NRP1-specific antisense oligonucleotides, and their use in the prevention and / or treatment of disease

Abstract

The present invention refers to oligonucleotides comprising 10 to 25 nucleotides, wherein at least one of the nucleotides is modified, and the oligonucleotide hybridizes with the pre-mRNA of neuropilin 1 (NRP1, CD304) of SEQ ID NO: 366 (GRCh38p13_Chr 10_33177492-33336262-1) or the mRNA of NRP1 of SEQ ID NO: 367 (RefSeq ID NM_003873.6). The present invention further relates to pharmaceutical compositions comprising such oligonucleotides. The pharmaceutical compositions and oligonucleotides are intended for use in methods of preventing and / or treating cancer, eye diseases, autoimmune disorders, and / or immune disorders.

Description

[Technical Field]

[0001] This invention refers to oligonucleotides, particularly antisense oligonucleotides, that comprise one or more modified nucleotides and hybridize with neuropilin (NRP) such as NRP1(CD304) premRNA or mRNA. Oligonucleotides inhibit the expression of NRP, NRP mRNA, and / or NRP premRNA and are used to prevent and / or treat cancer, eye diseases, autoimmune disorders, and / or immune disorders. [Background technology]

[0002] Neuropilin 1 (NRP1) is a multi-domain receptor involved in highly versatile signaling pathways that regulate cell migration, angiogenesis, cell survival, metastasis, and cell proliferation. Therefore, NRP1 has been shown to function as a co-receptor for a wide range of growth factor receptors (Prud'homme et al., Oncotarget, 2012, 3(9):921-939). Common binding partners of NRP1 include, for example, TGF-β receptors I and II, vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and plexin (semaphorin receptor) (Prud'homme et al., Oncotarget, 2012, 3(9):921-939). NRP1 expression is associated with regulatory T cells (T reg It has been reported in a wide variety of cells, including immune system cells (Prud'homme et al., Oncotarget, 2012, 3(9):921-939). Generally, neuropyrin is overexpressed in several human tumor types, including carcinoma, melanoma, glioblastoma, leukemia, and lymphoma. Overexpression of NRP1 correlates with more aggressive clinical tumor behavior (Prud'homme et al., Oncotarget, 2012, 3(9):921-939).

[0003] NRP1 has also been shown to be involved in vascular maturation. Previous studies have shown that immature blood vessels depend on and respond to vascular endothelial growth factor (VEGF), a common ligand for NRP1 (Pan et al., Cancer Cell, 2007, 11(1), 53-67). Therefore, inhibiting NRP1 expression prevents abnormal vascular maturation and forces them into an immature, VEGF-dependent state. Thus, combination therapy with anti-NRP1 and anti-VEGF is beneficial for the treatment of angiogenesis-related ocular diseases and tumors.

[0004] Functionally, NRP1 is associated with immunosuppression. Several studies have shown that regulatory T cells (T) reg ) shows that it expresses NRP1 on its surface (Hansen, W., Oncoimmunology, 2013, 2(2), e230399). VEGF-producing tumor cells exhibit NRP1 expression due to the interaction between NRP1 and VEGF. reg It attracts. VEGF promotes tumor angiogenesis, but T reg For example, it interferes with the antitumor immune response by secreting immunosuppressive cytokines (Hansen, et al., Oncoimmunology, 2013, 2(2), e230399). Inhibition of NRP1 affects the tumor microenvironment. reg It prevents infiltration and, therefore, improves the anti-tumor immune response.

[0005] Few antibodies have been developed to inhibit NRP1 activity. The anti-human NRP1 monoclonal antibody MNRP1685A (Genentech) specifically inhibits the VEGF-binding domain of NRP1. This antibody was used in a Phase I clinical trial to treat patients with progressive solid tumors (Weekes, et al., Investigational New Drug, 2014, 32(4), 653-660). However, relatively high concentrations and repeated administration of the antibody are required to successfully block NRP1.

[0006] Furthermore, the anti-VEGF antibody Aflibercept (Sanofi) is used as a common therapy for pathological retinal neovascularization, such as neovascular (wet) age-related macular degeneration (AMD), diabetic retinopathy, and retinopathy in prematurity. However, the application of Aflibercept is limited because it cannot inhibit the binding of non-classical ligands to NRP1, which acts as angiogenic growth factor (e.g., TGF-β, PDGF, semaphorin, HGF), and exhibits only low activity against mature blood vessels. Successfully blocking NRP1 activity requires relatively high concentrations and repeated administration via monthly intravitreous injections. Since these therapies are very inconvenient for patients, there is a need to develop improved therapies that allow for less frequent application.

[0007] Furthermore, one small molecule, EG00229 (Tocris), functions as a receptor antagonist for NRP1. EG00229 has been shown to inhibit the binding of VEGF-A to the b1 domain of NRP1, at least in vitro. EG00229 enhances the chemotherapy sensitivity of A549 cells (Jarvis et al., Journal of Medicinal Chemistry, 2010, 53(5), 2215-2226), but its clinical efficacy has not yet been determined in vivo.

[0008] US7,087,580 refers to oligonucleotides that hybridize with human neuropilin 1, including first-generation modifications and mutations such as substitutions, insertions, and deletions.

[0009] NRP1 contains several partially overlapping binding sites for different ligands and co-receptors. Conventional approaches using single antibodies and / or small molecules are unable to block all interaction sites of such multi-domain receptors, or block them very little. Antibody-based therapies require the administration of more than one antibody. Therefore, a safe and effective agent that simultaneously inhibits the complete function mediated by receptors such as NRP1 would be a crucial addition for the treatment of patients suffering from diseases or conditions affected by the activity of NRP1 and its pro-angiogenic ligands.

[0010] The oligonucleotides of the present invention have been highly successful in inhibiting the expression and activity of NRP1. The mode of action of oligonucleotides differs from that of antibodies or small molecules, and oligonucleotides offer significant advantages, for example, in the following respects: (i) Blocking each of the multiple functions and activities of the target, (ii) Due to their small molecular size, the penetration of tumor tissue within solid tumors, (iii) combinations of oligonucleotides with each other or with antibodies or small molecules, and (iv) Inhibition of intracellular effects that are not accessible by the antibody or cannot be inhibited via small molecules.

[0011] The oligonucleotides of the present invention are advantageous compared to previous generations of oligonucleotides due to their higher stability, stronger target affinity and potency, and independence from delivery reagents for achieving targeted suppression within cells. [Overview of the Initiative]

[0012] The present invention refers to oligonucleotides comprising or consisting of 10 to 25 nucleotides, wherein at least one nucleotide has a modification selected from the group consisting of cross-linked nucleic acids such as LNA, ENA, cET, 2'-fluoro-modified nucleotide, 2'O-methyl-modified nucleotide, 2'O-methoxyethyl-modified nucleotide, FANA, and combinations thereof, and the oligonucleotide hybridizes with the transcript or mRNA of neuropilin 1 (NRP1) of SEQ ID NO: 367 and / or the pre-mRNA of NRP1 of SEQ ID NO: 366. The modification is located in the 5' and / or 3' terminal sequences of the oligonucleotide, for example, a sequence of 5 nucleotides.

[0013] The oligonucleotides of the present invention hybridize outside or within the hybridization active region at positions 6450-6749, 12150-12449, 134250-134549, 36150-36449, 17550-17849, 138750-139049, 32550-32849, or combinations thereof. The oligonucleotides include, for example, SEQ ID NOs. 5, 120, 19, 163, 129, 364, 330, 158, 156, or combinations thereof, or consist thereof. For example, the oligonucleotides are: +A*+T*+A*T*T*T*A*G*G*T*C*C*A*+G*+C*+G(A15091HI; Sequence number 5), +T*+C*+G*T*T*G*G*A*A*A*T*G*C*C*A*T*+G*+C*+A(A15219HI; Sequence number 120), +T*+A*+C*A*T*G*G*T*A*A*C*G*C*+C*+T*+T(A15108HI; Sequence number 19), +A*+T*+A*T*T*T*A*G*G*T*C*C*+A*+G*+C*+G(A15486HI; Sequence number 5), +A*+T*A*T*T*T*A*G*G*T*C*C*+A*+G*+C*+G(A15487HI; Sequence number 5), +G*+G*+T*T*A*T*T*G*A*C*T*G*C*T*C*T*+C*+T*+A (A15262HI; SEQ ID NO: 163), +G*+T*+T*T*C*C*T*T*A*G*C*T*T*A*G*T*+G*+C*+C (A15228HI; SEQ ID NO: 129), +A*+A*T*+A*T*T*T*A*G*G*T*C*C*+A*G*+C*+G (A15466HI; SEQ ID NO: 364), +G*+T*+T*A*C*T*C*C*G*T*T*C*C*T*T*+C*+A*+G (A15430HI; SEQ ID NO: 330), +T*+A*+G*G*T*A*C*G*A*G*C*A*T*+C*+G*+G (A15257HI; SEQ ID NO: 158), +A*+A*+T*A*T*T*T*A*G*G*T*C*C*A*+G*+C*+G (A15465HI; SEQ ID NO: 364), +A*T*+A*T*T*T*A*G*G*T*C*C*A*+G*+C*+G (A15473HI; SEQ ID NO: 5), +T*+T*+C*G*T*A*T*T*T*A*A*A*C*T*C*+T*+A*+C (A15255HI; SEQ ID NO: 156) and selected from the group consisting of combinations thereof, wherein + represents an LNA nucleotide and * represents a phosphorothioate (PTO) bond between nucleotides or a mesyl-phosphoramidate or a combination thereof.

[0014] Furthermore, the oligonucleotides of the present invention have, for example, 80-99%, 85-98%, 90-95%, or 93% sequence identity to any one of the oligonucleotides of SEQ ID NO: 1 to SEQ ID NO: 364. The oligonucleotide contains, for example, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 nucleotides of any one of the oligonucleotides of SEQ ID NO: 1 to SEQ ID NO: 364, or consists of them. The oligonucleotides of the present invention inhibit the expression of NRP1, NRP1 mRNA, NRP1 pre-mRNA, or combinations thereof, for example, at nanomolar or micromolar concentrations.

[0015] The present invention further refers to a pharmaceutical composition comprising the oligonucleotide of the present invention and a pharmaceutically acceptable carrier, excipient, diluent, stimulant such as an adjuvant, or a combination thereof. Optionally, the pharmaceutical composition further comprises a therapeutic active agent such as a chemotherapeutic agent, another oligonucleotide, an antagonistic protein, an antibody, and / or a small molecule effective for the treatment of tumors or eye diseases.

[0016] The pharmaceutical composition and oligonucleotide of the present invention each inhibit the activity of a receptor, such as a growth receptor selected from the group consisting of, for example, TGF-β receptor I (TβRI), TGF-β receptor II (TβRII), VEGF, HGF, PDGF, and SEMA3 (Plexin), or a combination thereof, or inhibit the activity of a signaling factor such as, for example, p38MAPK, ERK1, ERK2, PI3K, Akt, NF-κB, pSMAD2, pSMAD3, Src, Pyk2, FAK, p-p130Cas, or a combination thereof. Furthermore, the pharmaceutical composition or oligonucleotide of the present invention inhibits, for example, the reg migration and / or function of T cells, myeloid-derived suppressor cells (MDSC), or any other immune cells, and combinations thereof to tumors.

[0017] The pharmaceutical compositions or oligonucleotides of the present invention are intended for use in the prevention and / or treatment of cancer, eye diseases, autoimmune disorders, and / or immune disorders. Eye diseases include, for example, age-related macular disease (AMD), diabetic retinopathy (DME), retinopathy of prematurity (Retinopathia praematurorum), or neovascular eye diseases such as corneal neovascularization. Cancers include, for example, bladder cancer, breast cancer, colorectal cancer, lung cancer, malignant melanoma, mesothelioma, lymphoma, skin cancer, bone cancer, prostate cancer, hepatocellular carcinoma, brain tumors, larynx, liver, gallbladder, pancreas, testes, rectum, parathyroid gland, thyroid gland, adrenal gland, nerve tissue, head and neck, colon, stomach, bronchi, kidney, basal cell carcinoma, neuroblastoma, squamous cell carcinoma, metastatic skin cancer, osteosarcoma, Ewing's sarcoma, reticular cell sarcoma, liposarcoma, leukemia, myeloma, giant cell tumor, small cell lung tumor, islet cell tumor, and primary encephaloma. These include ulcers, meningiomas, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy cell tumors, adenomas, hyperplasia, medullary carcinomas, enteric ganglia, Wilm's tumor, seminomas, ovarian tumors, leiomyomas, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, focal skin lesions, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumors, polycythemia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforme, leukemia, or epidermal carcinoma.

[0018] The pharmaceutical composition or oligonucleotide of the present invention is administered, for example, topically or systemically.

[0019] All documents cited or referenced herein ("Cited Documents"), and all documents cited or referenced in the Cited Documents, as well as instructions, descriptions, product specifications, and product sheets of any manufacturer of any product mentioned herein, are incorporated herein by reference and may be used in the practice of the Invention. More specifically, all referenced documents are incorporated by reference to the same extent that each individual document is specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawing]

[0020] [Figure 1] The RefSeq ID NM_003873.6 for NRP1 mRNA (SEQ ID NO: 367) is shown. [Figure 2] The results of screening for human NRP1-specific ASOs in SKOV-3 cells are shown. [Figure 3] This paper illustrates the screening results for human NRP1-specific ASOs in U87-MG cells. [Figure 4] The results of screening for human NRP1-specific ASOs in Panc1 cells are shown. [Figure 5] This paper illustrates the screening results for human NRP1-specific ASOs in CD4+ T cells. [Figure 6] This study illustrates concentration-dependent knockdown of NRP1 mRNA in SKOV-3 cells by a selected NRP1-specific ASO. [Figure 7] This study illustrates concentration-dependent knockdown of NRP1 mRNA in CD4+ T cells by a selected NRP1-specific ASO. [Figure 8] This report describes the monitoring of serological indicators for the acute toxicity of selected human NRP1-specific ASOs in Balb / c mice. [Figure 9] This study describes the knockdown of NRP1 mRNA in SKOV-3 cells by selected NRP1-specific ASOs. To compare the knockdown efficacy of ASOs with the same base sequence but different LNA modification patterns, SKOV-3 cells were treated with ASOs (A15486HI (SEQ ID NO: 5), A15479HI (SEQ ID NO: 5), A15487HI (SEQ ID NO: 5), A15091HI (SEQ ID NO: 5), and A15478HI (SEQ ID NO: 5) at a concentration of 5 μM for 3 days without the addition of transfection reagents. After the 3-day treatment period, the cells were lysed, and NRP1 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher). NRP1 expression values ​​were normalized to HPRT1 values ​​and compared with mock-treated samples. [Figure 10]This shows concentration-dependent knockdown of NRP1 mRNA in Panc1 cells using selected NRP1-specific ASOs (A15091HI (SEQ ID NO: 5), A15486HI (SEQ ID NO: 5), and A15487HI (SEQ ID NO: 5)). [Figure 11] This illustrates the knockdown of NRP1 mRNA in SKOV-3 cells by selected NRP1-specific ASOs (A15091HI (SEQ ID NO: 5), A15486HI (SEQ ID NO: 5), and A15487HI (SEQ ID NO: 5)). [Modes for carrying out the invention]

[0021] This invention provides human, monkey, mouse, and rat-specific oligonucleotides that hybridize with the mRNA sequences of neuropyrins such as NRP1 from human mice and / or monkeys and / or rats, thereby inhibiting NRP1 expression and activity, respectively. As a multi-domain receptor, NRP1 binds to several different types of ligands and receptors associated with cell migration, angiogenesis, cell survival, metastasis, and cell proliferation. Many ligands of NRP1 act as pro-angiogenic growth factors. Therefore, inhibiting NRP1 expression allows for simultaneous targeting of a broad spectrum of different NRP1 activities, thereby significantly increasing the feasibility of therapeutic success. These represent interesting and highly efficient tools for use in methods of preventing and / or treating cancer, eye diseases, autoimmune disorders, and / or immune disorders.

[0022] The elements of the present invention will be described in more detail below. While these elements are listed in specific embodiments, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and embodiments described should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of disclosed elements. Furthermore, any permutations and combinations of all elements described in this application should be considered disclosed by this description unless otherwise indicated by the context.

[0023] Throughout this specification and the claims, unless otherwise required by context, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to mean the inclusion of a specified member, component, or step, or a group of members, components, or steps, but not the exclusion of any other member, component, or step, or a group of members, components, or steps. The terms “a,” “an,” and “the,” and similar references used in the context describing the invention (particularly in the context of the claims), should be interpreted as encompassing both singular and plural forms unless otherwise indicated herein or unless clearly inconsistent with the context. The enumeration of value ranges herein is intended merely as a simplified way of referring individually to each distinct value within that range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually described herein. All methods described herein may be performed in any preferred order unless otherwise indicated herein or unless clearly inconsistent with the context. Any and all examples or illustrative language provided herein (e.g., “etc.”, “for example”) are intended solely to better illustrate the invention and not to limit the scope of the invention to any other claimed invention. Nothing in this specification should be construed as indicating any unclaimed element essential to the practice of the invention. The term “about” refers to the figures shown and + / - 10% of these figures.

[0024] The oligonucleotides of the present invention are, for example, antisense oligonucleotides consisting of or containing 10 to 25 nucleotides, 15 to 20 nucleotides, 12 to 18 nucleotides, or 14 to 17 nucleotides. The oligonucleotides consist of or contain 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides. The oligonucleotides of the present invention include, for example, at least one modified nucleotide. The modified nucleotides are, for example, cross-linked nucleotides such as locked nucleic acids (LNA, e.g., 2',4'-LNA such as β-D-LNA and / or α-LNA), cET, ENA, 2'-fluoro-modified nucleotides, 2'O-methyl, 2'O-methoxyethyl-modified nucleotides, or combinations thereof. The oligonucleotides of the present invention include, for example, nucleotides having one or more, two or more, three or more, or four or more of the same or different modifications. Furthermore, the oligonucleotides of the present invention optionally include a modified phosphate skeleton, the phosphate being, for example, a phosphorothioate, a methylphosphonate, a mesyl-phosphoamidate (MsPA; e.g., WO2021 / 030778), or a combination thereof. Optionally, the cytosine of the oligonucleotide contains one or more methyl groups.

[0025] For example, mesyl-phosphoroamidate is introduced as a stereorandom and / or stereoselective bond to the gap and / or one or both wings of a gapmer oligonucleotide. Optionally, all phosphate or phosphorothioate bonds of the oligonucleotide are replaced by mesyl-phosphoroamidate, or all phosphate or phosphorothioate bonds of the gap, or all phosphate or phosphorothioate bonds of one or both wings are replaced by mesyl-phosphoroamidate. For example, 1 to 5 phosphate or phosphorothioate bonds of the gap are replaced by mesyl-phosphoroamidate, and / or 3 to 5 phosphate or phosphorothioate bonds near the 5' end of the gap are replaced by mesyl-phosphoroamidate. Mesyl phosphoramidates are introduced into oligonucleotides, for example, using the Staudinger reaction between methanesulfonyl azide (MsN3, 0.5 M solution in 1:1 acetonitrile / toluene) and a trivalent phosphite intermediate produced by a phosphoramidite bond (e.g., Anderson BA et al., Nucleic Acid Research, 2021, Vol.49, No.16, 9026-9041).

[0026] Furthermore, phosphorothioates can be introduced, for example, into the gap and / or one or both wings of a gapmer oligonucleotide in a stereorandom and / or stereoselective manner.

[0027] According to the present invention, a “gapmer” means an oligonucleotide having a central region containing multiple nucleosides supporting RNase H cleavage located between the 5' and 3' regions. The position of the central region refers to the order of the nucleosides in the central region, which are counted starting from the 5' end of the central region. Thus, the nucleoside furthest 5' end of the central region is at position 1 of the central region. The “central region” may be referred to as the “gap,” and the “5' region” and “3' region” may be referred to as the “wings.”

[0028] The oligonucleotides of the present invention are, for example, antisense oligonucleotides (e.g., DNA), siRNA, or miRNA.

[0029] The "reduction" according to the present invention includes inhibiting effects such as the expression of different percentages and amounts.

[0030] This invention relates to providing oligonucleotides, such as antisense oligonucleotides, that mediate the restriction of available NRP1 mRNA for protein expression. To restrict protein expression, the oligonucleotides require the presence of a complementary nucleic acid sequence representing a hybridization target that enables the formation of a heteroduplex. The oligonucleotides of this invention hybridize with the pre-mRNA of SEQ ID NO: 366 (GRCh38p13_Chr 10_33177492-33336262-1) and / or the mRNA of SEQ ID NO: 367 (RefSeq ID NM_003873.6). GRCh38p13_Chr 10_33177492-33336262-1 and RefSeq ID NM_003873.6 specify the exact versions of the sequences. The formation of a heteroduplex between oligonucleotides and target RNA leads to the recruitment of enzymes such as RNaseH, which result in the degradation or inactivation of the target RNA, or the termination of transcription, thus limiting the amount of NRP1 mRNA available for protein expression.

[0031] The oligonucleotides of the present invention include, for example, one, two, three, or four or more modified nucleotides at the 3' and / or 5' ends of the oligonucleotide, and / or at any position within the oligonucleotide. The modified nucleotides follow, for example, a row of 1, 2, 3, 4, 5, or 6 modified nucleotides, or the modified nucleotides are combined with one, two, three, or four or more unmodified nucleotides. The modified nucleotides are, for example, in a sequence of five nucleotides at the 3' and / or 5' ends of the oligonucleotide. This sequence does not need to begin with the first nucleotide at the 3' or 5' end of the oligonucleotide. Table 1 below presents embodiments of oligonucleotides containing modified nucleotides, e.g., LNA indicated by (+) and phosphorothioate (PTO) indicated by (*). Alternatively, in the oligonucleotides of Table 1, one or more PTO bonds are replaced with mesyl phosphoramidates. Oligonucleotides consisting of or containing the sequences in Table 1 may contain any other modified nucleotides, as well as any other combination of modified and unmodified nucleotides. The oligonucleotides in Table 1 hybridize with human, monkey, mouse, and / or rat NRP1, preferably human NRP1 mRNA. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] Table 1-8 Table 1-9 Table 1-10 Table 1-11

[0032] The ASO of the present invention hybridizes with, for example, the hybridization active region of SEQ ID NO: 366 and / or 367. Such regions are the positions of SEQ ID NO: 366, 1650-1949, 3150-3449, 4050-4349, 4650-4949, 4950-5249, 5550-5849, 6150-6449, 6450-6749, 7650-7949, 9450-9749, 9750-10049, 10950-11249, 12150-12449, 12450-12749, 13350-13649, 14250-14549, 14550-14849, 15450-15749, 15750-16049, 17550-1 7849, 17850~18149, 18150~18449, 18450~18749, 19050~19349, 20250~20549, 21150~21449, 21450~21749, 23250~23549, 23550~23849, 25050~25349, 26550~26849, 28650~28949, 28950~29249, 30750~31049, 31050~31349, 31650~31949, 31950~32249, 32550~32849, 32850~33149, 3 3750~34049, 34650~34949, 36150~36449, 36750~37049, 37350~37649, 37650~37949, 38250~38549, 38850~39149, 39750~40049, 40050~40349, 41850~42149, 42150~42449, 42750~43049, 45450~45749, 46350~46649, 49050~49349, 51150~51449, 52050~52349, 52350~52649, 53550~ 53849, 54450~54749, 55650~55949, 56250~56549, 57450~57749, 57750~58049, 58050~58349, 59250~59549, 59850~60149, 61950~62249, 62550~62849, 62850~63149, 63150~63449, 63750~64049, 64350~64649, 64650~64949, 64950~65249, 65250~65549, 66150~66449, 67050~67349,67650~67949、68250~68549、69450~69749、69750~70049、70350~70649、71550~71849、71850~72149、72150~72449、72450~72749、73650~73949、74850~75149、75450~75749、76650~76949、76950~77249、77250~77549、77550~77849、78150~78449、78450~78749、78750~79049、79350~79649、79650~79949、80250~80549、80550~80849、82050~82349、82350~82649、83250~83549、83850~84149、85350~85649、86250~86549、86850~87149、88350~88649、88650~88949、89850~90149、90150~90449、91650~91949、91950~92249、92250~92549、92850~93149、93150~93449、94650~94949、94950~95249、95250~95549、96150~96449、96450~96749、102150~102449、109350~109649、109950~110249、110550~110849、111150~111449、111750~112049、112050~112349、113850~114149、114150~114449、114450~114749、114750~115049、116850~117149、118950~119249、120150~120449、121350~121649、121650~121949、122850~123149、123150~123449、124950~125249、126150~126449、128550~128849、128850~129149、129150~129449、129750~130049、130050~130349、131850~132149、132750~133049、133050~133349、133350~133649、134250~134549、134550~134849、134850~135149、136350~136649、137550~137849, 138450~138749, 138750~139049, 139050~139349, 139350~139649, 139650~139949, 140550~140849, 142050~142349, 142950~143249, 143850~144149, 144450~144749, 145950~146249, 146250~146549, 147450~147749, 148350~148649, 148 The regions include or consist of 650-148949, 149850-150149, 150150-150449, 151650-151949, 152550-152849, 154350-154649, 154950-155249, 155850-156149, 156150-156449, 156450-156749, 156750-157049, 157050-157349, 157950-158249, and / or 158550-158849. Oligonucleotides, particularly antisense oligonucleotides (ASOs), that hybridize in these regions are shown in Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14]

[0033] The oligonucleotides of the present invention hybridize with, for example, the human NRP1 premRNA and / or mRNA of SEQ ID NO: 366 and / or SEQ ID NO: 367. Such oligonucleotides are called NRP1 antisense oligonucleotides. The oligonucleotides of the present invention are, for example, antisense oligonucleotides, which are shown in Table 1. The present invention further refers to oligonucleotides such as antisense oligonucleotides having sequence homology of about 80% to 99%, about 85% to 98%, about 90% to 95%, about 90% to 99%, about 93% to about 99%, about 94% to about 99%, about 95% to about 99%, and at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with respect to the oligonucleotides in Table 1.

[0034] Furthermore, the present invention refers to oligonucleotides such as antisense oligonucleotides that contain or consist of at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 nucleotides from any one of the oligonucleotides of SEQ ID NOs: 1 to SEQ ID NOs: 364.

[0035] Each nucleotide in the sequence may be modified, and the ASO of the present invention preferably contains a core of 6 to 8 unmodified nucleotides. The ASO of the present invention contains, for example, one or more modified nucleotides, e.g., 1, 2, 3, 4, or 5 nucleotides, at the 5' and / or 3' ends of the oligonucleotide, i.e., on the 5' and / or 3' sides of the core. The 5' and 3' ends are modified to be identical or different. If the 5' and 3' ends are modified identically, the nucleotides are modified at the same positions counted from the 5' and 3' ends, respectively, having the same modification, e.g., LNA modification (in either case, counting starts at 1 from the end). If the 5' and 3' ends are modified differently, the positions of the modified nucleotides and / or the types of modifications at the 5' and 3' ends are different, and the types of nucleotide modifications are the same (e.g., LNA) or different. Modified nucleotides, such as LNA-modified nucleotides, do not need to be consecutive but can be separated by one or more unmodified nucleotides. Exemplary modification patterns of the 5' and 3' ends of the ASO of the present invention are described, where unmodified nucleotides are indicated by "_", and the number on either side of "_" refers to the number of modified nucleotides, such as LNA-modified nucleotides in a row. Modified nucleotides are located at any position of the 5' and / or 3' ends of the ASO, as shown, for example, in Table 3 below. [Table 3] Table 4 below shows the specific locations of LNA modifications at the 5' and 3' ends of each ASO. [Table 4]

[0036] In some embodiments, the oligonucleotides of the present invention reduce the amount of NRP1 mRNA and / or NRP1 protein expression by, for example, about 30% to 100%, 35% to 99%, 40% to 98%, 45% to 97%, 50% to 96%, 55% to 95%, 60% to 90%, 65% to 85%, 70% to 80%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The expression of NRP1 mRNA and / or protein is reduced, for example, in cells, tissues, organs, or subjects.

[0037] The reduction in NRP1 mRNA and / or NRP1 protein expression levels is determined, for example, by comparing the expression levels of NRP1 mRNA and / or NRP1 protein in a sample treated with the oligonucleotide of the present invention with the corresponding untreated control. The untreated control is, for example, an untreated sample such as cells, blood, urine, or saliva, in which NRP1, NRP1 mRNA, NRP1 premRNA expression, or a combination thereof is administered to a subject before administration of the oligonucleotide of the present invention. The untreated sample is collected from the subject, for example, before administration of the oligonucleotide of the present invention.

[0038] The oligonucleotides of the present invention reduce the amount of NRP1 mRNA and / or NRP1 protein expression at nanomolar or micromolar concentrations, for example, about 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.

[0039] The oligonucleotides of the present invention are used, for example, at concentrations of about 1, 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500, or 740 nM, or at concentrations of 1, 2.2, 3, 5, 6.6, or 10 μM.

[0040] The present invention also refers to pharmaceutical compositions comprising the oligonucleotides of the present invention and pharmaceutically acceptable carriers, excipients, and / or diluents. Optionally, the pharmaceutical composition further comprises, for example, chemotherapeutic agents, other oligonucleotides from or different from the present invention, fusion proteins, antibodies, and / or small molecules, such as antagonist proteins.

[0041] In some embodiments, the oligonucleotides or pharmaceutical compositions of the present invention are for use in methods of preventing and / or treating disorders. In some embodiments, the use of the oligonucleotides or pharmaceutical compositions of the present invention in methods of preventing and / or treating disorders is in combination with radiotherapy and / or laser therapy. Radiotherapy may be further used in combination with chemotherapeutic agents (e.g., platinum, gemcitabine).

[0042] The disorder is characterized by abnormal NRP1 activity, such as hyperactivity or NRP1 mRNA and / or protein imbalance, i.e., NRP1 mRNA and / or protein levels being abnormal (e.g., increased) compared to levels in normal, healthy cells, tissues, organs, or subjects, or by abnormal NRP1 activity. NRP1 levels increase, for example, due to increased amounts of NRP1 mRNA and / or NRP1 protein expression. Overactivity of NRP1 function is caused, for example, by the activation of mutations or by unknown genetic, epigenetic, or environmental mechanisms.

[0043] NRP1 mRNA and protein levels are measured by any standard method known to those skilled in the art, such as immunohistochemistry, Western blotting, quantitative real-time PCR, or QuantiGene assay, respectively.

[0044] The oligonucleotides and pharmaceutical compositions containing oligonucleotides of the present invention each have an inhibitory effect on NRP1 expression for, for example, 1, 2, 3, 4, 5, or 6 days, 1, 2, or 3 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or 1 or 2 years, respectively. For example, the therapeutic effect of the oligonucleotides of the present invention corresponds to the duration of the inhibitory effect.

[0045] The oligonucleotides or pharmaceutical compositions of the present invention are administered topically or systemically, for example, intravitreously, intramammarially, or subconjunctivally, for example, by injection, eye drops, orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, intraventricularly, percutaneously, and / or rectally. Alternatively, or in combination, ex vivo-treated immune cells are administered. Oligonucleotides are administered alone or in combination with other oligonucleotides of the present invention, optionally in combination with other oligonucleotides, fusion proteins, antibodies, small molecules, and / or other compounds such as antagonist proteins such as chemotherapeutic agents (e.g., platinum, gemcitabine). Furthermore, two or more oligonucleotides of the present invention are administered, for example, simultaneously, for example, in a pharmaceutical composition, or separately or together at staggered intervals.

[0046] In some embodiments, other oligonucleotides (i.e., not part of the present invention), antagonist proteins such as fusion proteins, antibodies, and / or small molecules are effective in preventing and / or treating cancer, eye diseases, autoimmune disorders, and / or immune disorders. Autoimmune disorders include, for example, autoimmune arthritis or gastrointestinal autoimmune disorders such as inflammatory bowel disease (IBD) or colitis, immune fatigue resulting from chronic viral infections such as HIV infection, cardiovascular disorders, inflammatory disorders such as chronic airway inflammation, bacterial, viral, and / or fungal infections such as sepsis, coronavirus infection or bovine tuberculosis infection, liver disorders, chronic kidney disorders, mental disorders (e.g., schizophrenia, bipolar disorder, Alzheimer's disease), cancer, or combinations thereof.

[0047] The oligonucleotides or pharmaceutical compositions of the present invention are used, for example, in methods for preventing and / or treating solid tumors or hematological malignancies. Examples of cancers that can be prevented and / or treated by the use of the oligonucleotides or pharmaceutical compositions of the present invention include bladder cancer, breast cancer, colorectal cancer, lung cancer, malignant melanoma, mesothelioma, lymphoma, skin cancer, bone cancer, prostate cancer, hepatocellular carcinoma, brain tumors, larynx, liver, gallbladder, pancreas, testes, rectum, parathyroid gland, thyroid gland, adrenal gland, nerve tissue, head and neck, colon, stomach, bronchi, kidney, basal cell carcinoma, neuroblastoma, squamous cell carcinoma, metastatic skin cancer, osteosarcoma, Ewing's sarcoma, reticular sarcoma, liposarcoma, leukemia, myeloma, and giant cell tumors. These include small cell lung tumors, islet cell tumors, primary brain tumors, meningiomas, acute and chronic lymphocytic and granulocytic tumors, acute and chronic myeloid leukemia, hairy cell tumors, adenomas, hyperplasia, medullary carcinomas, enteric ganglia, Wilm's tumor, seminomas, ovarian tumors, leiomyomas, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, focal skin lesions, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumors, polycythemia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforme, leukemia, or epidermoid carcinoma.

[0048] In some embodiments, two or more oligonucleotides of the invention are administered together, simultaneously, for example, in a pharmaceutical composition, or separately, or at staggered intervals. In other embodiments, one or more oligonucleotides of the invention are administered together, simultaneously, for example, in a pharmaceutical composition, or separately, or at staggered intervals, with another compound such as another oligonucleotide (i.e., not part of the invention), a fusion protein, an antibody, a small molecule, and / or an antagonist protein such as a chemotherapeutic agent. In some embodiments of these combinations, the oligonucleotide inhibits the expression and activity of a receptor such as a growth receptor and another oligonucleotide (i.e., not part of the invention), an antagonist protein such as a fusion protein, and the antibody and / or small molecule inhibits (antagonizes) the same or different growth receptor or inhibits (antagonizes) a signaling factor. The growth receptor is, for example, the TGF-β receptor I (TβRI), the TGF-β receptor II (TβRII), or the receptor for VEGF, HGF, PDGF, and / or SEMA3 (Plexin). The signaling factor is, for example, p38MAPK, ERK1, ERK2, PI3K, Akt, NF-κB, pSMAD2, pSMAD3, Src, Pyk2, FAK, and / or p-p130Cas.

[0049] In the case of an eye disease such as AMD or DME, the oligonucleotide of the invention can be used in combination with an antagonist protein such as an anti-VEGF antibody or a fusion protein, laser therapy and / or corticosteroids such as cortisol (C 21 H 30 O5), corticosterone (C 21 H 30 O4), cortisone (C 21 H 28 O5) and / or aldosterone (C 21 H 28 O5).

[0050] The subject of the invention is, for example, a mammal (e.g., human, monkey, dog, cat, horse, cow, pig), a bird, or a fish.

Example

[0051] The following examples illustrate different embodiments of the present invention, but the present invention is not limited to these embodiments.

[0052] Example 1: Design of NRP1 antisense oligonucleotides in humans, mice, and rats To design an ASO with specificity for the exon region within the human NRP1 gene, we used the NRP1 mRNA sequence with RefSeq ID NM_003873.6 (SEQ ID NO: 367; Figure 1). For ASOs with specificity to the intron region within the human NRP1 gene, the NRP1 premRNA sequence (GRCh38p13_Chr 10_33177492-1 (SEQ ID NO: 366)) was used. "H" after the ASO ID indicates a human NRP1-specific sequence that binds to the exon region of the premRNA, "HMI" after the ASO ID indicates a human / mouse cross-reactive NRP1 sequence that binds to the intron region of the premRNA, and "HI" after the ASO ID indicates a human NRP1-specific sequence that binds to the intron region of the premRNA. 15, 16, 17, 18, 19, and 20mer were designed according to in-house standards, and neg1 (described in WO2014 / 154843 A1) was used as a control oligonucleotide in all experiments (e.g., in Table 1).

[0053] Example 2: Targeted knockdown efficacy screening of human NRP1-specific ASOs To investigate the knockdown efficacy of computer-designed NRP1-specific ASOs, three efficacy screening rounds were conducted in human cell lines. Cells were treated with each ASO at a concentration of 5 μM for 3 days without the addition of transfection reagents. After the 3-day treatment period, cells were lysed, and NRP1 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher). NRP1 expression values ​​were normalized to HPRT1 values ​​and compared to mock-treated samples. The results are shown in Figures 3, 4, and 5, and Tables 5, 6, and 7. As illustrated in Figure 3 and Table 5, treatment of SKOV-3 cells with 160 out of 398 tested ASOs (0.2%) resulted in over 50% target inhibition (represented by residual NRP1 mRNA expression of less than 0.5 compared to mock-treated cells). [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0054] The knockdown efficacy of NRP1-specific ASOs was further tested in U87-MG cells. As shown in Figure 4 and Table 6, treatment with 38 of the 120 ASOs tested (31.7%) resulted in over 50% target inhibition (represented by residual NRP1 mRNA expression of less than 0.5 compared to mock-treated cells). [Table 6-1] [Table 6-2]

[0055] The knockdown efficacy of NRP1-specific ASOs was further tested in Panc1 cells. As shown in Figure 5 and Table 7, treatment with 154 of the 257 ASOs tested (59.9%) resulted in over 50% target inhibition (represented by residual NRP1 mRNA expression of less than 0.5 compared to mock-treated cells). The control oligonucleotide did not inhibit NRP1 expression in any of the three cell lines. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0056] Example 3: CD4 + Investigation of targeted knockdown in T cells using selected human NRP1-specific ASOs. Nine human NRP1-specific ASOs were selected and cultured under conditions that preferentially activate and expand Treg cells in human CD4. + The knockdown efficacy of NRP1-specific ASOs in T cells was investigated. Therefore, CD4 + T cells were isolated from PBMCs and treated with the indicated ASO at a concentration of 5 μM for 3 days. As shown in Figure 6, compared to mock-treated cells, over 40% NRP1 knockdown was observed in 8 out of 9 ASOs tested (88.9%). In contrast, treatment with the control oligo did not adversely affect NRP1 expression. [Table 8]

[0057] Example 4: Investigation of concentration-dependent target knockdown by selected human NRP1-specific ASOs. Concentration-dependent knockdown of NRP1 mRNA expression by NRP1-specific ASO was performed in SKOV-3 cells and CD4 cells. + We investigated at the mRNA level in T cells, and each IC 50 The values ​​were calculated. Therefore, SKOV-3 cells were treated with each ASO at concentrations of 5000 nM, 1667 nM, 556 nM, 185 nM, 62 nM, 21 nM, and 7 nM for 3 days. CD4 + T cells were treated with each ASO at the same concentration for 3 days. After the treatment period, the cells were lysed, and NRP1 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher). NRP1 expression values ​​were normalized to HPRT1 values ​​and compared with mock-treated samples (Figures 7 and 8, and Tables 9 and 10). SKOV-3 (Figure 7) and CD4 + Concentration-dependent knockdown of NRP1 mRNA after treatment with all tested NRP1-specific ASOs in T cells (Figure 8) was observed for ICs ranging from 119 nM (A15091HI in SKOV-3 cells, Table 9) to 1101 nM (A15166HI in CD4+ T cells, Table 10). 50 Observed by value. [Table 9] [Table 10]

[0058] Example 5: Investigation of acute toxicity of human NRP1-specific ASO To determine the acute toxicity of the antisense oligonucleotide A15005HMR (SEQ ID NO: 368, described in US20190330640A1) and the antisense oligonucleotides A15091HI (SEQ ID NO: 5), A15108HI (SEQ ID NO: 19), and A15166HI (SEQ ID NO: 69) of the present invention, female Balb / c mice were subcutaneously injected with selected human NRP1-specific ASOs at a dose of 20 mg per kg of body weight for 5 consecutive days. General appearance was monitored daily, and serological parameters ALAT (alanine aminotransferase), ASAT (aspartate transferase), and LDH (lactate dehydrogenase) were analyzed on days 5, 9, and 12, as shown in Figure 9. After treatment of mice with the ASOs of the present invention, little increase in ALAT, ASAT, or LDH was observed, but substantial increases were observed in mice treated with A15005HMR. Furthermore, all mice treated with A15005HMR died before the second measurement on day 9, but none of the mice treated with ASO from the present invention died.

[0059] Example 6: Computer-assisted analysis of potential off-target binding sites The results of the computer analysis are shown in Table 11 below. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6] [Table 11-7] [Table 11-8] [Table 11-9]

[0060] To test the off-target effects of the oligonucleotides of the present invention, two different databases were screened in computer. These databases were RefSecRNA, which contains the sequences of spliced ​​RNA, and ENSEMBL, which contains the sequences of unspliced ​​RNA. The oligonucleotides shown in Table 11, with the exception of A15005HMR, have no potential off-target binding sites with zero mismatches, i.e., 100% sequence complementarity (0 mm) to the off-target sequence, or one mismatch, i.e., ((n-1) / n*100)% sequence complementarity (1 mm) to the off-target sequence. The number of potential off-target sites for the oligonucleotides of the present invention with two mismatches, i.e., ((n-2) / n*100)% sequence complementarity (2 mm), is limited to a maximum of 30 (see Table 11, RefSeq(gene Id), 2 mm).

[0061] Example 7: Concentration-dependent knockdown of NRP1 mRNA expression by NRP1-specific ASO We investigated the concentration-dependent knockdown of NRP1 mRNA expression by NRP1-specific ASOs with the same base sequence but different LNA modification patterns at the mRNA level in Panc1 and SKOV-3 cells, and examined the IC of each cell type. 50The values ​​were calculated. Therefore, cells were treated for 3 days with ASO (A15091HI (SEQ ID NO: 5), A15486HI (SEQ ID NO: 5), or A15487HI (SEQ ID NO: 5), or Neg1 control) at concentrations of 5000 nM, 1667 nM, 550 nM, 185 nM, 62 nM, 21 nM, and 7 nM, respectively. After the treatment period, the cells were lysed, and NRP1 and HPRT1 mRNA expression was analyzed using the QuantiGene Singleplex assay (ThermoFisher). NRP1 expression values ​​were normalized to HPRT1 values ​​and compared with mock-treated samples (Figures 10 and 11, and Tables 13 and 14). Concentration-dependent knockdown of NRP1 mRNA after treatment with all tested NRP1-specific ASOs in Panc1 (Figure 10) and SKOV-3 cells (Figure 11) was observed for ICs ranging from 287 nM (A15486HI in Panc1 cells, Table 13) to 184 nM (A15486HI in SKOV-3 cells, Table 14). 50 Observed by value.

[0062] Table 12 below lists the mean NRP1 mRNA expression levels in ASO-treated SKOV-3 cells compared to mock-treated cells. Expression levels are normalized relative to HPRT1. [Table 12]

[0063] Table 13 below lists the selected NRP1-specific ASOs and their respective ICs. 50 The mean NRP1 mRNA inhibition and IC values ​​in Panc1 cells treated with different concentrations of ASO concentration-dependent inhibition of NRP1 mRNA expression in Panc1 cells, based on the values. 50 A list of values ​​is shown. [Table 13]

[0064] Table 14 below lists the selected NRP1-specific ASOs and their respective ICs. 50The mean NRP1 mRNA inhibition levels and IC values ​​in SKOV-3 cells treated with different concentrations of ASO concentration-dependent inhibition of NRP1 mRNA expression in Panc1 cells. 50 A list of values ​​is shown. [Table 14]

Claims

1. An oligonucleotide comprising 10 to 25 nucleotides, wherein at least one nucleotide has a modification selected from the group consisting of cross-linked nucleic acids such as LNA, ENA, cET, 2'-fluoro-modified nucleotide, 2'O-methyl-modified nucleotide, 2'O-methoxyethyl-modified nucleotide, FANA, and combinations thereof, and the oligonucleotide hybridizes with the transcript or mRNA of neuropilin 1 (NRP1) of SEQ ID NO: 367 and / or the pre-mRNA of NRP1 of SEQ ID NO:

366.

2. The oligonucleotide according to claim 1, wherein the modification is located at the 5' and / or 3' end of the oligonucleotide.

3. The oligonucleotide according to claim 1 or 2, wherein the oligonucleotide hybridizes outside or within the hybridization active region at positions 6450-6749, 12150-12449, 134250-134549, 36150-36449, 17550-17849, 138750-139049, 32550-32849, or a combination thereof.

4. The oligonucleotide according to any one of claims 1 to 3, wherein the oligonucleotide includes SEQ ID NO: 5, SEQ ID NO: 120, SEQ ID NO: 19, SEQ ID NO: 163, SEQ ID NO: 129, SEQ ID NO: 364, SEQ ID NO: 330, SEQ ID NO: 158, SEQ ID NO: 156, or a combination thereof.

5. The oligonucleotide is +A*+T*+A*T*T*T*A*G*G*T*C*C*A*+G*+C*+G (A15091HI; SEQ ID NO: 5), +T*+C*+G*T*T*G*G*A*A*A*T*G*C*C*A*T*+G*+C*+A (A15219HI; SEQ ID NO: 120), +T*+A*+C*A*T*G*G*T*A*A*C*G*C*+C*+T*+T (A15108HI; SEQ ID NO: 19), +A*+T*+A*T*T*T*A*G*G*T*C*C*+A*+G*+C*+G (A15486HI; SEQ ID NO: 5), +A*+T*A*T*T*T*A*G*G*T*C*C*+A*+G*+C*+G (A15487HI; SEQ ID NO: 5), +G*+G*+T*T*A*T*T*G*A*C*T*G*C*T*C*T*+C*+T*+A (A15262HI; SEQ ID NO: 163), +G*+T*+T*T*C*C*T*T*A*G*C*T*T*A*G*T*+G*+C*+C (A15228HI; SEQ ID NO: 129), +A*+A*T*+A*T*T*T*A*G*G*T*C*C*+A*G*+C*+G (A15466HI; SEQ ID NO: 364), +G*+T*+T*A*C*T*C*C*G*T*T*C*C*T*T*+C*+A*+G (A15430HI; SEQ ID NO: 330), +T*+A*+G*G*T*A*C*G*A*G*C*A*T*+C*+G*+G(A15257HI; Sequence ID 158), +A*+A*+T*A*T*T*T*A*G*G*T*C*C*A*+G*+C*+G (A15465HI; SEQ ID NO: 364), +A*T*+A*T*T*T*A*G*G*T*C*C*A*+G*+C*+G (A15473HI; SEQ ID NO: 5), +T*+T*+C*G*T*A*T*T*T*A*A*A*C*T*C*+T*+A*+C (A15255HI; SEQ ID NO: 156) An oligonucleotide according to any one of claims 1 to 4, selected from the group consisting of and combinations thereof, wherein + represents an LNA nucleotide and * represents a phosphorothioate (PTO) bond between nucleotides.

6. The oligonucleotide according to claim 4 or 5, wherein the oligonucleotide has 80-99%, 85-98%, 90-95%, or 93% sequence identity with any one of the oligonucleotides of SEQ ID NO: 1 to SEQ ID NO:

364.

7. The oligonucleotide according to any one of claims 4 to 6, wherein the oligonucleotide comprises at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 nucleotides from any one oligonucleotide of SEQ ID NOs: 1 to 364.

8. The oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide inhibits the expression of NRP1, NRP1 mRNA, NRP1 premRNA, or a combination thereof at a nanomolar or micromolar concentration.

9. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant or other stimulant, or a combination thereof.

10. The pharmaceutical composition according to claim 9, further comprising a therapeutic activator.

11. A pharmaceutical composition according to claim 9 or 10, or an oligonucleotide according to any one of claims 1 to 8, which further inhibits the activity of a growth receptor selected from the group consisting of TGF-β receptor I (TβRI), TGF-β receptor II (TβRII), VEGF, HGF, PDGF, and SEMA3 (Plexin), or a combination thereof, or further inhibits the activity of a signaling factor such as p38MAPK, ERK1, ERK2, PI3K, Akt, NF-κB, pSMAD2, pSMAD3, Src, Pyk2, FAK, p-p130Cas, or a combination thereof.

12. T ｒｅｇ A pharmaceutical composition according to any one of claims 9 to 11, or an oligonucleotide according to any one of claims 1 to 8 or 11, which inhibits the migration of cells to a tumor.

13. A pharmaceutical composition according to any one of claims 9 to 12, or an antisense oligonucleotide according to any one of claims 1 to 8, 11, or 12, for use in the prevention and / or treatment of cancer, eye diseases, autoimmune disorders, and / or immune disorders.

14. The pharmaceutical composition for use or antisense oligonucleotide according to claim 13, wherein the eye disease is age-related macular disease (AMD), diabetic retinopathy (DME), retinopathy of prematurity (Retinopatisia praematurorum), or a neovascular eye disease such as corneal neovascularization.

15. The aforementioned cancers include bladder cancer, breast cancer, colorectal cancer, lung cancer, malignant melanoma, mesothelioma, lymphoma, skin cancer, bone cancer, prostate cancer, hepatocellular carcinoma, brain tumor, larynx, liver, gallbladder, pancreas, testis, rectum, parathyroid gland, thyroid gland, adrenal gland, nerve tissue, head and neck, colon, stomach, bronchi, kidney, basal cell carcinoma, neuroblastoma, squamous cell carcinoma, metastatic skin cancer, osteosarcoma, Ewing's sarcoma, reticular sarcoma, liposarcoma, leukemia, myeloma, giant cell tumor, small cell lung tumor, islet cell tumor, primary brain tumor, meningioma, acute and chronic lymphocytic and granulomatous cancers. A pharmaceutical composition or antisense oligonucleotide for use according to claim 13, which is a granulocytic tumor, acute and chronic myeloid leukemia, hairy cell tumor, adenoma, hyperplasia, medullary carcinoma, enteric ganglion neuroma, Wilm's tumor, seminoma, ovarian tumor, leiomyoma, cervical dysplasia, retinoblastoma, soft tissue sarcoma, malignant carcinoid, focal skin lesion, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, anaplastic astrocytoma, glioblastoma multiforme, leukemia, or epidermoid carcinoma.

16. A pharmaceutical composition or antisense oligonucleotide for use according to any one of claims 13 to 15, wherein the oligonucleotide or composition is administerable topically or systemically.

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