91000 to 91499, position 92000 to 92499, position 92500 to 92999, position 93500 to 93999, position 94500 to 94999 of SEQ ID NO.2 or a combination thereof.

The inhibitor and antisense oligonucleotide, respectively, of the present invention comprises for example a sequence selected from the group consisting of SEQ ID NO.13, SEQ ID NO.64, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.29, SEQ ID NO.27, SEQ ID NO.79, one of SEQ ID NO.3 to SEQ ID NO.12, one of SEQ ID NO.14 to SEQ ID NO.19, one of SEQ ID NO.22 to SEQ ID NO.26, SEQ ID NO.28, one of SEQ ID NO.30 to SEQ ID NO.63, one of SEQ ID NO.65 to SEQ ID NO.78, one of SEQ ID NO.80 to SEQ ID NO.221 and a combination thereof.

The inhibitor and antisense oligonucleotide, respectively, of the present invention, wherein the antisense oligonucleotide is for example selected from the group consisting of

(A34011HM; SEQ ID NO. 13)

+G*+T*+A*A*G*T*T*G*C*T*C*G*G*T*+G*+G*+T,

(A34062Hi; SEQ ID NO. 64)

+C*+A*+C*G*G*C*T*T*G*T*C*T*A*T*+C*+A*+G,

(A34018H; SEQ ID NO. 20)

+T*+T*+G*T*A*G*T*A*T*T*G*G*C*+G*+G*+C,

(A34019H; SEQ ID NO. 21)

+C*+T*+T*G*T*A*G*T*A*T*T*G*G*C*+G*+G*+C,

(A34027H; SEQ ID NO. 29)

+C*+G*+C*A*A*T*A*C*T*G*T*T*G*A*+A*+C*+C,

(A34025HM; SEQ ID NO. 27)

+C*+G*+T*T*T*G*G*T*A*A*A*G*G*C*+T*+A*+T,

(A34077Hi; SEQ ID NO. 79)

+T*+C*+G*T*A*T*C*T*A*C*T*G*T*C*+T*+A*+A,

(A34010H; SEQ ID NO. 12)

+C*+T*+T*A*T*C*A*C*G*T*T*T*A*C*+G*+C*+T,

(A34012H; SEQ ID NO. 14)

+G*+A*+T*G*C*G*G*T*T*G*T*A*A*G*+T*+T*+G,

(A34113HM; SEQ ID NO. 115)

+T*+G*+C*T*C*G*G*T*G*G*T*A*A*C*+T*+G*+T,

(A34114HM; SEQ ID NO. 116)

+A*+A*+G*T*T*G*C*T*C*G*G*T*G*G*+T*+A*+A,

(A34115H; SEQ ID NO. 117)

+T*+G*+A*T*G*C*G*G*T*T*G*T*A*A*+G*+T*+T,

(A34137Hi; SEQ ID NO. 139)

+A*+A*+C*A*C*T*G*C*T*G*G*T*A*T*+T*+C*+G,

(A34063Hi; SEQ ID NO. 65)

+A*+G*+C*T*T*C*C*T*T*T*A*A*G*C*+G*+A*+C,

(A34026H; SEQ ID NO. 28)

+C*+G*+T*T*C*T*T*G*G*C*G*C*C*A*+C*+A*+T,

(A34122HM; SEQ ID NO. 124)

+C*+A*+C*G*T*T*T*G*G*T*A*A*A*G*+G*+C*+T,

(A34075Hi; SEQ ID NO. 77)

+C*+G*+C*C*A*G*C*T*T*A*C*C*T*T*+G*+A*+T,

(A34189Hi; SEQ ID NO. 191)

+T*+G*+T*C*G*C*C*A*G*C*T*T*A*C*+C*+T*+T

and a combination thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage between the nucleotides.

The inhibitor of the present invention inhibits for example the expression of MTDH at a nanomolar or micromolar concentration.

The present invention further refers to a pharmaceutical composition comprising an inhibitor of the present invention and a pharmaceutically acceptable carrier, excipient, dilutant or a combination thereof. Optionally, the pharmaceutical composition further comprises another active agent, another oligonucleotide, an antibody, a peptide-based therapeutic, a protein-based therapeutic and/or a small molecule.

The Inhibitor and the pharmaceutical composition, respectively, of the present invention are for example for use in a method of preventing and/or treating a disorder, where an MTDH imbalance is involved. The disorder is for example a tumor such as a malignant or benign tumor, which is for example selected from the group consisting of breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforma, leukemia, epidermoid carcinoma and a kidney disease such as diabetic nephropathy.

The inhibitor or the pharmaceutical composition of the present invention is for example suitable to be administered locally or systemically.

All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

DESCRIPTION OF FIGURES

FIG. 1A and FIG. 1B show efficacy screening experiments EFO-21 (FIG. 1A) and SKOV-3 (FIG. 1B). The cells were treated with the respective human specific antisense oligonucleotide at a concentration of 5 μM for three days without the use of a transfection reagent. MTDH expression values are normalized to HPRT1 values and residual MTDH expression as compared to mock-treated cells (set to 1) is depicted in FIG. 1A and FIG. 1B.

FIG. 2 shows the dose-dependent knockdown of MTDH mRNA expression by human specific MTDH antisense oligonucleotides in EFO-21 cells. The cells were treated for three days with the respective antisense oligonucleotide at the following concentrations without the use of a transfection reagent: 6 μM, 1.5 μM, 375 nM, 94 nM, 24 nM, 6 nM, 1.5 nM. The MTDH expression values are normalized to HPRT1 values and residual MTDH expression as compared to mock-treated cells (set to 1) is depicted.

FIG. 3A and FIG. 3B depicts efficacy screening experiments Renca (FIG. 3A) and 4T1 (FIG. 3B). The cells were treated with the respective mouse specific antisense oligonucleotide at a concentration of 5 μM for three days without the use of a transfection reagent. MTDH expression values are normalized to HPRT1 values and residual MTDH expression as compared to mock-treated cells (set to 1) is depicted in FIG. 3A and FIG. 3B.

FIG. 4 shows the dose-dependent knockdown of MTDH mRNA expression by mouse specific MTDH antisense oligonucleotides in 4T1 cells. The cells were treated for three days with the respective antisense oligonucleotide at the following concentrations without the use of a transfection reagent: 6 μM, 1.5 μM, 375 nM, 94 nM, 24 nM, 6 nM, 1.5 nM. The MTDH expression values are normalized to HPRT1 values.

DETAILED DESCRIPTION

The present invention provides for the first time human and murine antisense oligonucleotides which hybridize with mRNA and pre-mRNA sequences of MTDH and inhibit the expression and activity, respectively, of MTDH. Thus, the oligonucleotides of the present invention represent an interesting and highly efficient tool for use in a method of preventing and/or treating disorders, where the MTDH expression and activity, respectively, is increased in comparison to a healthy subject. The MTDH expression for example is involved in the induction of the disease and/or mediates resistance to another therapy. The oligonucleotide of the present invention hybridizes for example with a nucleic acid sequence of MTDH of SEQ ID NO.1 (human mRNA), SEQ ID NO. 2 (human pre-mRNA), SEQ ID NO.223 (mouse mRNA) and/or SEQ ID NO.224 (mouse pre-mRNA), wherein the oligonucleotide inhibits at least 50% of the MTDH expression.

In the following, the elements of the present invention will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Throughout this specification and the claims, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”, “for example”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

An oligonucleotide of the present invention is for example an antisense oligonucleotide (ASO) consisting of or comprising 10 to 25 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 12 to 18 nucleotides, or 15 to 17 nucleotides. The oligonucleotides for example consist of or comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. The oligonucleotides of the present invention comprise at least one nucleotide which is modified. The modified nucleotide is for example a bridged nucleotide such as a locked nucleic acid (LNA, e.g., 2′,4′-LNA), cET, ENA, a 2′Fluoro modified nucleotide, a 2′O-Methyl modified nucleotide or a combination thereof. In some embodiments, the oligonucleotide of the present invention comprises nucleotides having the same or different modifications. In some embodiments the oligonucleotide of the present invention comprises a modified phosphate backbone, wherein the phosphate is for example a phosphorothioate.

The oligonucleotide of the present invention comprises the one or more modified nucleotide at the 3′- and/or 5′-end of the oligonucleotide and/or at any position within the oligonucleotide, wherein modified nucleotides follow in a row of 1, 2, 3, 4, 5, or 6 modified nucleotides, or a modified nucleotide is combined with one or more unmodified nucleotides. The following Table 1 presents embodiments of oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). The oligonucleotides consisting of or comprising the sequences of Table 1 may comprise any other modified nucleotide and/or any other combination of modified and unmodified nucleotides. Oligonucleotides of Table 1 hybridize with exonic regions of the mRNA of human MTDH (SEQ ID NO.1; NM_178812.3) or with intronic regions of the pre-mRNA of human MTDH (SEQ ID NO.2; chr8:976353340-97738900), indicated by “i” in the following Table 1:

TABLE 1

List of antisense oligonucleotides hybridizing with human MTDH mRNA for

example of SEQ ID 1 and / or intronic regions of the human MTDH pre-mRNA (Hi) for

example of SEQ ID 2. Some antisense oligonucleotides also hybridize with mouse MTDH

mRNA (HM). The antisense oligonucleotides were designed according to in house criteria

and neg1 (described in WO2014154843 A1) was used as a control oligonucleotide in all

experiments.

Seq

ID
Name
Antisense Sequence 5′-3′
Antisense Sequence 5-3′ with PTO (*) and LNA (+)

3
A34001H
GAACAATGGCGGCTTG
+G*+A*+A*C*A*A*T*G*G*C*G*G*C*+T*+T*+G

4
A34002H
TCGGCGGAACAATGGC
+T*+C*+G*G*C*G*G*A*A*C*A*A*T*+G*+G*+C

5
A34003H
CTCGGCGGAACAATGGC
+C*+T*+C*G*G*C*G*G*A*A*C*A*A*T*+G*+G*+C

6
A34004H
ACGCAGTGGAATAGTCG
+A*+C*+G*C*A*G*T*G*G*A*A*T*A*G*+T*+C*+G

7
A34005H
AGGCCGACCGAGAGCAT
+A*+G*+G*C*C*G*A*C*C*G*A*G*A*G*+C*+A*+T

8
A34006H
AGCTCGGTGCGCAGAAA
+A*+G*+C*T*C*G*G*T*G*C*G*C*A*G*+A*+A*+A

9
A34007H
GTACCGTTTCGGCTCCA
+G*+T*+A*C*C*G*T*T*T*C*G*G*C*T*+C*+C*+A

10
A34008H
TCGCTCCGGAGATTCTT
+T*+C*+G*C*T*C*C*G*G*A*G*A*T*T*+C*+T*+T

11
A34009H
GCCACTTCAACAGTCCG
+G*+C*+C*A*C*T*T*C*A*A*C*A*G*T*+C*+C*+G

12
A34010H
CTTATCACGTTTACGCT
+C*+T*+T*A*T*C*A*C*G*T*T*T*A*C*+G*+C*+T

13
A34011HM
GTAAGTTGCTCGGTGGT
+G*+T*+A*A*G*T*T*G*C*T*C*G*G*T*+G*+G*+T

14
A34012H
GATGCGGTTGTAAGTTG
+G*+A*+T*G*C*G*G*T*T*G*T*A*A*G*+T*+T*+G

15
A34013H
GAACGGTCACTCCAACT
+G*+A*+A*C*G*G*T*C*A*C*T*C*C*A*+A*+C*+T

16
A34014H
CTTCGGCTGGATCACTA
+C*+T*+T*C*G*G*C*T*G*G*A*T*C*A*+C*+T*+A

17
A34015H
GTACTTCGGCTGGATCA
+G*+T*+A*C*T*T*C*G*G*C*T*G*G*A*+T*+C*+A

18
A34016H
ACGAGTACTTCGGCTGG
+A*+C*+G*A*G*T*A*C*T*T*C*G*G*C*+T*+G*+G

19
A34017H
TTTGACGAGTACTTCGG
+T*+T*+T*G*A*C*G*A*G*T*A*C*T*T*+C*+G*+G

20
A34018H
TTGTAGTATTGGCGGC
+T*+T*+G*T*A*G*T*A*T*T*G*G*C*+G*+G*+C

21
A34019H
CTTGTAGTATTGGCGGC
+C*+T*+T*G*T*A*G*T*A*T*T*G*G*C*+G*+G*+C

22
A34020H
ACGTTTCTCGTCTGGCT
+A*+C*+G*T*T*T*C*T*C*G*T*C*T*G*+G*+C*+T

23
A34021H
TCACGTTTCTCGTCTGG
+T*+C*+A*C*G*T*T*T*C*T*C*G*T*C*+T*+G*+G

24
A34022H
GATCGTTCTTGTTCTAT
+G*+A*+T*C*G*T*T*C*T*T*G*T*T*C*+T*+A*+T

25
A34023H
TACGCACTACAGGTTAA
+T*+A*+C*G*C*A*C*T*A*C*A*G*G*T*+T*+A*+A

26
A34024H
CATATTCTACGCACTAC
+C*+A*+T*A*T*T*C*T*A*C*G*C*A*C*+T*+A*+C

27
A34025HM
CGTTTGGTAAAGGCTAT
+C*+G*+T*T*T*G*G*T*A*A*A*G*G*C*+T*+A*+T

28
A34026H
CGTTCTTGGCGCCACAT
+C*+G*+T*T*C*T*T*G*G*C*G*C*C*A*+C*+A*+T

29
A34027H
CGCAATACTGTTGAACC
+C*+G*+C*A*A*T*A*C*T*G*T*T*G*A*+A*+C*+C

30
A34028H
ACCTCAACTTAGCAACG
+A*+C*+C*T*C*A*A*C*T*T*A*G*C*A*+A*+C*+G

31
A34029H
TCGGTACAGATAGGTAG
+T*+C*+G*G*T*A*C*A*G*A*T*A*G*G*+T*+A*+G

32
A34030H
TCAATTGTCGGTACAGA
+T*+C*+A*A*T*T*G*T*C*G*G*T*A*C*+A*+G*+A

33
A34031H
TTGCTCAATTGTCGGTA
+T*+T*+G*C*T*C*A*A*T*T*G*T*C*G*+G*+T*+A

34
A34032H
TGCCGATAGGAAGTTTC
+T*+G*+C*C*G*A*T*A*G*G*A*A*G*T*+T*+T*+C

35
A34033H
GCTCAGATGCCGATAGG
+G*+C*+T*C*A*G*A*T*G*C*C*G*A*T*+A*+G*+G

36
A34034H
CATCGTCCTGTTAGAGT
+C*+A*+T*C*G*T*C*C*T*G*T*T*A*G*+A*+G*+T

37
A34035Hi
CCGTTCTCTACTGCCGC
+C*+C*+G*T*T*C*T*C*T*A*C*T*G*C*+C*+G*+C

38
A34036Hi
CTCGGCTTTCGACTAAG
+C*+T*+C*G*G*C*T*T*T*C*G*A*C*T*+A*+A*+G

39
A34037Hi
TCGTCCTATACTTCCTG
+T*+C*+G*T*C*C*T*A*T*A*C*T*T*C*+C*+T*+G

40
A34038Hi
TTATTCTAGCGGTGACG
+T*+T*+A*T*T*C*T*A*G*C*G*G*T*G*+A*+C*+G

41
A34039Hi
CTTCCGAGCGCAGTCTT
+C*+T*+T*C*C*G*A*G*C*G*C*A*G*T*+C*+T*+T

42
A34040Hi
CTAGGTCACCGCACTTC
+C*+T*+A*G*G*T*C*A*C*C*G*C*A*C*+T*+T*+C

43
A34041Hi
GAAGCGCGTCTAGACCT
+G*+A*+A*G*C*G*C*G*T*C*T*A*G*A*+C*+C*+T

44
A34042Hi
CGTTCCGGCCTCTGTTG
+C*+G*+T*T*C*C*G*G*C*C*T*C*T*G*+T*+T*+G

45
A34043Hi
CGTATTAGGTAACCGAC
+C*+G*+T*A*T*T*A*G*G*T*A*A*C*C*+G*+A*+C

46
A34044Hi
CTCGTTCGTTGAACTCG
+C*+T*+C*G*T*T*C*G*T*T*G*A*A*C*+T*+C*+G

47
A34045Hi
CCAGTAAATCGGTGCCT
+C*+C*+A*G*T*A*A*A*T*C*G*G*T*G*+C*+C*+T

48
A34046Hi
AAGTGATACGCACTAGA
+A*+A*+G*T*G*A*T*A*C*G*C*A*C*T*+A*+G*+A

49
A34047Hi
GATCTTAATTCGCTGGA
+G*+A*+T*C*T*T*A*A*T*T*C*G*C*T*+G*+G*+A

50
A34048Hi
CTTCGTACTGGCTTACT
+C*+T*+T*C*G*T*A*C*T*G*G*C*T*T*+A*+C*+T

51
A34049Hi
TACGTGTATACCTTGCA
+T*+A*+C*G*T*G*T*A*T*A*C*C*T*T*+G*+C*+A

52
A34050Hi
AACCACTTACCGATCAG
+A*+A*+C*C*A*C*T*T*A*C*C*G*A*T*+C*+A*+G

53
A34051Hi
CGTGAAGGCCTATCCAG
+C*+G*+T*G*A*A*G*G*C*C*T*A*T*C*+C*+A*+G

54
A34052Hi
TTATAACGCATGTCGGA
+T*+T*+A*T*A*A*C*G*C*A*T*G*T*C*+G*+G*+A

55
A34053Hi
AAGCATTCGCCGGAATC
+A*+A*+G*C*A*T*T*C*G*C*C*G*G*A*+A*+T*+C

56
A34054Hi
TATAAGCATTCGCCGGA
+T*+A*+T*A*A*G*C*A*T*T*C*G*C*C*+G*+G*+A

57
A34055Hi
GCTAACGCTAGGTTTAC
+G*+C*+T*A*A*C*G*C*T*A*G*G*T*T*+T*+A*+C

58
A34056Hi
TTGCAATATAAGCGACT
+T*+T*+G*C*A*A*T*A*T*A*A*G*C*G*+A*+C*+T

59
A34057Hi
GGTCAGGCATAACACTC
+G*+G*+T*C*A*G*G*C*A*T*A*A*C*A*+C*+T*+C

60
A34058Hi
GAATAAGCTGCGTTCAC
+G*+A*+A*T*A*A*G*C*T*G*C*G*T*T*+C*+A*+C

61
A34059Hi
TATGAGCTATAACCGCC
+T*+A*+T*G*A*G*C*T*A*T*A*A*C*C*+G*+C*+C

62
A34060Hi
CCAAGATATGGCTCCGA
+C*+C*+A*A*G*A*T*A*T*G*G*C*T*C*+C*+G*+A

63
A34061Hi
GTGTGTGACCACTAGTA
+G*+T*+G*T*G*T*G*A*C*C*A*C*T*A*+G*+T*+A

64
A34062Hi
CACGGCTTGTCTATCAG
+C*+A*+C*G*G*C*T*T*G*T*C*T*A*T*+C*+A*+G

65
A34063Hi
AGCTTCCTTTAAGCGAC
+A*+G*+C*T*T*C*C*T*T*T*A*A*G*C*+G*+A*+C

66
A34064Hi
TTACTAGTGCGTTGAGA
+T*+T*+A*C*T*A*G*T*G*C*G*T*T*G*+A*+G*+A

67
A34065Hi
TTTACGTTAGGCCTCTG
+T*+T*+T*A*C*G*T*T*A*G*G*C*C*T*+C*+T*+G

68
A34066Hi
CAGACGCTGCGGAACTA
+C*+A*+G*A*C*G*C*T*G*C*G*G*A*A*+C*+T*+A

69
A34067Hi
CCAGATACGGTTCTCAC
+C*+C*+A*G*A*T*A*C*G*G*T*T*C*T*+C*+A*+C

70
A34068Hi
TGATCCATCGTCCAAGT
+T*+G*+A*T*C*C*A*T*C*G*T*C*C*A*+A*+G*+T

71
A34069Hi
GTATGCTTAATAGGCCG
+G*+T*+A*T*G*C*T*T*A*A*T*A*G*G*+C*+C*+G

72
A34070Hi
CATACAGCCGTGTCTAC
+C*+A*+T*A*C*A*G*C*C*G*T*G*T*C*+T*+A*+C

73
A34071Hi
CAGTAGTACCTTGTACG
+C*+A*+G*T*A*G*T*A*C*C*T*T*G*T*+A*+C*+G

74
A34072Hi
CTATACTTTGACACGGA
+C*+T*+A*T*A*C*T*T*T*G*A*C*A*C*+G*+G*+A

75
A34073Hi
GAGCTTTCCGAACATAC
+G*+A*+G*C*T*T*T*C*C*G*A*A*C*A*+T*+A*+C

76
A34074Hi
CACCGAACACCTATGTA
+C*+A*+C*C*G*A*A*C*A*C*C*T*A*T*+G*+T*+A

77
A34075Hi
CGCCAGCTTACCTTGAT
+C*+G*+C*C*A*G*C*T*T*A*C*C*T*T*+G*+A*+T

78
A34076Hi
GTGCTCATACGCTCCTA
+G*+T*+G*C*T*C*A*T*A*C*G*C*T*C*+C*+T*+A

79
A34077Hi
TCGTATCTACTGTCTAA
+T*+C*+G*T*A*T*C*T*A*C*T*G*T*C*+T*+A*+A

80
A34078Hi
GTACTCTAACCGTCTTT
+G*+T*+A*C*T*C*T*A*A*C*C*G*T*C*+T*+T*+T

81
A34079Hi
TTGTCTCCGAGCCTTAT
+T*+T*+G*T*C*T*C*C*G*A*G*C*C*T*+T*+A*+T

82
A34080Hi
GCCGTCCATAAGCCATG
+G*+C*+C*G*T*C*C*A*T*A*A*G*C*C*+A*+T*+G

83
A34081Hi
TAGTAGCACAGAGGCGA
+T*+A*+G*T*A*G*C*A*C*A*G*A*G*G*+C*+G*+A

84
A34082Hi
CGTGCAGCTTGTAGTCT
+C*+G*+T*G*C*A*G*C*T*T*G*T*A*G*+T*+C*+T

85
A34083Hi
TCTAACGTACAAACGCT
+T*+C*+T*A*A*C*G*T*A*C*A*A*A*C*+G*+C*+T

86
A34084Hi
ATGTACTCGTGCTCTGG
+A*+T*+G*T*A*C*T*C*G*T*G*C*T*C*+T*+G*+G

87
A34085Hi
CCGACCGATTGAGGCCA
+C*+C*+G*A*C*C*G*A*T*T*G*A*G*G*+C*+C*+A

88
A34086Hi
GATACCACGTGTTGCTA
+G*+A*+T*A*C*C*A*C*G*T*G*T*T*G*+C*+T*+A

89
A34087Hi
TACCGATGTTCAATGCC
+T*+A*+C*C*G*A*T*G*T*T*C*A*A*T*+G*+C*+C

90
A34088Hi
TTAGTTTATTCCGGATC
+T*+T*+A*G*T*T*T*A*T*T*C*C*G*G*+A*+T*+C

91
A34089Hi
TTTAGGAGCCGAGTATA
+T*+T*+T*A*G*G*A*G*C*C*G*A*G*T*+A*+T*+A

92
A34090Hi
GAGTCTGTTAACGACAA
+G*+A*+G*T*C*T*G*T*T*A*A*C*G*A*+C*+A*+A

93
A34091Hi
TACCATAGCCGCTCTTA
+T*+A*+C*C*A*T*A*G*C*C*G*C*T*C*+T*+T*+A

94
A34092Hi
GAATAGATACGTGCCAT
+G*+A*+A*T*A*G*A*T*A*C*G*T*G*C*+C*+A*+T

95
A34093Hi
GGTGGTCTCACGAACTA
+G*+G*+T*G*G*T*C*T*C*A*C*G*A*A*+C*+T*+A

96
A34094Hi
GATGATTCTAGTAACCG
+G*+A*+T*G*A*T*T*C*T*A*G*T*A*A*+C*+C*+G

97
A34095Hi
CGTGTATAAGTGGAGGT
+C*+G*+T*G*T*A*T*A*A*G*T*G*G*A*+G*+G*+T

98
A34096Hi
ACCTATACGCATACAAG
+A*+C*+C*T*A*T*A*C*G*C*A*T*A*C*+A*+A*+G

99
A34097Hi
CCAGCGATTGTACATAT
+C*+C*+A*G*C*G*A*T*T*G*T*A*C*A*+T*+A*+T

100
A34098Hi
GTGTCTACGTCATCCAT
+G*+T*+G*T*C*T*A*C*G*T*C*A*T*C*+C*+A*+T

101
A34099Hi
AGCCGGAGGCGCTTAAT
+A*+G*+C*C*G*G*A*G*G*C*G*C*T*T*+A*+A*+T

102
A34100Hi
CCTTTGCCAATACGTTA
+C*+C*+T*T*T*G*C*C*A*A*T*A*C*G*+T*+T*+A

103
A34101Hi
TTTGGTGCGGTAGCTTG
+T*+T*+T*G*G*T*G*C*G*G*T*A*G*C*+T*+T*+G

104
A34102Hi
CTAGGACACCATGGTAC
+C*+T*+A*G*G*A*C*A*C*C*A*T*G*G*+T*+A*+C

105
A34103Hi
ATTGCGGATTGCGTCAC
+A*+T*+T*G*C*G*G*A*T*T*G*C*G*T*+C*+A*+C

106
A34104Hi
ACAGATGTTCGGCCAGC
+A*+C*+A*G*A*T*G*T*T*C*G*G*C*C*+A*+G*+C

107
A34105H
GAACAATGGCGGCTTGG
+G*+A*+A*C*A*A*T*G*G*C*G*G*C*T*+T*+G*+G

108
A34106H
GGAACAATGGCGGCTTG
+G*+G*+A*A*C*A*A*T*G*G*C*G*G*C*+T*+T*+G

109
A34107H
CTCGGCGGAACAATGG
+C*+T*+C*G*G*C*G*G*A*A*C*A*A*+T*+G*+G

110
A34108H
CCTCGGCGGAACAATGG
+C*+C*+T*C*G*G*C*G*G*A*A*C*A*A*+T*+G*+G

111
A34109HM
TAGGCCGACCGAGAGCA
+T*+A*+G*G*C*C*G*A*C*C*G*A*G*A*+G*+C*+A

112
A34110HM
TAGGCCGACCGAGAGC
+T*+A*+G*G*C*C*G*A*C*C*G*A*G*+A*+G*+C

113
A34111H
TACCGTTTCGGCTCCAG
+T*+A*+C*C*G*T*T*T*C*G*G*C*T*C*+C*+A*+G

114
A34112H
CCACTTCAACAGTCCGC
+C*+C*+A*C*T*T*C*A*A*C*A*G*T*C*+C*+G*+C

115
A34113HM
TGCTCGGTGGTAACTGT
+T*+G*+C*T*C*G*G*T*G*G*T*A*A*C*+T*+G*+T

116
A34114HM
AAGTTGCTCGGTGGTAA
+A*+A*+G*T*T*G*C*T*C*G*G*T*G*G*+T*+A*+A

117
A34115H
TGATGCGGTTGTAAGTT
+T*+G*+A*T*G*C*G*G*T*T*G*T*A*A*+G*+T*+T

118
A34116HM
AGACCATTCATCATCGA
+A*+G*+A*C*C*A*T*T*C*A*T*C*A*T*+C*+G*+A

119
A34117H
ACGAGTACTTCGGCTG
+A*+C*+G*A*G*T*A*C*T*T*C*G*G*+C*+T*+G

120
A34118H
TGACGAGTACTTCGGCT
+T*+G*+A*C*G*A*G*T*A*C*T*T*C*G*+G*+C*+T

121
A34119H
GGCTATTTTTGACGAGT
+G*+G*+C*T*A*T*T*T*T*T*G*A*C*G*+A*+G*+T

122
A34120H
CACGTTTCTCGTCTGGC
+C*+A*+C*G*T*T*T*C*T*C*G*T*C*T*+G*+G*+C

123
A34121H
ACGCACTACAGGTTAAG
+A*+C*+G*C*A*C*T*A*C*A*G*G*T*T*+A*+A*+G

124
A34122HM
CACGTTTGGTAAAGGCT
+C*+A*+C*G*T*T*T*G*G*T*A*A*A*G*+G*+C*+T

125
A34123H
TTTGCTCAATTGTCGGT
+T*+T*+T*G*C*T*C*A*A*T*T*G*T*C*+G*+G*+T

126
A34124H
GATGCCGATAGGAAGTT
+G*+A*+T*G*C*C*G*A*T*A*G*G*A*A*+G*+T*+T

127
A34125H
TGGCTCAGATGCCGATA
+T*+G*+G*C*T*C*A*G*A*T*G*C*C*G*+A*+T*+A

128
A34126H
ATCGTCCTGTTAGAGTA
+A*+T*+C*G*T*C*C*T*G*T*T*A*G*A*+G*+T*+A

129
A34127Hi
CTCTCGGCTTTCGACTA
+C*+T*+C*T*C*G*G*C*T*T*T*C*G*A*+C*+T*+A

130
A34128Hi
CCTCTCGGCTTTCGACT
+C*+C*+T*C*T*C*G*G*C*T*T*T*C*G*+A*+C*+T

131
A34129Hi
CGTCCTATACTTCCTGA
+C*+G*+T*C*C*T*A*T*A*C*T*T*C*C*+T*+G*+A

132
A34130Hi
CTAGCGGTGACGGTTC
+C*+T*+A*G*C*G*G*T*G*A*C*G*G*+T*+T*+C

133
A34131Hi
GTGCTTATTCTAGCGGT
+G*+T*+G*C*T*T*A*T*T*C*T*A*G*C*+G*+G*+T

134
A34132Hi
AAGCGCGTCTAGACCT
+A*+A*+G*C*G*C*G*T*C*T*A*G*A*+C*+C*+T

135
A34133Hi
TCAAAGCGTATTAGGTA
+T*+C*+A*A*A*G*C*G*T*A*T*T*A*G*+G*+T*+A

136
A34134Hi
CACCTCAAAGCGTATTA
+C*+A*+C*C*T*C*A*A*A*G*C*G*T*A*+T*+T*+A

137
A34135Hi
ACACCTCAAAGCGTATT
+A*+C*+A*C*C*T*C*A*A*A*G*C*G*T*+A*+T*+T

138
A34136Hi
TCGTTGAACTCGCCTTA
+T*+C*+G*T*T*G*A*A*C*T*C*G*C*C*+T*+T*+A

139
A34137Hi
AACACTGCTGGTATTCG
+A*+A*+C*A*C*T*G*C*T*G*G*T*A*T*+T*+C*+G

140
A34138Hi
ACTCGTTCGTTGAACTC
+A*+C*+T*C*G*T*T*C*G*T*T*G*A*A*+C*+T*+C

141
A34139Hi
CTCGTTCGTTGAACTC
+C*+T*+C*G*T*T*C*G*T*T*G*A*A*+C*+T*+C

142
A34140Hi
CAGACTCGTTCGTTGAA
+C*+A*+G*A*C*T*C*G*T*T*C*G*T*T*+G*+A*+A

143
A34141Hi
ACAGACTCGTTCGTTGA
+A*+C*+A*G*A*C*T*C*G*T*T*C*G*T*+T*+G*+A

144
A34142Hi
ACAGACTCGTTCGTTG
+A*+C*+A*G*A*C*T*C*G*T*T*C*G*+T*+T*+G

145
A34143Hi
CACAAATCTACCACGCC
+C*+A*+C*A*A*A*T*C*T*A*C*C*A*C*+G*+C*+C

146
A34144Hi
GTAAATCGGTGCCTGAA
+G*+T*+A*A*A*T*C*G*G*T*G*C*C*T*+G*+A*+A

147
A34145Hi
AGTAAATCGGTGCCTGA
+A*+G*+T*A*A*A*T*C*G*G*T*G*C*C*+T*+G*+A

148
A34146Hi
CGTACTGGCTTACTGAA
+C*+G*+T*A*C*T*G*G*C*T*T*A*C*T*+G*+A*+A

149
A34147Hi
TTCGTACTGGCTTACTG
+T*+T*+C*G*T*A*C*T*G*G*C*T*T*A*+C*+T*+G

150
A34148Hi
TCTACGTGTATACCTTG
+T*+C*+T*A*C*G*T*G*T*A*T*A*C*C*+T*+T*+G

151
A34149Hi
CCACCAGTATTGGATAG
+C*+C*+A*C*C*A*G*T*A*T*T*G*G*A*+T*+A*+G

152
A34150Hi
CGCCGGAATCTGTATTC
+C*+G*+C*C*G*G*A*A*T*C*T*G*T*A*+T*+T*+C

153
A34151Hi
ATTCGCCGGAATCTGTA
+A*+T*+T*C*G*C*C*G*G*A*A*T*C*T*+G*+T*+A

154
A34152Hi
AGCATTCGCCGGAATC
+A*+G*+C*A*T*T*C*G*C*C*G*G*A*+A*+T*+C

155
A34153Hi
TAAGCATTCGCCGGAAT
+T*+A*+A*G*C*A*T*T*C*G*C*C*G*G*+A*+A*+T

156
A34154Hi
AAGCATTCGCCGGAAT
+A*+A*+G*C*A*T*T*C*G*C*C*G*G*+A*+A*+T

157
A34155Hi
ATAAGCATTCGCCGGAA
+A*+T*+A*A*G*C*A*T*T*C*G*C*C*G*+G*+A*+A

158
A34156Hi
TAAGCATTCGCCGGAA
+T*+A*+A*G*C*A*T*T*C*G*C*C*G*+G*+A*+A

159
A34157Hi
ATAAGCATTCGCCGGA
+A*+T*+A*A*G*C*A*T*T*C*G*C*C*+G*+G*+A

160
A34158Hi
TGTATAAGCATTCGCCG
+T*+G*+T*A*T*A*A*G*C*A*T*T*C*G*+C*+C*+G

161
A34159Hi
GTATAAGCATTCGCCG
+G*+T*+A*T*A*A*G*C*A*T*T*C*G*+C*+C*+G

162
A34160Hi
ACGCTAGGTTTACTTAT
+A*+C*+G*C*T*A*G*G*T*T*T*A*C*T*+T*+A*+T

163
A34161Hi
AACGCTAGGTTTACTTA
+A*+A*+C*G*C*T*A*G*G*T*T*T*A*C*+T*+T*+A

164
A34162Hi
AGCTAACGCTAGGTTTA
+A*+G*+C*T*A*A*C*G*C*T*A*G*G*T*+T*+T*+A

165
A34163Hi
GCGTTCACATCCTAGTC
+G*+C*+G*T*T*C*A*C*A*T*C*C*T*A*+G*+T*+C

166
A34164Hi
AAGACTCTACTCACACG
+A*+A*+G*A*C*T*C*T*A*C*T*C*A*C*+A*+C*+G

167
A34165Hi
TACTAGTGCGTTGAGAA
+T*+A*+C*T*A*G*T*G*C*G*T*T*G*A*+G*+A*+A

168
A34166Hi
ATTACTAGTGCGTTGAG
+A*+T*+T*A*C*T*A*G*T*G*C*G*T*T*+G*+A*+G

169
A34167Hi
TCATTACTAGTGCGTTG
+T*+C*+A*T*T*A*C*T*A*G*T*G*C*G*+T*+T*+G

170
A34168Hi
GAACATGCCATCGAAAC
+G*+A*+A*C*A*T*G*C*C*A*T*C*G*A*+A*+A*+C

171
A34169Hi
GATACGGTTCTCACATT
+G*+A*+T*A*C*G*G*T*T*C*T*C*A*C*+A*+T*+T

172
A34170Hi
AGATACGGTTCTCACAT
+A*+G*+A*T*A*C*G*G*T*T*C*T*C*A*+C*+A*+T

173
A34171Hi
CAGATACGGTTCTCACA
+C*+A*+G*A*T*A*C*G*G*T*T*C*T*C*+A*+C*+A

174
A34172Hi
ACTTTCACTTAGTTACG
+A*+C*+T*T*T*C*A*C*T*T*A*G*T*T*+A*+C*+G

175
A34173Hi
TAGCACACGGCACAAGC
+T*+A*+G*C*A*C*A*C*G*G*C*A*C*A*+A*+G*+C

176
A34174Hi
ATAGCACACGGCACAAG
+A*+T*+A*G*C*A*C*A*C*G*G*C*A*C*+A*+A*+G

177
A34175Hi
ACATGATCCATCGTCCA
+A*+C*+A*T*G*A*T*C*C*A*T*C*G*T*+C*+C*+A

178
A34176Hi
ATACATGATCCATCGTC
+A*+T*+A*C*A*T*G*A*T*C*C*A*T*C*+G*+T*+C

179
A34177Hi
TAACTCTTATTCGGTCC
+T*+A*+A*C*T*C*T*T*A*T*T*C*G*G*+T*+C*+C

180
A34178Hi
CTAACTCTTATTCGGTC
+C*+T*+A*A*C*T*C*T*T*A*T*T*C*G*+G*+T*+C

181
A34179Hi
CAGCCGTGTCTACCTAA
+C*+A*+G*C*C*G*T*G*T*C*T*A*C*C*+T*+A*+A

182
A34180Hi
ACAGCCGTGTCTACCTA
+A*+C*+A*G*C*C*G*T*G*T*C*T*A*C*+C*+T*+A

183
A34181Hi
ACATACAGCCGTGTCTA
+A*+C*+A*T*A*C*A*G*C*C*G*T*G*T*+C*+T*+A

184
A34182Hi
GAATTACATACAGCCGT
+G*+A*+A*T*T*A*C*A*T*A*C*A*G*C*+C*+G*+T

185
A34183Hi
GTACGCAGAAGGTATTC
+G*+T*+A*C*G*C*A*G*A*A*G*G*T*A*+T*+T*+C

186
A34184Hi
GGCATGAGCTTTCCGAA
+G*+G*+C*A*T*G*A*G*C*T*T*T*C*C*+G*+A*+A

187
A34185Hi
TCATACGCTCCTATCTG
+T*+C*+A*T*A*C*G*C*T*C*C*T*A*T*+C*+T*+G

188
A34186Hi
TACGCTCCTATCTGTGC
+T*+A*+C*G*C*T*C*C*T*A*T*C*T*G*+T*+G*+C

189
A34187Hi
ATACGCTCCTATCTGTG
+A*+T*+A*C*G*C*T*C*C*T*A*T*C*T*+G*+T*+G

190
A34188Hi
CATACGCTCCTATCTGT
+C*+A*+T*A*C*G*C*T*C*C*T*A*T*C*+T*+G*+T

191
A34189Hi
TGTCGCCAGCTTACCTT
+T*+G*+T*C*G*C*C*A*G*C*T*T*A*C*+C*+T*+T

192
A34190Hi
GCTCATACGCTCCTATC
+G*+C*+T*C*A*T*A*C*G*C*T*C*C*T*+A*+T*+C

193
A34191Hi
AAGGTGCTCATACGCTC
+A*+A*+G*G*T*G*C*T*C*A*T*A*C*G*+C*+T*+C

194
A34192Hi
CCAAAGGTGCTCATACG
+C*+C*+A*A*A*G*G*T*G*C*T*C*A*T*+A*+C*+G

195
A34193Hi
GCCAAAGGTGCTCATAC
+G*+C*+C*A*A*A*G*G*T*G*C*T*C*A*+T*+A*+C

196
A34194Hi
CGAGCCTTATTTCTACA
+C*+G*+A*G*C*C*T*T*A*T*T*T*C*T*+A*+C*+A

197
A34195Hi
CAATGCAGTAAGCGCTC
+C*+A*+A*T*G*C*A*G*T*A*A*G*C*G*+C*+T*+C

198
A34196Hi
ACGTACAAACGCTCTTT
+A*+C*+G*T*A*C*A*A*A*C*G*C*T*C*+T*+T*+T

199
A34197Hi
TAACGTACAAACGCTCT
+T*+A*+A*C*G*T*A*C*A*A*A*C*G*C*+T*+C*+T

200
A34198Hi
CCTACCGATGTTCAATG
+C*+C*+T*A*C*C*G*A*T*G*T*T*C*A*+A*+T*+G

201
A34199Hi
GAGCCGAGTATACATAA
+G*+A*+G*C*C*G*A*G*T*A*T*A*C*A*+T*+A*+A

202
A34200Hi
TAGGAGCCGAGTATACA
+T*+A*+G*G*A*G*C*C*G*A*G*T*A*T*+A*+C*+A

203
A34201Hi
GTCTGTTAACGACAAAG
+G*+T*+C*T*G*T*T*A*A*C*G*A*C*A*+A*+A*+G

204
A34202Hi
AAGAGTCTGTTAACGAC
+A*+A*+G*A*G*T*C*T*G*T*T*A*A*C*+G*+A*+C

205
A34203Hi
TAGCCGCTCTTAAGTAA
+T*+A*+G*C*C*G*C*T*C*T*T*A*A*G*+T*+A*+A

206
A34204Hi
AATAGATACGTGCCATG
+A*+A*+T*A*G*A*T*A*C*G*T*G*C*C*+A*+T*+G

207
A34205Hi
TGGTGGTCTCACGAACT
+T*+G*+G*T*G*G*T*C*T*C*A*C*G*A*+A*+C*+T

208
A34206Hi
TTACCATAGCCGCTCTT
+T*+T*+A*C*C*A*T*A*G*C*C*G*C*T*+C*+T*+T

209
A34207Hi
GTCTTTACCATAGCCGC
+G*+T*+C*T*T*T*A*C*C*A*T*A*G*C*+C*+G*+C

210
A34208Hi
CCTATACGCATACAAGT
+C*+C*+T*A*T*A*C*G*C*A*T*A*C*A*+A*+G*+T

211
A34209Hi
GGAACTAGTATCTGTAC
+G*+G*+A*A*C*T*A*G*T*A*T*C*T*G*+T*+A*+C

212
A34210Hi
CAGTGTGTCTACGTCAT
+C*+A*+G*T*G*T*G*T*C*T*A*C*G*T*+C*+A*+T

213
A34211Hi
TCAGTGTGTCTACGTCA
+T*+C*+A*G*T*G*T*G*T*C*T*A*C*G*+T*+C*+A

214
A34212Hi
AAGCCGGAGGCGCTTAA
+A*+A*+G*C*C*G*G*A*G*G*C*G*C*T*+T*+A*+A

215
A34213Hi
AGCCGGAGGCGCTTAA
+A*+G*+C*C*G*G*A*G*G*C*G*C*T*+T*+A*+A

216
A34214Hi
CCAGCTACCACGTGCGG
+C*+C*+A*G*C*T*A*C*C*A*C*G*T*G*+C*+G*+G

217
A34215Hi
TTGCGGATTGCGTCACT
+T*+T*+G*C*G*G*A*T*T*G*C*G*T*C*+A*+C*+T

218
A34216Hi
TTGCGGATTGCGTCAC
+T*+T*+G*C*G*G*A*T*T*G*C*G*T*+C*+A*+C

219
A34217Hi
GAGATTGCGGATTGCGT
+G*+A*+G*A*T*T*G*C*G*G*A*T*T*G*+C*+G*+T

220
A34218Hi
AGATTGCGGATTGCGT
+A*+G*+A*T*T*G*C*G*G*A*T*T*G*+C*+G*+T

221
A34219Hi
CGTCAGTAATTTGGAGT
+C*+G*+T*C*A*G*T*A*A*T*T*T*G*G*+A*+G*+T

222
Control

+C*+G*+T*T*T*A*G*G*C*T*A*T*G*T*A*+C*+T*+T

oligo

(Neg1)

The oligonucleotides of the present invention hybridize for example with mRNA of human MTDH of SEQ ID NO.1 and/or introns of the pre-mRNA of human MTDH of SEQ ID NO.2. Such oligonucleotides are called MTDH antisense oligonucleotides. In some embodiments, the oligonucleotides hybridize within a hybridizing active area which is one or more region(s) on the MTDH mRNA, e.g., of SEQ ID NO.1 and/or the MTDH pre-mRNA, e.g., of SEQ ID NO.2, where hybridization with an oligonucleotide highly likely results in a potent knockdown of the MTDH expression. In the present invention surprisingly several hybridizing active regions were identified for example selected from hybridizing active regions for example selected from position 8500 to 8999, position 9000 to 9499, position 9500 to 9999, position 1000 to 10499, position 10500 to 10999, position 11000 to 11499, position 13000 to 13499, position 14000 to 14499, position 15500 to 15999, position 16500 to 16999, position 17500 to 17999, position 18000 to 18499, position 20500 to 20999, position 21000 to 21499, position 22500 to 22999, position 24000 to 24499, position 25000 to 25499, position 25500 to 25999, position 27000 to 27499, position 29000 to 29499, position 29500 to 29999, position 32000 to 32499, position 37000 to 37499, position 37500 to 37999, position 43500 to 43999, position 44500 to 44999, position 45500 to 45999, position 46500 to 46999, position 47000 to 47499, position 49000 to 49499, position 50500 to 50999, position 52000 to 52499, position 52500 to 52999, position 54000 to 54499, position 55500 to 55999, position 61500 to 61999, position 64000 to 64499, position 64500 to 64999, position 65000 to 65499, position 65500 to 65999, position 68000 to 68499, position 68500 to 68999, position 71500 to 71999, position 72000 to 72499, position 74500 to 74999, position 76000 to 76499, position 77000 to 77499, position 77500 to 77999, position 78000 to 78499, position 80500 to 80999, position 81000 to 81499, position 81500 to 81999, position 82000 to 82499, position 83500 to 83999, position 85000 to 85499, position 86000 to 86499, position 87500 to 87999, position 88500 to 88999, position 89000 to 89499, position 89500 to 89999, position 90000 to 90499, position 90500 to 90999, position 91000 to 91499, position 92000 to 92499, position 92500 to 92999, position 93500 to 93999, position 94500 to 94999 or a combination thereof (including the terminal figures of the ranges) of MTDH pre-mRNA for example of SEQ ID NO.2. Antisense oligonucleotides hybridizing with these regions are indicated in the following Table 2:

First position

Region of SEQ ID NO.2/ASO name
on SEQ ID NO. 2
SEQ ID NO.

Region 8500-8999

A34001H
8844
3

A34002H
8850
4

A34003H
8850
5

A34105H
8843
107

A34106H
8844
108

A34107H
8851
109

A34108H
8851
110

Region 9000-9499

A34004H
9104
6

A34005H
9237
7

A34006H
9258
8

A34007H
9292
9

A34008H
9486
10

A34109HM
9238
111

A34110HM
9239
112

A34111H
9291
113

Region 9500-9999

A34035Hi
9564
37

A34036Hi
9649
38

A34037Hi
9732
39

A34038Hi
9837
40

A34039Hi
9938
41

A34040Hi
9999
42

A34127Hi
9651
129

A34128Hi
9652
130

A34129Hi
9731
131

A34130Hi
9833
132

A34131Hi
9841
133

Region 10000-10499

A34041Hi
10050
43

A34042Hi
10127
44

A34043Hi
10162
45

A34044Hi
10215
46

A34045Hi
10321
47

A34132Hi
10050
134

A34133Hi
10168
135

A34134Hi
10172
136

A34135Hi
10173
137

A34136Hi
10210
138

A34138Hi
10216
140

A34139Hi
10216
141

A34140Hi
10219
142

A34141Hi
10220
143

A34142Hi
10221
144

A34143Hi
10249
145

A34144Hi
10318
146

A34145Hi
10319
147

Region 10500-10999

A34047Hi
10538
49

A34048Hi
10576
50

A34049Hi
10635
51

A34051Hi
10902
53

A34146Hi
10573
148

A34147Hi
10575
149

A34148Hi
10637
150

Region 11000-11499

A34052Hi
11270
54

A34053Hi
11316
55

A34054Hi
11319
56

A34055Hi
11418
57

A34150Hi
11309
152

A34151Hi
11312
153

A34152Hi
11316
154

A34153Hi
11317
155

A34154Hi
11317
156

A34155Hi
11318
157

A34156Hi
11318
158

A34157Hi
11319
159

A34158Hi
11321
160

A34159Hi
11321
161

A34160Hi
11414
162

A34161Hi
11415
163

A34162Hi
11419
164

Region 13000-13499

A34059Hi
13208
61

Region 14000-14499

A34164Hi
14032
166

Region 15500-15999

A34061Hi
15672
63

Region 16500-16999

A34064Hi
16779
66

A34165Hi
16778
167

A34166Hi
16780
168

A34167Hi
16782
169

Region 17500-17999

A34168Hi
17990
170

Region 18000-18499

A34065Hi
18193
67

Region 20500-20999

A34172Hi
20810
174

Region 21000-21499

A34069Hi
21257
71

Region 22500-22999

A34076Hi
22958
78

A34185Hi
22954
187

A34186Hi
22951
188

A34187Hi
22952
189

A34188Hi
22953
190

A34190Hi
22956
192

A34191Hi
22961
193

A34192Hi
22964
194

A34193Hi
22965
195

Region 24000-24499

A34078Hi
24443
80

Region 25000-25499

A34079Hi
25369
81

A34194Hi
25362
196

Region 25500-25999

A34009H
25742
11

A34112H
25741
114

Region 27000-27499

A34050Hi
27086
52

Region 29000-29499

A34058Hi
29054
60

A34163Hi
29045
165

Region 29500-29999

A34060Hi
29798
62

Region 32000-32499

A34062Hi
32217
64

A34063Hi
32290
65

Region 37000-37499

A34067Hi
37441
69

A34169Hi
37438
171

A34170Hi
37439
172

A34171Hi
37440
173

Region 37500-37999

A34068Hi
37598
70

A34070Hi
37675
72

A34071Hi
37753
73

A34173Hi
37539
175

A34174Hi
37540
176

A34175Hi
37601
177

A34176Hi
37603
178

A34177Hi
37657
179

A34178Hi
37658
180

A34179Hi
37671
181

A34180Hi
37672
182

A34181Hi
37676
183

A34182Hi
37681
184

A34183Hi
37741
185

Region 43500-43999

A34083Hi
43635
85

A34084Hi
43723
86

A34196Hi
43631
198

A34197Hi
43633
199

Region 44500-44999

A34085Hi
44960
87

Region 45500-45999

A34087Hi
45815
89

A34198Hi
45817
200

Region 46500-46999

A34089Hi
46545
91

A34199Hi
46540
201

A34200Hi
46543
202

Region 47000-47499

A34090Hi
47441
92

A34201Hi
47439
203

A34202Hi
47443
204

Region 49000-49499

A34091Hi
49327
93

A34203Hi
49322
205

A34206Hi
49328
208

A34207Hi
49332
209

Region 50500-50999

A34096Hi
50651
98

A34208Hi
50650
210

Region 52000-52499

A34010H
52132
12

A34113HM
52206
115

A3414HM
52210
116

A34115H
52222
117

A34011HM
52212
13

A34012H
52221
14

A34137Hi
52428
139

Region 52500-52999

A34046Hi
52534
48

A34149Hi
52809
151

Region 54000-54499

A34056Hi
54009
58

A34057Hi
54285
59

Region 55500-55999

A34013H
55816
15

Region 61500-61999

A34066Hi
61845
68

Region 64000-64499

A34116HM
64490
118

A34072Hi
64330
74

Region 64500-64999

A34073Hi
64958
75

A34184Hi
64963
186

Region 65000-65499

A34074Hi
65234
76

Region 65500-65999

A34075Hi
65535
77

A34077Hi
65685
79

A34189Hi
65538
191

Region 68000-68499

A34080Hi
68180
82

A34195Hi
68481
197

Region 68500-68999

A34081Hi
68654
83

A34082Hi
68927
84

Region 71500-71999

A34086Hi
71718
88

Region 72000-72499

A34088Hi
72396
90

Region 74500-74999

A34092Hi
74688
94

A34093Hi
74876
95

A34094Hi
74933
96

A34204Hi
74687
206

A34205Hi
74877
207

Region 76000-76499

A34095Hi
76285
97

Region 77000-77499

A34097Hi
77266
99

Region 77500-77999

A34209Hi
77568
211

Region 78000-78499

A34098Hi
78105
100

A34099Hi
78457
101

A34210Hi
78109
212

A34211Hi
78110
213

A34212Hi
78458
214

A34213Hi
78458
215

Region 80500-80999

A34100Hi
80503
102

Region 81000-81499

A34214Hi
81204
216

Region 81500-81999

A34101Hi
81622
103

Region 82000-82499

A34102Hi
82216
104

Region 83500-83999

A34117H
83822
119

A34118H
83823
120

A34119H
83832
121

A34014H
83814
16

A34015H
83817
17

A34016H
83821
18

A34017H
83825
19

Region 85000-85499

A34103Hi
85364
105

A34215Hi
85363
217

A34216Hi
85364
218

A34217Hi
85367
219

A34218Hi
85367
220

Region 86000-86499

A34219Hi
86343
221

Region 87500-87999

A34018H
87626
20

A34019H
87626
21

Region 88500-88999

A34104Hi
88632
106

Region 89000-89499

A34120H
89314
122

A34020H
89313
22

A34021H
89315
23

Region 89500-89999

A34022H
89562
24

Region 90000-90499

A34121H
90312
123

A34023H
90313
25

A34024H
90320
26

Region 90500-90999

A34122HM
90556
124

A34025HM
90554
27

A34026H
90661
28

A34027H
90746
29

Region 91000-91499

A34028H
91356
30

Region 92000-92499

A34029H
92491
31

A34030H
92498
32

Region 92500-92999

A34123H
92503
125

A34031H
92502
33

Region 93500-93999

A34124H
93935
126

A34125H
93942
127

A34032H
93933
34

A34033H
93940
35

Region 94500-94999

A34126H
94647
128

A34034H
94648
36

Table 2 shows some hybridizing active regions and antisense oligonucleotides hybridizing in this region.

The following Table 3 presents embodiments of oligonucleotides comprising modified nucleotides for example LNA which are indicated by (+) and phosphorothioate (PTO) indicated by (*). The oligonucleotides consisting of or comprising the sequences of Table 3 may comprise any other modified nucleotide and/or any other combination of modified and unmodified nucleotides. Oligonucleotides of Table 3 hybridize with exonic regions of the mRNA of mouse MTDH (SEQ ID NO.223; NM_001357926.1) or with intronic regions of the pre-mRNA of mouse MTDH (SEQ ID NO.224; chr15:34076600-34149700), indicated by “i” in the following Table 3:

TABLE 3

List of antisense oligonucleotides hybridizing with mouse MTDH mRNA for

example of SEQ ID NO. 223 and / or intronic regions of the mouse MTDH pre-mRNA (Mi)

for example of SEQ ID NO. 224. The antisense oligonucleotides were designed according

to in house criteria and neg1 (described in WO2014154843 A1) was used as a control

oligonucleotide in all experiments.

Seq

ID
Name
Antisense Sequence 5′-3′
Antisense Sequence 5′-3′ with PTO (*) and LNA

225
A34001M
CGAGCGCATCGCGGCCA
+C*+G*+A*G*C*G*C*A*T*C*G*C*G*G*+C*+C*+A

226
A34002M
CCGCTGGAACAGTCGTG
+C*+C*G*C*T*G*G*A*A*C*A*G*T*C*+G*+T*+G

227
A34003M
CCGTCAGAGAGACTCGC
+C*+C*+G*T*C*A*G*A*G*A*G*A*C*T*+C*+G*+C

228
A34004M
CCGACCGAGAGCAACTC
+C*+C*+G*A*C*C*G*A*G*A*G*C*A*A*+C*+T*+C

229
A34005M
AAACCTAGGCCGACCGA
+A*+A*+A*C*C*T*A*G*G*C*C*G*A*C*+C*+G*+A

230
A34006M
AAACCTAGGCCGACCG
+A*+A*+A*C*C*T*A*G*G*C*C*G*A*+C*+C*+G

231
A34007M
AAAACCTAGGCCGACCG
+A*+A*+A*A*C*C*T*A*G*G*C*C*G*A*+C*+C*+G

232
A34008M
CCGTGCGCAGAAAACCT
+C*+C*+G*T*G*C*G*C*A*G*A*A*A*A*+C*+C*+T

233
A34009M
AACTCCGTGCGCAGAAA
+A*+A*+C*T*C*C*G*T*G*C*G*C*A*G*+A*+A*+A

234
A34010M
TACCGCTTCGGCTCTAG
+T*+A*+C*C*G*C*T*T*C*G*G*C*T*C*+T*+A*+G

235
A34011M
TACCGCTTCGGCTCTA
+T*+A*+C*C*G*C*T*T*C*G*G*C*T*+C*+T*+A

236
A34012M
GTACCGCTTCGGCTCTA
+G*+T*+A*C*C*G*C*T*T*C*G*G*C*T*+C*+T*+A

237
A34013M
AGCCGTAACCTAGAAGG
+A*+G*+C*C*G*T*A*A*C*C*T*A*G*A*+A*+G*+G

238
A34014M
CTCCTTCGCTTCTTGCG
+C*+T*+C*C*T*T*C*G*C*T*T*C*T*T*G*+C*+G

239
A34015M
CAACAGTCCGTCCATTT
+C*+A*+A*C*A*G*T*C*C*G*T*C*C*A*+T*+T*+T

240
A34016M
TCAACAGTCCGTCCATT
+T*+G*+A*A*C*A*G*T*C*C*G*T*C*C*+A*+T*+T

241
A34017M
GTACTTCAACAGTCCGT
+G*+T*+A*C*T*T*C*A*A*C*A*G*T*C*+C*+G*+T

242
A34018M
GTACTTCAACAGTCCG
+G*+T*+A*C*T*T*C*A*A*C*A*G*T*+C*+C*+G

243
A34019M
GGTACTTCAACAGTCCG
+G*+G*+T*A*C*T*T*C*A*A*C*A*G*T*+C*+C*+G

244
A34020HM
ATCATGGCGTGAACTGT
+A*+T*C*A*T*G*G*C*G*T*G*A*A*C*+T*+G*+T

245
A34021M
CGCTGTTGTCGTTTCTC
+C*+G*+C*T*G*T*T*G*T*C*G*T*T*T*+C*+T*+C

246
A34022M
GTTTACGCTGTTGTCG
+G*+T*+T*T*A*C*G*C*T*G*T*T*G*+T*+C*+G

247
A34023M
TCACGTTTACGCTGTTG
+T*+G*+A*C*G*T*T*T*A*C*G*C*T*G*+T*+T*+G

248
A34024M
TATCAOGTTTACGCTGT
+T*+A*+T*C*A*C*G*T*T*T*A*C*G*C*+T*+G*+T

249
A34025HM
TGTAAGTTGCTCGGTGG
+T*+G*+T*A*A*G*T*T*G*C*T*C*G*G*+T*+G*+G

250
A34026M
AGTTGTAAGTTGCTCGG
+A*+G*+T*T*G*T*A*A*G*T*T*G*C*T*+C*+G*+G

251
A34027M
GAAAATATTGAGCGATC
+G*+A*+A*A*A*T*A*T*T*G*A*G*C*G*+A*+T*+C

252
A34028HM
GTTGATTACGGCTAA
+G*+T*+T*G*A*T*T*A*C*G*G*C*+T*+A*+A

253
A34029HM
GGTTGATTACGGCTAA
+G*+G*+T*T*G*A*T*T*A*C*G*G*C*+T*+A*+A

254
A34030M
AGGTTGATTACGGCTAA
+A*+G*+G*T*T*G*A*T*T*A*C*G*G*C*+T*+A*+A

255
A34031M
ATCATCGATATAAGGTT
+A*+T*+C*A*T*C*G*A*T*A*T*A*A*G*+G*+T*+T

256
A34032M
GTAATAGATGGCTCGGC
+G*+T*+A*A*T*A*G*A*T*G*G*C*T*C*+G*+G*+C

257
A34033M
TAACATCGGTGGCACTT
+T*+A*+A*C*A*T*C*G*G*T*G*G*C*A*+C*+T*+T

258
A34034M
GATCATTCTCGTTCAAT
+G*+A*+T*C*A*T*T*C*T*C*G*T*T*C*+A*+A*+T

259
A34035M
AAGGTCGTGCATAAGAT
+A*+A*+G*G*T*C*G*T*G*C*A*T*A*A*+G*+A*+T

260
A34036M
AAGGTCGTGCATAAGA
+A*+A*+G*G*T*C*G*T*G*C*A*T*A*+A*+G*+A

261
A34037M
TCTAAGGTCGTGCATAA
+T*+G*+T*A*A*G*G*T*C*G*T*G*C*A*+T*+A*+A

262
A34038M
TCTAAGGTCGTGCATA
+T*+C*+T*A*A*G*G*T*C*G*T*G*C*+A*+T*+A

263
A34039M
GGTCTAAGGTCGTGCAT
+G*+G*+T*C*T*A*A*G*G*T*C*G*T*G*+C*+A*+T

264
A34040M
TCCGTCATATGCTCAAA
+T*+G*+C*G*T*C*A*T*A*T*G*C*T*C*+A*+A*+A

265
A34041M
CTCCGTCATATGCTCAA
+C*+T*+C*C*G*T*C*A*T*A*T*G*C*T*+C*+A*+A

266
A34042M
TAACCGGAGGTCTCCAC
+T*+A*+A*C*C*G*G*A*G*G*T*C*T*C*C*+A*+C

267
A34043M
AGATCATACGTCATTCA
+A*+G*+A*T*C*A*T*A*C*G*T*C*A*T*+T*+C*+A

268
A34044M
ACAGATCATACGTCATT
+A*+C*+A*G*A*T*C*A*T*A*C*G*T*C*+A*+T*+T

269
A34045M
TGTACATGGCCGAGCAT
+T*+G*+T*A*C*A*T*G*G*C*C*G*A*G*+C*+A*+T

270
A34046HM
CGTTTGGTAAAGGCTAT
+C*+G*+T*T*T*G*G*T*A*A*A*G*G*C*+T*+A*+T

271
A34047M
CCTTCTACCGGCGTGG
+C*+C*+T*T*C*T*A*C*C*G*G*C*G*+T*+G*+G

272
A34048M
CCTTCTACCGGCGTG
+C*+C*+T*T*C*T*A*C*C*G*G*C*+G*+T*+G

273
A34049M
CCTCCTTCTACCGGCG
+C*+C*+T*C*C*T*T*C*T*A*C*C*G*+G*+C*+G

274
A34050Mi
GGACATTGGCTCAATC
+G*+G*+A*C*A*T*T*G*G*C*T*C*A*+A*+T*+C

275
A34051Mi
ACGCATCTGAGTACTTG
+A*+C*+G*C*A*T*C*T*G*A*G*T*A*C*+T*+T*+G

276
A34052Mi
TTGCTTACGCATCTGAG
+T*+T*+G*C*T*T*A*C*G*C*A*T*C*T*+G*+A*+G

277
A34053Mi
ACGAGGTTAAGAAGGTG
+A*+C*+G*A*G*G*T*T*A*A*G*A*A*G*+G*+T*+G

278
A34054Mi
TACCAAACTCCGAGTGT
+T*+A*+C*C*A*A*A*C*T*C*C*G*A*G*+T*+G*+T

279
A34055Mi
TGATTGATTCCGAGAT
+T*+G*+A*T*T*G*A*T*T*C*C*G*A*+G*+A*+T

280
A34056Mi
ACGTAGCTTACATAAGT
+A*+C*+G*T*A*G*C*T*T*A*C*A*T*A*+A*+G*+T

281
A34057Mi
AACGATATAGCTAGCAC
+A*+A*+C*G*A*T*A*T*A*G*C*T*A*G*+C*+A*+C

282
A34058Mi
GTAAGTAAGAATACGAT
+G*+T*+A*A*G*T*A*A*G*A*A*T*A*C*+G*+A*+T

283
A34059Mi
ACGATGGTAGGAATTTA
+A*+C*+G*A*T*G*G*T*A*G*G*A*A*T*+T*+T*+A

284
A34060Mi
CACGTTGGTCAATAGTA
+C*+A*+C*G*T*T*G*G*T*C*A*A*T*A*G*+T*+A

285
A34061Mi
GACGAGGAGTTAACAAA
+G*+A*+C*G*A*G*G*A*G*T*T*A*A*C*+A*+A*+A

286
A34062Mi
TAGTATATTAGCCACTC
+T*+A*+G*T*A*T*A*T*T*A*G*C*C*A*+C*+T*+C

287
A34063Mi
AGACGGATTGOTGATAT
+A*+G*+A*C*G*G*A*T*T*G*C*T*G*A*+T*+A*+T

288
A34064Mi
TGTAGACGGATTGCTGA
+T*+G*+T*A*G*A*C*G*G*A*T*T*G*C*+T*+G*+A

289
A34065Mi
GTTATGTAGACGGATT
+G*+T*+T*A*T*G*T*A*G*A*C*G*G*+A*+T*+T

290
A34066Mi
CATGCGCATTAAAGAGT
+C*+A*+T*G*C*G*C*A*T*T*A*A*A*G*+A*+G*+T

291
A34067Mi
AATTGCTCTACGGCTAC
+A*+A*+T*T*G*C*T*C*T*A*C*G*G*C*+T*+A*+C

292
A34068Mi
ACCAATTGCTCTACGGC
+A*+C*+C*A*A*T*T*G*C*T*C*T*A*C*+G*+G*+C

293
A34069Mi
TTTCTTAGGAACCGGAT
+T*+T*+T*G*T*T*A*G*G*A*A*C*C*G*+G*+A*+T

294
A34070Mi
CATCGCTAGACGCCTCG
+C*+A*+T*C*G*C*T*A*G*A*C*G*C*C*+T*+O*+G

295
A34071Mi
AACAGCCTCATCGCTAG
+A*+A*+C*A*G*C*C*T*C*A*T*C*G*C*+T*+A*+G

296
A34072Mi
GACTTGTCGTCTGCAAA
+G*+A*C*T*T*G*T*C*G*T*C*T*G*C*+A*+A*+A

297
A34073Mi
GTAGTATACAGTTGGTG
+G*+T*+A*G*T*A*T*A*C*A*G*T*T*G*+G*+T*+G

298
A34074Mi
AACACCGCCTTAGTCAT
+A*+A*+C*A*C*C*G*C*C*T*T*A*G*T*+C*+A*+T

299
A34075Mi
ACCTTATAAGTTCGTAA
+A*+C*+C*T*T*A*T*A*A*G*T*T*C*G*+T*+A*+A

300
A34076Mi
GGCGAACCTTATAAGT
+G*+G*+C*G*A*A*C*C*T*T*A*T*A*+A*+G*+T

301
A34077Mi
GCTTGTGCGGTGCTCTC
+G*+C*+T*T*G*T*G*C*G*G*T*G*C*T*+C*+T*+C

302
A34078Mi
CCTCGATGTTAGATGTT
+C*+C*+T*C*G*A*T*G*T*T*A*G*A*T*+G*+T*+T

303
A34079Mi
ACTACTCGGATTTCACT
+A*+C*+T*A*C*T*C*G*G*A*T*T*T*C*+A*+C*+T

304
A34080Mi
TTCGGACTATTAAACCA
+T*+T*+G*G*G*A*C*T*A*T*T*A*A*A*+C*+C*+A

305
A34081Mi
TTCGCTCACAGCAGACC
+T*+T*C*G*C*T*C*A*C*A*G*C*A*G*A*+C*+C

306
A34082Mi
AGTATTAATATAGCGGT
+A*+G*+T*A*T*T*A*A*T*A*T*A*G*C*+G*+G*+T

307
A34083Mi
GCCGATGAATATTCAGT
+G*+C*+C*G*A*T*G*A*A*T*A*T*T*C*+A*+G*+T

308
A34084Mi
ATGTATTATGAGCCGAT
+A*+T*+G*T*A*T*T*A*T*G*A*G*C*C*+G*+A*+T

309
A34085Mi
GTGCAACAAGTGATTAC
+G*+T*+G*C*A*A*C*A*A*G*T*G*A*T*+T*+A*+C

310
A34086Mi
GTTAAGTTGGTCCAAGA
+G*+T*+T*A*A*G*T*T*G*G*T*C*C*A*+A*+G*+A

311
A34087Mi
TCGACTGATCTGTTCCA
+T*+C*+G*A*C*T*G*A*T*C*T*G*T*T*+C*+C*+A

312
A34088Mi
CCGTTCGACTGATCTGT
+C*+C*+G*T*T*C*G*A*C*T*G*A*T*C*+T*+G*+T

313
A34089Mi
TACACCGTTCGACTGA
+T*+A*+C*A*C*C*G*T*T*C*G*A*C*+T*+G*+A

314
A34090Mi
TTTCTACACCGTTCGA
+T*+T*+T*G*T*A*C*A*C*C*G*T*T*+C*+G*+A

315
A34091Mi
TCGGCAAACCATTCACT
+T*+C*+G*G*C*A*A*A*C*C*A*T*T*C*+A*+C*+T

316
A34092Mi
GCTAAGTTAACGCTTCC
+G*+C*+T*A*A*G*T*T*A*A*C*G*C*T*+T*+C*+C

317
A34093Mi
TGGTCTATGCTAGAGGT
+T*+G*+G*T*C*T*A*T*G*C*T*A*G*A*+G*+G*+T

318
A34094Mi
CTCGTGGTATGACTAAG
+C*+T*+C*G*T*G*G*T*A*T*G*A*C*T*+A*+A*+G

319
A34095Mi
AAGCATCCGACCTTTGG
+A*+A*+G*C*A*T*C*C*G*A*C*C*T*T*+T*+G*+G

320
A34096Mi
TTCGGTAGCTAGTCTGC
+T*+T*+C*G*G*T*A*G*C*T*A*G*T*C*+T*+G*+C

321
A34097Mi
GGAAGTGGTCCACGATA
+G*+G*+A*A*G*T*G*G*T*C*C*A*C*G*+A*+T*+A

322
A34098Mi
AGGCGACGGAGGAGTTA
+A*+G*+G*C*G*A*C*G*G*A*G*G*A*G*+T*+T*+A

323
A34099Mi
TATACTAACGCAGATCC
+T*+A*+T*A*C*T*A*A*C*G*C*A*G*A*+T*+C*+C

324
A34100Mi
ATATACTAACGCAGATC
+A*+T*+A*T*A*C*T*A*A*C*G*C*A*G*+A*+T*+C

325
A34101Mi
GTAGCAGCGTGTACATT
+G*+T*+A*G*C*A*G*C*G*T*G*T*A*C*+A*+T*+T

326
A34102Mi
AGTGGTAGCAGCGTGTA
+A*+G*+T*G*G*T*A*G*C*A*G*C*G*T*G*+T*+A

327
A34103Mi
CTCTACTATCGCACCAG
+C*+T*+C*T*A*C*T*A*T*C*G*C*A*C*+C*+A*+G

328
A34104Mi
GCTCTACTATCGCACC
+C*+C*+T*C*T*A*C*T*A*T*C*G*C*+A*+C*+C

222
Control

+C*+G*+T*T*T*A*G*G*C*T*A*T*G*T*A*+C*+T*+T

Oligo

(Neg1)

The oligonucleotide of the present invention inhibits for example at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of MTDH expression such as the, e.g., human, rat or murine, MTDH expression. The inhibition of the MTDH expression is determined compared to an untreated cell, tissue, organ or subject, i.e., an untreated control. The untreated cell, tissue, organ or subject is the same type of cell, tissue, organ or subject as the cell, tissue, organ or subject to which an oligonucleotide of the present invention was added, i.e., a corresponding untreated cell, tissue, organ or subject which is for example a corresponding untreated control. This belongs to standard proceedings of a skilled person as shown in the examples. The oligonucleotides of the present invention are for example active and inhibit expression for example in a cell, tissue, organ, or a subject. The oligonucleotide of the present invention inhibits the expression of MTDH at a nanomolar or micromolar concentration for example in a concentration of 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 nM, or 1, 10 or 100 μM.

The oligonucleotide of the present invention is for example used in a concentration of 1, 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500, or 740 nM, or 1, 2.2, 3, 5, 6.6 or 10 μM.

In some embodiments the present invention refers to a pharmaceutical composition comprising an oligonucleotide of the present invention and a pharmaceutically acceptable carrier, excipient and/or dilutant. The pharmaceutical composition further comprises for example a chemotherapeutic, another disease specific active agent such as insulin, angiotensin-converting enzyme inhibitor, angiotensin receptor blocker, another oligonucleotide, an antibody, a peptide-based therapeutic, a protein-based therapeutic, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe and/or a small molecule which is for example effective in tumor treatment, or treatment of a kidney disease such as diabetic nephropathy.

In some embodiments, the oligonucleotide or the pharmaceutical composition of the present invention is for use in a method of preventing and/or treating a disorder. In some embodiments, the use of the oligonucleotide or the pharmaceutical composition of the present invention in a method of preventing and/or treating a disorder is combined with radiotherapy. The radiotherapy may be further combined with a chemotherapy (e.g., platinum, gemcitabine). The disorder is for example characterized by an MTDH imbalance, i.e., the MTDH level is increased in comparison to the level in a normal, healthy cell, tissue, organ or subject or MTDH expression for example is involved in the induction of the disease and/or mediates resistance to another therapy. The MTDH level is for example increased by an increased MTDH expression and activity, respectively. The MTDH level can be measured by any standard method such as immunohistochemistry, western blot, quantitative real time PCR or QuantiGene assay known to a person skilled in the art.

An oligonucleotide or a pharmaceutical composition of the present invention is for example administered locally or systemically for example orally, sublingually, nasally, inhaled, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumoral, intrathecal, transdermal, and/or rectal. Alternatively or in combination ex vivo treated immune cells are administered. The oligonucleotide is administered alone or in combination with another antisense oligonucleotide of the present invention and optionally in combination with another compound such as another oligonucleotide, an antibody, a carbohydrate-modified antibody, a peptide-based therapeutic, a protein-based therapeutic, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a small molecule and/or a chemotherapeutic (e.g., platinum, gemcitabine) and/or another disease specific agent such as insulin, angiotension-converting enzyme inhibitor, and/or angiotensin receptor blocker. In some embodiments, the other oligonucleotide (i.e., not being part of the present invention), the antibody, a peptide-based therapeutic, a protein-based therapeutic, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, and/or the small molecule are effective in preventing and/or treating diabetes kidney disease such as diabetic nephropathy, artheriosclerosis, a nephrological disorder and/or cancer. An oligonucleotide or a pharmaceutical composition of the present invention is for example used in a method of preventing and/or treating a solid tumor or a hematologic tumor. Examples of cancers preventable and/or treatable by use of the oligonucleotide or pharmaceutical composition of the present invention are breast cancer, lung cancer, malignant melanoma, lymphoma, skin cancer, bone cancer, prostate cancer, liver cancer, brain cancer, cancer of the larynx, gall bladder, pancreas, testicular, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, reticulum cell sarcoma, liposarcoma, myeloma, giant cell tumor, small-cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, intestinal ganglioneuromas, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiform a, leukemia, or epidermoid carcinoma.

Another example of a disease preventable and/or treatable by use of the oligonucleotide or pharmaceutical composition of the present invention other than cancer is for example diabetic, nephropathy.

In some embodiments two or more oligonucleotides of the present invention are administered together, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. In other embodiments, one or more oligonucleotides of the present invention are administered together with another compound such as another oligonucleotide (i.e., not being part of the present invention), an antibody, a peptide-based therapeutic, a protein-based therapeutic, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe, a small molecule and/or a chemotherapeutic, at the same time point for example in a pharmaceutical composition or separately, or on staggered intervals. In some embodiments of these combinations, the antisense oligonucleotide inhibits the expression and activity, respectively, of MTDH and the other oligonucleotide (i.e., not being part of the present invention), the antibody, a peptide-based therapeutic, a protein-based therapeutic, a HERA fusion protein, a ligand trap, a Fab fragment, a nanobody, a BiTe and/or small molecule inhibits (antagonist) MTDH or inhibits or stimulates (agonist) an immune suppressive factor and/or an immune stimulatory factor or inhibits another target that is involved in cancer progression and/or metastasis. The immune suppressive factor is for example selected from the group consisting of IDO1, IDO2, CTLA-4, PD-1, PD-L1, LAG-3, VISTA, A2AR, CD39, CD73, STAT3, TDO2, TIM-3, TIGIT, TGF-beta, BTLA, MICA, NKG2A, KIR, CD160, MTDH, Xbp1 and a combination thereof. The immune stimulatory factor is for example selected from the group consisting of 4-1BB, Ox40, KIR, GITR, CD27, 2B4 and a combination thereof. The factor involved in cancer progression and/or metastasis is for example selected from the group consisting of SND1, HER-2, BRAF, VEGF, EGFR1, EGFR2, BCR/ABL, ABL, MET, ALK, JAK2, BTK, miR-223, CCL18, CCL20, Lcn2, CCL5/CCR9, DDR2, PHD2, IL6, SDF-1/CXCL12 and a combination thereof.

The immune suppressive factor is a factor whose expression and/or activity is for example increased in a cell, tissue, organ or subject. The immune stimulatory factor is a factor whose level is increased or decreased in a cell, tissue, organ or subject depending on the cell, tissue, organ or subject and its individual conditions. The factor involved in cancer progression and/or metastasis is a factor whose level is increased or decreased in a cell, tissue, organ or subject depending on the cell, tissue, organ or subject and its individual conditions in comparison to a healthy subject or is for example involved in the induction of the disease and/or mediates resistance to another therapy.

An antibody in combination with the oligonucleotide or the pharmaceutical composition of the present invention is for example an anti-PD-1 antibody, an anti-PD-L1 antibody, or a bispecific antibody. A small molecule in combination with the oligonucleotide or the pharmaceutical composition of the present invention are for example Sunitinib, Alecitinib, Afatinib, Ibrutinib, Imatinib, Lenvatinib, or Epacadostat. A chemotherapy in combination with the oligonucleotide or the pharmaceutical composition of the present invention is for example platinum or gemcitabine.

A subject of the present invention is for example a mammalian such as a dog, cat, horse, cow, pig etc., a bird or a fish.

EXAMPLES

The following examples illustrate different embodiments of the present invention, but the invention is not limited to these examples. The following experiments are performed on cells endogenously expressing MTDH, i.e., the cells do not represent an artificial system comprising transfected reporter constructs. Such artificial systems generally show a higher degree of inhibition and lower IC₅₀values than endogenous systems which are closer to therapeutically relevant in vivo systems. Further, in the following experiments no transfecting agent is used, i.e., gymnotic delivery is performed. Transfecting agents are known to increase the activity of an oligonucleotide which influences the IC₅₀value (see for example Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, No. 5, 2012). As artificial systems using a transfecting agent are hardly or impossible to translate into therapeutic approaches and no transfection formulation has been approved so far for oligonucleotides, the following experiments are performed without any transfecting agent.

Example 1: Efficacy Screen of MTDH Antisense Oligonucleotides in Human Cancer Cell Lines

In order to investigate the knockdown efficacy of the in silico designed MTDH antisense oligonucleotides, efficacy screening experiments were performed in two cell lines, namely EFO-21 and SKOV-3. Therefore, cells were treated with the respective antisense oligonucleotide at a concentration of 5 μM for three days without the use of a transfection reagent. Cells were lysed after three days treatment period, MTDH and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the MTDH expression values were normalized to HPRT1 values. As shown in FIG. 1A) and Table 4, treatment of EFO-21 cells with 35 of 104 (34%) antisense oligonucleotides resulted in a knockdown efficacy of >80% (represented by a residual MTDH mRNA expression of <0.2 compared to mock-treated cells). Treatment with the control oligonucleotide neg1 had no relevant impact on MTDH mRNA expression. As shown in FIG. 1 B) and Table 5, treatment of SKOV-3 cells with nine of 104 (9%) antisense oligonucleotides resulted in a knockdown efficacy of >70% (represented by a residual MTDH mRNA expression of <0.3 compared to mock-treated cells). Treatment with the control oligo neg1 had no relevant impact on MTDH mRNA expression. Tables 4 and 5 are shown in the following:

TABLE 4

List of the mean MTDH mRNA expression values in antisense

oligonucleotide-treated EFO-21 cells compared to mock-

treated cells. Expression values are normalized to HPRT1.

Residual MTDH mRNA expression

ASO
(compared to mock-treated cells)
SEQ ID NO.

A34011HM
0.04
13

A34062Hi
0.05
64

A34082Hi
0.05
84

A34084Hi
0.05
86

A34048Hi
0.05
50

A34053Hi
0.08
55

A34061Hi
0.08
63

A34088Hi
0.09
90

A34027H
0.09
29

A34077Hi
0.10
79

A34065Hi
0.11
67

A34012H
0.11
14

A34026H
0.11
28

A34079Hi
0.12
81

A34094Hi
0.12
96

A34044Hi
0.13
46

A34052Hi
0.13
54

A34060Hi
0.13
62

A34095Hi
0.14
97

A34074Hi
0.14
76

A34018H
0.14
20

A34019H
0.14
21

A34047Hi
0.15
49

A34086Hi
0.15
88

A34068Hi
0.15
70

A34073Hi
0.16
75

A34071Hi
0.16
73

A34041Hi
0.17
43

A34025HM
0.17
27

A34076Hi
0.17
78

A34022H
0.19
24

A34050Hi
0.19
52

A34072Hi
0.19
74

A34066Hi
0.20
68

A34064Hi
0.20
66

A34038Hi
0.22
40

A34054Hi
0.22
56

A34083Hi
0.22
85

A34100Hi
0.23
102

A34030H
0.23
32

A34021H
0.23
23

A34078Hi
0.23
80

A34045Hi
0.23
47

A34039Hi
0.24
41

A34058Hi
0.24
60

A34029H
0.25
31

A34081Hi
0.25
83

A34067Hi
0.27
69

A34043Hi
0.28
45

A34091Hi
0.28
93

A34010H
0.29
12

A34016H
0.29
18

A34023H
0.30
25

A34031H
0.31
33

A34096Hi
0.31
98

A34098Hi
0.32
100

A34024H
0.32
26

A34090Hi
0.33
92

A34092Hi
0.34
94

A34080Hi
0.34
82

A34008H
0.34
10

A34032H
0.36
34

A34046Hi
0.36
48

A34034H
0.36
36

A34075Hi
0.37
77

A34049Hi
0.37
51

A34017H
0.37
19

A34087Hi
0.38
89

A34035Hi
0.39
37

A34057Hi
0.40
59

A34104Hi
0.42
106

A34056Hi
0.43
58

A34070Hi
0.46
72

A34059Hi
0.50
61

A34040Hi
0.51
42

A34013H
0.51
15

A34089Hi
0.52
91

A34037Hi
0.53
39

A34028H
0.54
30

A34055Hi
0.54
57

A34042Hi
0.54
44

A34009H
0.55
11

A34036Hi
0.58
38

A34007H
0.60
9

A34003H
0.63
5

A34069Hi
0.63
71

A34001H
0.63
3

A34033H
0.64
35

A34014H
0.64
16

A34063Hi
0.67
65

A34093Hi
0.71
95

A34051Hi
0.72
53

A34015H
0.74
17

A34020H
0.74
22

A34006H
0.76
8

A34103Hi
0.77
105

A34097Hi
0.77
99

A34102Hi
0.82
104

A34101Hi
0.84
103

A34002H
0.86
4

A34004H
0.93
6

A34005H
1.10
7

A34085Hi
1.18
87

Control oligo
1.19
222

(Neg1)

A34099Hi
1.20
101

TABLE 5

List of the mean MTDH mRNA expression values in antisense

oligonucleotide-treated SKOV-3 cells compared to mock-

treated cells. Expression values are normalized to HPRT1.

Residual MTDH mRNA expression

ASO
(compared to mock-treated cells)
SEQ ID NO.

A34011HM
0.10
13

A34026H
0.18
28

A34084Hi
0.19
86

A34025HM
0.22
27

A34082Hi
0.25
84

A34062Hi
0.26
64

A34077Hi
0.28
79

A34019H
0.28
21

A34012H
0.30
14

A34060Hi
0.31
62

A34052Hi
0.31
54

A34080Hi
0.32
82

A34061Hi
0.32
63

A34047Hi
0.33
49

A34018H
0.33
20

A34073Hi
0.33
75

A34058Hi
0.33
60

A34083Hi
0.33
85

A34044Hi
0.34
46

A34078Hi
0.35
80

A34100Hi
0.35
102

A34027H
0.35
29

A34079Hi
0.35
81

A34076Hi
0.35
78

A34065Hi
0.36
67

A34092Hi
0.37
94

A34021H
0.37
23

A34043Hi
0.38
45

A34030H
0.38
32

A34049Hi
0.38
51

A34057Hi
0.39
59

A34074Hi
0.39
76

A34048Hi
0.39
50

A34046Hi
0.40
48

A34072Hi
0.41
74

A34029H
0.42
31

A34038Hi
0.42
40

A34091Hi
0.43
93

A34023H
0.43
25

A34064Hi
0.43
66

A34104Hi
0.44
106

A34081Hi
0.45
83

A34053Hi
0.45
55

A34031H
0.45
33

A34045Hi
0.45
47

A34068Hi
0.45
70

A34071Hi
0.47
73

A34020H
0.47
22

A34036Hi
0.47
38

A34054Hi
0.47
56

A34066Hi
0.48
68

A34067Hi
0.48
69

A34086Hi
0.49
88

A34050Hi
0.49
52

A34024H
0.49
26

A34022H
0.49
24

A34032H
0.50
34

A34051Hi
0.50
53

A34098Hi
0.51
100

A34013H
0.51
15

A34007H
0.51
9

A34056Hi
0.51
58

A34070Hi
0.51
72

A34016H
0.53
18

A34094Hi
0.54
96

A34004H
0.54
6

A34085Hi
0.56
87

A34041Hi
0.57
43

A34037Hi
0.58
39

A34010H
0.59
12

A34039Hi
0.60
41

A34017H
0.61
19

A34090Hi
0.62
92

A34028H
0.62
30

A34033H
0.63
35

A34088Hi
0.64
90

A34093Hi
0.65
95

A34009H
0.67
11

A34097Hi
0.68
99

A34095Hi
0.69
97

A34002H
0.71
4

A34069Hi
0.71
71

A34008H
0.72
10

A34035Hi
0.72
37

A34102Hi
0.73
104

A34040Hi
0.73
42

A34003H
0.73
5

A34059Hi
0.73
61

A34015H
0.74
17

A34001H
0.74
3

A34099Hi
0.75
101

A34063Hi
0.76
65

A34014H
0.77
16

A34103Hi
0.80
105

A34055Hi
0.84
57

A34005H
0.84
7

A34042Hi
0.84
44

A34006H
0.86
8

Control oligo
0.87
222

(Neg1)

A34087Hi
0.87
89

A34075Hi
0.88
77

A34034H
0.88
36

A34101Hi
0.94
103

A34089Hi
1.02
91

A34096Hi
1.02
98

Example 2: Determination of IC₅₀Values of Selected MTDH Antisense Oligonucleotides in Human Cancer Cells

The dose-dependent knockdown of MTDH mRNA expression by MTDH antisense oligonucleotides in EFO-21 cells was investigated and the respective IC₅₀values were calculated. Therefore, EFO-21 cells were treated for three days with the respective antisense oligonucleotide at the following concentrations without the use of a transfection reagent: 6 μM, 1.5 μM, 375 nM, 94 nM, 24 nM, 6 nM, 1.5 nM. After the treatment period, cells were lysed, MTDH and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the MTDH expression values were normalized to HPRT1 values. Residual MTDH mRNA expression as compared to mock-treated cells is depicted. A dose-dependent knockdown of MTDH with all tested antisense oligonucleotides was observed (FIG. 2 and Table 6) with IC₅₀values in the submicromolar range (Table 6). Table 6 is presented in the following:

TABLE 6

Dose-dependent inhibition of MTDH mRNA expression in EFO-21 cells by

selected MTDH antisense oligonucleotides and respective IC₅₀values.

IC₅₀
Residual MTDH mRNA expression

ASO
(nM)
6 μM
1.5 μM
375 nM
94 nM
24 nM
6 nM
1.5 nM

A34011HM
180.1
0.016
0.102
0.288
0.711
0.948
1.008
1.342

A34018H
975.2
0.102
0.391
0.516
0.705
0.808
0.904
1.111

A34019H
742.6
0.098
0.282
0.538
0.775
0.819
0.955
0.982

A34025HM
586.1
0.131
0.371
0.636
0.967
1.197
1.052
0.873

A34027H
375.4
0.134
0.332
0.558
0.898
1.240
1.178
1.062

A34052Hi
1041
0.169
0.413
0.915
1.052
1.381
1.125
1.005

A34062Hi
346.6
0.102
0.206
0.557
0.731
1.121
1.188
1.043

A34077Hi
687.3
0.122
0.343
0.703
1.008
1.238
1.101
1.155

Example 3: Efficacy Screen of MTDH Antisense Oligonucleotides in Mouse Cancer Cell Lines

In order to investigate the knockdown efficacy of the in silico designed MTDH antisense oligonucleotide, efficacy screening experiments were performed in two cell lines, namely Renca and 4T1. Therefore, cells were treated with the respective antisense oligonucleotide at a concentration of 5 μM for three days without the use of a transfection reagent. Cells were lysed after three days treatment period, MTDH and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the MTDH expression values were normalized to HPRT1 values. As shown in FIG. 3A) and Table 7, treatment with 21 of 104 (20%) antisense oligonucleotides resulted in a knockdown efficacy of >80% (represented by a residual MTDH mRNA expression of <0.2 compared to mock-treated cells). Treatment with the control oligo neg1 had no relevant impact on MTDH mRNA expression. As shown in FIG. 3 B) and Table 8, treatment with 40 of 104 (38%) antisense oligonucleotides resulted in a knockdown efficacy of >80% (represented by a residual MTDH mRNA expression of <0.2 compared to mock-treated cells). Treatment with the control oligo neg1 had no relevant impact on MTDH mRNA expression. Tables 7 and 8 are shown in the following:

TABLE 7

List of the mean MTDH mRNA expression values in antisense

oligonucleotide-treated Renca cells compared to mock-treated

cells. Expression values are normalized to HPRT1.

Residual MTDH mRNA expression

ASO
(compared to mock-treated cells)
SEQ ID NO.

A34073Mi
0.06
297

A34095Mi
0.07
319

A34039M
0.09
263

A34029HM
0.09
253

A34082Mi
0.10
306

A34050Mi
0.10
274

A34023M
0.11
247

A34030M
0.12
254

A34064Mi
0.12
288

A34036M
0.12
260

A34026M
0.13
250

A34052Mi
0.13
276

A34025HM
0.13
249

A34097Mi
0.13
321

A34068Mi
0.14
292

A34063Mi
0.15
287

A34051Mi
0.16
275

A34038M
0.16
262

A34055Mi
0.18
279

A34065Mi
0.19
289

A34022M
0.19
246

A34021M
0.20
245

A34084Mi
0.21
308

A34037M
0.21
261

A34035M
0.22
259

A34028HM
0.22
252

A34059Mi
0.22
283

A34088Mi
0.23
312

A34102Mi
0.24
326

A34087Mi
0.25
311

A34096Mi
0.27
320

A34090Mi
0.29
314

A34034M
0.30
258

A34041M
0.32
265

A34077Mi
0.34
301

A34040M
0.35
264

A34080Mi
0.35
304

A34061Mi
0.36
285

A34094Mi
0.36
318

A34101Mi
0.36
325

A34060Mi
0.37
284

A34093Mi
0.38
317

A34086Mi
0.39
310

A34010M
0.39
234

A34091Mi
0.41
315

A34103Mi
0.44
327

A34072Mi
0.47
296

A34089Mi
0.47
313

A34011M
0.47
235

A34009M
0.47
233

A34081Mi
0.48
305

A34008M
0.48
232

A34074Mi
0.50
298

A34066Mi
0.51
290

A34079Mi
0.52
303

A34057Mi
0.53
281

A34083Mi
0.55
307

A34054Mi
0.55
278

A34012M
0.55
236

A34046HM
0.56
270

A34031M
0.57
255

A34053Mi
0.58
277

A34024M
0.58
248

A34002M
0.59
226

A34085Mi
0.59
309

A34076Mi
0.61
300

A34003M
0.62
227

A34092Mi
0.64
316

A34067Mi
0.67
291

A34104Mi
0.69
328

A34075Mi
0.71
299

A34033M
0.71
257

A34056Mi
0.74
280

A34098Mi
0.75
322

A34027M
0.76
251

A34078Mi
0.77
302

A34020HM
0.78
244

A34017M
0.78
241

A34062Mi
0.79
286

A34004M
0.79
228

A34019M
0.80
243

A34032M
0.80
256

A34013M
0.81
237

A34005M
0.81
229

A34071Mi
0.82
295

A34069Mi
0.84
293

A34100Mi
0.87
324

A34042M
0.87
266

A34048M
0.89
272

A34001M
0.90
225

A34043M
0.90
267

A34006M
0.91
230

A34014M
0.91
238

A34044M
0.92
268

A34007M
0.93
231

A34058Mi
0.99
282

A34016M
1.03
240

A34018M
1.03
242

A34047M
1.05
271

A34070Mi
1.07
294

A34049M
1.07
273

A34099Mi
1.09
323

A34015M
1.11
239

A34045M
1.15
269

Control oligo
1.33
222

(Neg1)

TABLE 8

List, of the mean MTDH mRNA expression values in antisense

oligonucleotide-treated 4T1 cells compared to mock-treated

cells. Expression values are normalized to HPRT1.

Residual MTDH mRNA expression

ASO
(compared to mock-treated cells)
SEQ ID NO.

A34095Mi
0.01
319

A34026M
0.02
250

A34073Mi
0.02
297

A34022M
0.02
246

A34025HM
0.03
249

A34023M
0.03
247

A34050Mi
0.04
274

A34082Mi
0.04
306

A34039M
0.04
263

A34052Mi
0.04
276

A34041M
0.04
265

A34087Mi
0.04
311

A34051Mi
0.05
275

A34036M
0.05
260

A34064Mi
0.05
288

A34029HM
0.06
253

A34021M
0.07
245

A34068Mi
0.07
292

A34055Mi
0.07
279

A34063Mi
0.07
287

A34088Mi
0.07
312

A34090Mi
0.08
314

A34038M
0.09
262

A34028HM
0.10
252

A34059Mi
0.11
283

A34035M
0.11
259

A34065Mi
0.11
289

A34097Mi
0.11
321

A34093Mi
0.12
317

A34084Mi
0.13
308

A34101Mi
0.13
325

A34037M
0.13
261

A34040M
0.14
264

A34102Mi
0.14
326

A34030M
0.15
254

A34089Mi
0.15
313

A34043M
0.16
267

A34103Mi
0.17
327

A34024M
0.18
248

A34096Mi
0.18
320

A34046HM
0.21
270

A34086Mi
0.22
310

A34061Mi
0.23
285

A34008M
0.23
232

A34060Mi
0.23
284

A34077Mi
0.26
301

A34034M
0.27
258

A34048M
0.28
272

A34011M
0.28
235

A34080Mi
0.29
304

A34074Mi
0.34
298

A34057Mi
0.35
281

A34083Mi
0.36
307

A34016M
0.36
240

A34044M
0.37
268

A34067Mi
0.37
291

A34015M
0.37
239

A34094Mi
0.38
318

A34032M
0.42
256

A34085Mi
0.42
309

A34018M
0.43
242

A34066Mi
0.44
290

A34091Mi
0.44
315

A34078Mi
0.45
302

A34069Mi
0.48
293

A34017M
0.49
241

A34076Mi
0.50
300

A34092Mi
0.50
316

A34014M
0.52
238

A34010M
0.53
234

A34020HM
0.53
244

A34009M
0.54
233

A34033M
0.55
257

A34072Mi
0.57
296

A34031M
0.58
255

A34012M
0.59
236

A34003M
0.60
227

A34013M
0.61
237

A34054Mi
0.63
278

A34019M
0.64
243

A34049M
0.66
273

A34079Mi
0.67
303

A34075Mi
0.70
299

A34104Mi
0.70
328

A34002M
0.71
226

A34099Mi
0.74
323

A34053Mi
0.74
277

A34056Mi
0.76
280

A34042M
0.76
266

A34058Mi
0.77
282

A34100Mi
0.79
324

A34027M
0.80
251

A34045M
0.81
269

A34070Mi
0.84
294

A34098Mi
0.86
322

A34001M
0.87
225

A34062Mi
0.87
286

A34006M
0.94
230

A34007M
1.00
231

A34071Mi
1.06
295

A34005M
1.07
229

Control oligo
1.10
222

(Neg1)

A34004M
1.14
228

A34081Mi
1.15
305

A34047M
1.16
271

Example 4: Determination of IC₅₀Values of Selected MTDH Antisense Oligonucleotides in Mouse Cancer Cells

The dose-dependent knockdown of MTDH mRNA expression by MTDH antisense oligonucleotides in 4T1 cells was investigated and the respective IC₅₀values were calculated. Therefore, 4T1 cells were treated for three days with the respective antisense oligonucleotide at the following concentrations without the use of a transfection reagent: 6 μM, 1.5 μM, 375 nM, 94 nM, 24 nM, 6 nM, 1.5 nM. After the treatment period, cells were lysed, MTDH and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher) and the MTDH expression values were normalized to HPRT1 values. Residual MTDH mRNA expression as compared to mock-treated cells is depicted. We observed a dose-dependent knockdown of MTDH with all tested antisense oligonucleotides (FIG. 4 and Table 9) with IC₅₀values in the submicromolar range (Table 9). Table 9 is shown in the following:

TABLE 9

Dose-dependent inhibition of MTDH mRNA expression in 4T1 cells by selected

MTDH antisense oligonucleotides and respective IC₅₀values.

IC₅₀
Residual MTDH mRNA expression

ASO
(nM)
6 μM
1.5 μM
375 nM
94 nM
24 nM
6 nM
1.5 nM

A34022M
410
0.20
0.24
0.53
0.80
0.72
0.85
0.81

A34023M
341
0.21
0.27
0.61
1.11
1.11
1.16
n.d.

A34051Mi
164
0.12
0.24
0.38
0.55
0.76
0.94
0.78

A34052Mi
138
0.11
0.20
0.36
0.52
0.63
0.79
n.d.

A34055Mi
586
0.19
0.33
0.52
0.65
0.78
0.77
0.75

A34059Mi
239
0.13
0.24
0.45
0.67
0.91
1.02
0.84

A34068Mi
72
0.16
0.21
0.33
0.60
0.95
1.23
n.d.

A34082Mi
257
0.07
0.12
0.35
0.83
0.89
0.92
n.d.

Inhibitor of metadherin expression

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