Oligonucleotides for reducing expression of leucine-rich repeat kinase 2 (LRRK2) and their use for preventing and / or treating human diseases

Abstract

The present invention relates to an oligonucleotide comprising from 10 to 25 nucleotides, at least one nucleotide having a modification wherein said oligonucleotide hybridizes to a transcript or mRNA of leucine-rich repeat kinase 2 (LRRK2) of SEQ ID NO. 1 and / or to a pre-mRNA of LRRK2 of SEQ ID NO. 2 and / or a fragment thereof. The invention further relates to a pharmaceutical composition comprising such oligonucleotides, and to the use of said oligonucleotides and pharmaceutical compositions, respectively, in methods for the prevention and / or treatment of human diseases.

Description

Technical Field

[0001] The present invention relates to oligonucleotides, in particular antisense oligonucleotides, which comprise one or more modified nucleotides and hybridize to LRRK2 mRNA and / or pre-mRNA. The oligonucleotides inhibit the expression of LRRK2, LRRK2 mRNA and / or LRRK2 pre-mRNA and are used to prevent and / or treat human diseases, such as Parkinson's disease (PD) and other neurodegenerative diseases. Background Art

[0002] Neurodegenerative diseases are caused by the progressive loss of neuronal structure or function in a process called neurodegeneration. Such neuronal damage may ultimately involve cell death. Neurodegenerative diseases include amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, and prion disease. Neurodegeneration can be found at the level of many different neuronal circuits in the brain.

[0003] Parkinson's disease (PD) is the second most common neurodegenerative disease, and approximately 1 in 100 people over the age of 60 suffer from the disease. Since the current standard of care is limited to symptomatic treatment, disease-modifying or neuroprotective therapies are highly desirable (Lees, Hardy, & Revesz, 2009). Pathological hallmarks of PD include accumulation of α-synuclein (α-syn) into Lewy bodies (LBs) and Lewy neurons (LNs), and progressive loss of dopamine-stimulated (DA) neurons in the substantia nigra pars compacta (SNpc) (Trojanowski & Lee, 1998).

[0004] Currently, a variety of drugs provide some relief from the symptoms of neurodegenerative diseases such as Parkinson's disease. However, there is a lack of effective / acceptable treatments for such diseases.

[0005] Understanding the genetics of PD has identified several genetic alleles that cause or are associated with disease onset and progression (Chang et al., 2017; Gasser, Hardy, & Mizuno, 2011; Hardy, 2010; Lunati, Lesage, & Brice, 2018). Human genetic evidence suggests that LRRK2 function is directly involved in the risk of developing PD, the underlying disease pathology and progression of PD, and therefore validates LRRK2 as an attractive therapeutic target. LRRK2 is a large protein of 2527 amino acids that is widely expressed throughout the body, with its highest levels detected in lung and kidney tissues. The LRKK2 protein consists of multiple functional domains, including a complex protein Ras (ROC)-type GTP binding domain, a ROC carboxyl-terminal (COR) domain, and a serine / threonine protein kinase domain (Cookson, 2017). Overall, LRRK2 appears to regulate aspects of intracellular vesicle trafficking, maintenance of cellular organelles including homoeosomes, endosomes, and lysosomes.

[0006] Altered LRRK2 function due to genetic, expressive or environmental factors, and similar mutations in or near the LRRK2 gene are directly involved in the development and progression of diseases, including synuclein diseases such as PD. Therefore, inhibiting its expression may provide an effective treatment opportunity for these diseases. Inhibiting LRRK2 expression can be achieved, for example, by targeted degradation of mRNA encoding LRRK2 or by blocking its translation.

[0007] The following two competing technologies are mainly described to specifically inhibit mRNA expression: siRNA and antisense oligonucleotides (ASOs). siRNA targets mRNA in the cytoplasm. In contrast, RNase H-dependent ASOs target mRNA and / or pre-mRNA in the nucleus. Thus, intronic regions can also be targeted by ASOs that are not accessible to siRNAs, making the targeting space of ASOs significantly larger.

[0008] In addition, due to its double-stranded nature, siRNA itself does not cross the cell membrane and the delivery system is required for its in vitro and in vivo activity. Although there are siRNA delivery systems that can effectively deliver siRNA to hepatocytes in vivo, there is currently no system that can deliver siRNA to extrahepatic tissues in vivo with sufficient efficacy. In contrast, modified ASOs can enter many cell types in vitro in sufficient quantities without transfection or delivery methods, thereby causing potent and sequence-specific target gene attenuation. In vivo, naked, unbound ASOs achieve target gene attenuation in several different relevant tissues after systemic administration. After direct administration, ASOs achieve potent and long-lasting target gene attenuation in the central nervous system (CNS), indicating the strong potential of ASOs for treating CNS diseases (such as neurodegenerative diseases) (e.g., Geary et al., Adv Drug Deliv Rev. 2015 June 29; 87: 46-51).

[0009] The present invention designs oligonucleotides, such as antisense oligonucleotides (ASOs), that cause a significant decrease in LRRK2 mRNA and protein expression in the CNS as a therapeutic treatment option for PD.

[0010] The mode of action of oligonucleotides is different from that of antibodies or small molecules, and oligonucleotides are highly advantageous in, for example, the following aspects:

[0011] (i) better penetration into tissues, such as brain tissue, compared to that of antibodies due to their substantially smaller size (e.g., Pizzo et al., J Physiol. 2018 Feb 1;596(3):445-475; Noguchi et al., MAbs. 2017 Oct;9(7):1210-1215),

[0012] (ii) prolonged retention and duration of action in the brain following direct administration,

[0013] (iii) blocking multiple functions and activities of the target separately,

[0014] (iv) combinations of oligonucleotides with each other or with antibodies or small molecules, and

[0015] (v) Inhibition of intracellular effects that are inaccessible to antibodies or cannot be inhibited by small molecules.

[0016] Therefore, there is a need for new and improved therapies for treating diseases such as PD that are able to reduce the amount of LRRK2 mRNA and protein with minimal toxic side effects. Summary of the invention

[0017] The present invention relates to an oligonucleotide comprising 10 to 25 nucleotides, at least one nucleotide having a modification selected from the group consisting of: a bridging nucleic acid such as LNA, ENA, cET, a 2'fluorine-modified nucleotide, a 2'O-methyl-modified nucleotide, a 2'O-methoxy-modified nucleotide, FANA and a combination thereof, wherein the modification is located in a sequence of 5 nucleotides at the 5' end and / or 3' end of the oligonucleotide, and wherein the oligonucleotide hybridizes with a transcript or mRNA of leucine-rich repeat kinase 2 (LRRK2) of SEQ ID NO. 1, and / or with a pre-mRNA of LRRK2 of SEQ ID NO. 2 and / or a fragment thereof. The fragment is produced by biological methods, including (but not limited to) alternative splicing. For example, the oligonucleotide hybridizes outside the hybridization active region of the following positions or within the hybridization active region of the following positions: position 102001 to 102500, position 27001 to 27500, position 80501 to 81000, position 24501 to 25000, position 114001 to 114500, position 92001 to 92500, position 99501 to 100000, position 67001 to 675 00, position 15001 to 15500, position 24001 to 24500, position 26501 to 27000, position 79501 ​​to 80000, position 87001 to 87500, position 120501 to 121000, position 142001 to 142500, position 135001 to 135500, position 71001 to 71500, position 140501 to 141000 or a combination thereof.

[0018] The oligonucleotide comprises or consists of, for example, SEQ ID NO. 210, SEQ ID NO. 69, SEQ ID NO. 164, SEQ ID NO. 254, SEQ ID NO. 222, SEQ ID NO. 186, SEQ ID NO. 204, SEQ ID NO. 142, SEQ ID NO. 209, SEQ ID NO. 255, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 60, SEQ ID NO. 66, SEQ ID NO. 162, SEQ ID NO. 171, SEQ ID NO. 224, SEQ ID NO. 241, SEQ ID NO. 250, SEQ ID NO. 149, SEQ ID NO. 256, SEQ ID NO. 239, or a combination thereof. The sequence comprises at least one modification.

[0019] For example, the oligonucleotide is selected from the group consisting of: +T*+A*+C*A*A*T*A*T*T*T*A*T*G*G*T*T*+C*+T*+C (A57208Hi; SEQ ID NO.210), +A*+A*+T*A*C*T*A*C*T*A*T*T*T*G*T*T*+T*+C*+C (A57067Hi; SEQ ID NO.69), +G*+T*+C*A*T*A*A*G*T*T*G*T*C*A*G*+T*+A*+T (A57162Hi; SEQ ID NO.164), +G*+A*+C*T*T*C*G*T*T*T*A*A*T*A*C*+C*+A*+G (A57252H; SEQ ID NO.254), +C*+T*+T*T*G*C*T*T*A*T*T*C*G*T*G*+A*+C*+T (A57220Hi; SEQ IDNO.222), +T*+T*+T*A*T*T*G*C*A*G*T*C*A*T*A*G*+T*+G*+A (A57184Hi; SEQ ID NO.186), +C*+T*+T*T*G*T*A*A*A*T*G*A*C*G*T*T*+T*+C*+C (A57202Hi; SEQ ID NO.204), +T*+G*+A*A*A*T*G*T*C*T*A*T*T*A*G*C*+A*+C*+A (A57140Hi, SEQ ID NO.142), +A*+C*+A*A*T*A*T*T*T*A*T*G*G*T*T*C*+T*+C*+A (A57207Hi; SEQ ID NO.209), +G*+G*+A*C*T*T*C*G*T*T*T*A*A*T*A*C*+C*+A*+G (A57253H; SEQ ID NO.255), +C*+G*+G*C*T*A*A*A*T*G*T*T*T*C*A*G*+T*+T*+C (A57043Hi; SEQ ID NO.45), +T*+C*+G*G*C*T*A*A*A*T*G*T*T*T*C*+A*+G*+T (A57044Hi; SEQ ID NO.46), +G*+T*+G*T*A*A*C*T*A*A*G*T*G*A*T*+C*+T*+C (A57058Hi; SEQ ID NO.60), +G*+A*+T*A*T*T*G*A*C*A*A*C*C*T*G*+C*+T*+G (A57064Hi; SEQ IDNO.66), +T*+G*+T*G*T*T*A*C*C*A*T*A*T*C*C*A*+T*+T*+T(A57160Hi; SEQ ID NO.162), +C*+T*+G*T*G*A*C*T*T*T*G*C*A*T*C*A*+A*+C*+T(A57169Hi; SEQ ID NO.171), +C*+A*+A*T*G*T*A*T*A*C*C*A*T*G*C*+A*+G*+T(A57222Hi; SEQ ID NO.224), +T*+T*+C*C*T*G*A*T*T*A*T*A*C*C*A*T*+A*+T*+G(A57239Hi; SEQ ID NO.241), +A*+T*+G*C*T*C*T*T*A*T*C*C*A*C*A*A*+I*+C*+C(A57248Hi; SEQ ID NO.250), +I*+I*+I*G*A*I*C*I*G*T*A*G*T*A*C*A*+T*+A*+C(A57147Hi; SEQ ID NO.149), +A*+T*+G*G*A*C*T*T*C*G*T*T*T*A*A*T*+A*+C*+C(A57254H; SEQ ID NO.256), +G*+G*+A*C*A*T*G*T*A*T*C*T*T*A*A*T*+A*+G*+T(A57237Hi; SEQ ID NO.239) and combinations thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage between nucleotides. Alternatively, the oligonucleotide has 80% to 99%, 85% to 98%, 90% to 95% or 93% sequence identity with any one of the oligonucleotides of SEQ ID NO.3 to SEQ ID NO.5301 of the present invention. .

[0020] In addition, the oligonucleotide comprises or consists 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 of any one of the oligonucleotides of, for example, SEQ ID NO. 3 to SEQ ID NO. 5301 of the present invention. The nucleotides are, for example, consecutive nucleotides.

[0021] The oligonucleotides of the invention inhibit the expression of, for example, LRRK2, LRRK2 mRNA, LRRK2 pre-mRNA, or a combination thereof, at nanomolar or micromolar concentrations.

[0022] The present invention further relates to a pharmaceutical composition comprising or consisting of an oligonucleotide of the present invention and a pharmaceutically acceptable carrier, excipient, diluent, stimulant (such as an adjuvant) or a combination thereof. The pharmaceutical composition may comprise an additional therapeutic agent. The oligonucleotide of the present invention and the pharmaceutical composition are, for example, used in a method for preventing and / or treating a human disease such as a neurodegenerative disease, respectively. The neurodegenerative disease is, for example, selected from the group consisting of: Parkinson's disease (PD), Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, multiple sclerosis, multiple system atrophy, dementia with Lewy bodies (DLB), pure autonomic failure (PAF), frontotemporal dementia and prion disease. The pharmaceutically acceptable carrier, excipient, adjuvant or a combination thereof or the therapeutic agent is, for example, administered separately before or after delivery of the oligonucleotide, or, for example, administered simultaneously via different delivery methods. The delivery method is, for example, oral, sublingual, nasal, subcutaneous, intravenous, intraperitoneal, intramuscular, intratumoral, intrathecal, intracerebroventricular, transdermal and / or rectal.

[0023] The oligonucleotides and pharmaceutical compositions of the invention are for example for local or systemic administration, respectively.

[0024] The present invention further relates to a method for preventing and / or treating human diseases, comprising administering a therapeutically effective amount of an oligonucleotide of the present invention or a pharmaceutical composition comprising the oligonucleotide. Human diseases (such as neurological diseases) may be selected from the group consisting of Parkinson's disease (PD), Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis, multiple sclerosis, multiple system atrophy, dementia with Lewy bodies (DLB), pure autonomic failure (PAF), frontotemporal dementia and prion disease. The oligonucleotide or pharmaceutical composition may be administered topically or systemically.

[0025] All documents cited or referenced herein ("herein-cited documents") and all documents cited or referenced in the herein-cited documents, as well as any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or incorporated by reference in any document herein are hereby incorporated by reference and may be used in the practice of the present invention. More specifically, all references are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 RefSeq ID NM_198578 of LRRK2 mRNA (SEQ ID NO. 1) is shown.

[0027] Figure 2GRCh38:12:40224997:40369285, in particular LRRK2_GRCh38:12:40224997:40369285:1_seqGenomicENST00000298910.12 (LRRK2 pre-mRNA of SEQ ID NO. 2) is depicted.

[0028] Figures 3A-3D depict the screening of human LRRK2-specific ASOs in A549 cells (human lung cancer).

[0029] Figures 4A to 4D show the screening of human LRRK2-specific ASOs in MDA-MB-231 cells (human breast cancer). DETAILED DESCRIPTION

[0030] The present invention provides oligonucleotides that hybridize to mRNA and / or pre-mRNA sequences of, for example, human-derived leucine-rich repeat kinase 2 (LRRK2). These oligonucleotides hybridize to introns and / or exons and / or exon-exon junctions and / or exon-intron junctions of LRRK2 pre-mRNA, for example, in neuronal cells or tumor cells or other mammalian cells. By recruiting enzymes such as ribonuclease H, pre-mRNA degradation and reduced levels of LRRK2 mRNA. Therefore, the production of LRRK2 protein is prevented and the level of LRRK2 protein is reduced in, for example, neuronal cells or tumor cells or other mammalian cells. Therefore, LRRK2 protein levels and their downstream effects are reduced. The oligonucleotides of the present invention represent promising and efficient tools in methods for preventing and / or treating disorders (e.g., neurodegenerative disorders), wherein LRRK2 protein expression, function and / or activity are associated with disease occurrence and progression, respectively.

[0031] The oligonucleotide of the present invention hybridizes, for example, to LRRK2 mRNA of SEQ ID NO. 1 (RefSeq ID NM_198578) and / or LRRK2 pre-mRNA of SEQ ID NO. 2 (GRCh38:12:40224997:40369285).

[0032] Hereinafter, 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 can be combined in any manner and in any number to produce additional embodiments. The embodiments and embodiments provided herein are not intended to limit the present invention to only the embodiments clearly described. This specification should be understood to support and encompass the embodiments that combine the embodiments clearly described with any number of disclosed elements. In addition, unless otherwise indicated by the context, any arrangement or combination of all described elements in this application should be considered to be disclosed by the description of the application.

[0033] Throughout this specification and the scope of the claims, unless the context requires otherwise, the word "comprising" and variations such as "including" and "having" should be understood to imply the inclusion of the stated members, integers or steps or groups of members, integers or steps, but not to exclude any other members, wholes or steps or groups of members, wholes or steps. Unless otherwise indicated herein or clearly contradicted by the context, the terms "one", "a kind" and "the" and similar references used in the context of describing the present invention (especially in the context of the scope of the claims) should be interpreted as covering both the singular and the plural. The description of the range of values ​​herein is merely intended to serve as a shorthand method for individually referring to each individual value within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification, and the reference degree is as if it were individually described herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or illustrative language (e.g., "such as", "for example") provided herein is intended only to better illustrate the present invention, and does not limit the scope of the present invention claimed in other ways. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0034] The oligonucleotide of the present invention is, for example, an antisense oligonucleotide consisting of or comprising about 10 to about 25 nucleotides, about 10 to about 15 nucleotides, about 15 to about 20 nucleotides, about 12 to about 19 nucleotides, or about 14 to about 18 nucleotides. The oligonucleotide, for example, consists of or comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. The oligonucleotide of the present invention comprises at least one modified nucleotide. The modified nucleotide may be, for example, a bridging nucleotide, such as a locked nucleic acid (LNA, e.g., 2', 4'-LNA), cET, ENA, a 2' fluorine-modified nucleotide, a 2' O-methyl-modified nucleotide, or a combination thereof. The oligonucleotide of the present invention comprises nucleotides having, for example, one or more, two or more, three or more, or four or more identical or different modifications. Additionally, the oligonucleotides of the invention optionally comprise a modified phosphate backbone, wherein the phosphate is, for example, a phosphodiester, a phosphorothioate, a methylphosphonate, a methylphosphorothioate, a phosphoramidate, a phosphorodiamidate, a thiophosphoramidate, a methylsulfonylphosphoramidate, or a combination thereof.

[0035] "Reduction" according to the present invention includes inhibition of effects, such as expression, in different percentages and amounts, respectively.

[0036] The present invention relates to providing an oligonucleotide, such as an antisense oligonucleotide, that mediates restriction of available LRKK2 mRNA for protein expression. In order to restrict protein expression, the oligonucleotide requires the presence of a complementary nucleic acid sequence representing a hybridization target that allows the formation of a heteroduplex. The oligonucleotide of the present invention hybridizes with the RNA of SEQ ID NO.1 and / or the pre-mRNA of SEQ ID NO.2. The formation of a heteroduplex between the oligonucleotide and the target RNA results in enzyme-mediated degradation such as RNase H or inactivation of the target RNA or termination of transcription, and thus limits the amount of available LRKK2 mRNA for protein expression.

[0037] In addition, the present invention relates to an oligonucleotide of the present invention, comprising one or more, two or more, three or more or four or more modified nucleotides, for example at the 3' end and / or 5' end of the oligonucleotide and / or at any position within the oligonucleotide, wherein the modified nucleotides are in a row of about 1, 2, 3, 4, 5 or 6 modified nucleotides, or the modified nucleotides are combined with one or more, two or more, three or more or four or more unmodified nucleotides. Table 1 below presents an embodiment of an oligonucleotide comprising a modified nucleotide, for example, an LNA indicated by (+) and a phosphorothioate (PTO) indicated by (*). An oligonucleotide consisting of or comprising a sequence of Table 1 may comprise any other modified nucleotides and any other combination of modified and unmodified nucleotides.

[0038] Table 1. Oligonucleotides that hybridize to human LRKK2 mRNA:

[0039] Table 1: List of human LRRK2-specific ASOs and control oligonucleotides. The "H" after the ASO name indicates a human LRRK2-specific sequence that binds to the exon region of the pre-mRNA, and the "Hi" after the ASO name indicates a human LRRK2-specific sequence that binds to the intron region of the pre-mRNA. 16, 17, 18 and 19-mers were designed according to internal guidelines, Neg1 (described in WO2014154843A1), R01002, R01011 and R01025 were used as non-targeting control oligonucleotides in some experiments.

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[0113] The ASO of the present invention hybridizes, for example, to a hybridization active region. Such a region comprises or consists of: for example, SEQ ID Position of NO.2 1 to 500, 501 to 1000, 1001 to 1500, 1501 to 2000, 2001 to 2500, 3501 to 4000, 4001 to 4500, 4501 to 5000, 5001 to 5500, 5001 to 6000, 6001 to 6500, 6501 to 7000, 7001 to 7500, 7501 to 8000, 8001 to 8500, 8501 to 9000, 9001 to 10000, 10501 to 11000, 11001 to 11500, 11501 to 12000, 12001 to 12500, 12501 to 13000, 13001 to 13500, 13501 to 1400, 14501 to 15000, 15001 to 15500, 15501 to 16000, 16001 to 16500, 16501 to 17000, 17001 to 17500, 17501 to 18000, 18001 to 18500, 18501 to 19000, 19001 to 19500, 20001 to 20500, 20501 to 21000, 21001 to 21500, 21501 to 22000, 22001 to 22500, 23001 to 23500, 23501 to 24000, 24001 to 24500, 24501 to 25 1 to 25000, 25001 to 25500, 26001 to 26500, 26501 to 27000, 27001 to 27500, 27501 to 28000, 28001 to 28500, 29001 to 29500, 29501 to 30000, 30001 to 30500, 30501 to 31000, 31001 to 31500, 32001 to 32500, 33001 to 33500, 33501 to 34000, 34001 to 34500, 34501 to 35000, 35001 to 35500, 35501 to 36000, 36001 to 36500, 36 501 to 37000, 37001 to 37500, 37501 to 38000, 38001 to 38500, 38501 to 39000, 39001 to 39500, 39501 to 40000, 40001 to 40500, 40501 to 41000, 42001 to 42500, 42501 to 43000, 43001 to 43500, 43501 to 44000, 44001 to 44500, 44501 to 45000, 45001 to 45500, 45501 to 46000, 46001 to 46500, 46501 to 47000, 47001 to 47500,47501 to 48000, 48001 to 48500, 48501 to 49000, 49001 to 49500, 49501 to 50000, 50001 to 50500, 50501 to 51000, 51001 to 51500, 51501 to 52000, 52001 to 52500, 5250 1 to 53000, 53001 to 53500, 53501 to 54000, 54001 to 54500, 54501 to 55000, 55001 to 55500, 56001 to 56500, 56501 to 57000, 57001 to 57500, 57501 to 58000, 58001 to 585 00, 58501 to 59000, 59001 to 59500, 59501 to 60000, 60001 to 60500, 60501 to 61000, 61001 to 61500, 61501 to 62000, 62001 to 62500, 62501 to 63000, 63001 to 63500, 6 3501 to 64000, 64001 to 64500, 64501 to 65000, 65001 to 65500, 65501 to 66000, 66001 to 66500, 66501 to 67000, 67001 to 67500, 67501 to 68000, 68001 to 68500, 68501 to 69000, 69001 to 69500, 69501 to 70000, 70001 to 70500, 70501 to 71000, 71001 to 71500, 71501 to 72000, 72001 to 72500, 72501 to 73000, 73001 to 73500, 73501 to 7400 0, 74001 to 74500, 74501 to 75000, 75001 to 75500, 75501 to 76000, 76001 to 76500, 76501 to 77000, 77001 to 77500, 77501 to 78000, 78001 to 78500, 78501 to 79000, 790 01 to 79500, 79501 ​​to 80000, 80001 to 80500, 80501 to 81000, 81001 to 81500, 81501 to 82000, 82001 to 82500, 82501 to 83000, 83001 to 83500, 83501 to 84000, 84001 to 8 4500、84501 to 85000、85001 to 85500、85501 to 86000、86001 to 86500、86501 to 87000、87001 to 87500、87501 to 88000、88001 to 88500、88501 to 89000、89001 to 89500、89501 to 90000, 90001 to 90500, 90501 to 91000, 91001 to 91500, 91501 to 92000, 92001 to 92500, 92501 to 93000, 93001 to 93500, 93501 to 94000, 94001 to 94500, 9450 1 to 95000, 95001 to 95500, 95501 to 96000, 96001 to 96500, 96501 to 97000, 97001 to 97500, 97501 to 98000, 98001 to 98500, 98501 to 99000, 99001 to 99500, 99501 to 10 0000, 100001 to 100500, 100501 to 101000, 101001 to 101500, 101501 to 102000, 102001 to 102500, 102501 to 103000, 103001 to 103500, 103501 to 104000, 104001 to 104500, 105001 to 105500, 105501 to 106000, 106001 to 106500, 106501 to 107000, 107001 to 107500, 108001 to 108500, 108501 to 109000, 109001 to 109500, 109501 to 110000, 110001 to 110500, 110501 to 111000, 111001 to 111500, 111501 to 112000, 112001 to 112500, 112501 to 113000, 113001 to 113500, 113501 to 114000, 1140 01 to 114500, 114501 to 115000, 115001 to 115500, 115501 to 116000, 116001 to 116500, 116501 to 117000, 117001 to 117500, 117501 to 118000, 118001 to 118500, 118 501 to 119000, 119001 to 119500, 119501 to 120000, 120001 to 120500, 120501 to 121000, 121001 to 121500, 122001 to 122500, 122501 to 123000, 123001 to 123500, 1 24001 to 124500, 124501 to 125000, 125001 to 126000, 126001 to 126500, 127001 to 127500, 129001 to 129500, 129501 to 130000, 130001 to 130500, 131001 to 131500,131501 to 132000, 132501 to 133000, 134001 to 134500, 134501 to 135000, 135001 to 135500, 135501 to 136000, 136001 to 136500, 136501 to 137000, 137001 to 137500, 137501 to 138000, 138001 to 138500, 138501 to 139000, 139001 to 139500, 139501 to 140000, 140001 to 140500, 140501 to 141000, 141001 to 141500, 141501 to 142000, 142001 to 142500, 142501 to 143000, 143001 to 143500, 143501 to 144000 or 144001 to 144173. The ASOs that hybridize in these regions are shown in Table 2 below:

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[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218] The oligonucleotides of the invention hybridize, for example, with the mRNA and / or pre-mRNA of human LRRK2 of SEQ ID NO. 1 and / or SEQ ID NO. 2. Such oligonucleotides are referred to as LRRK2 antisense oligonucleotides. The oligonucleotides of the invention, which are, for example, antisense oligonucleotides, are shown in Table 1. The invention further relates to oligonucleotides, such as antisense oligonucleotides, having 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%, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology with the oligonucleotides of Table 1.

[0219] In addition, the present invention relates to oligonucleotides, such as antisense oligonucleotides comprising or consisting 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 of any one of the oligonucleotides of SEQ ID NO.3 to SEQ ID NO.5301.

[0220] Each nucleotide of the sequence may be modified, wherein the ASO of the invention preferably comprises a core of 6 to 8 unmodified nucleotides. The ASO of the invention comprises, for example, one or more modified nucleotides, for example, 1, 2, 3, 4 or 5 nucleotides at the 5' and / or 3' end of the oligonucleotide, i.e., on the 5' side and / or 3' side of the core. The 5' and 3' ends may be modified identically or differently. If the 5' and 3' ends are identically modified, the nucleotides are modified at the same position counted from the 5' and 3' ends, respectively (counting starts at 1 from the end in each case), which are identically modified, for example LNA-modified. If the 5' and 3' ends are differently modified, the position and / or modification type of the modified nucleotides at the 5' and 3' ends are different; the nucleotide modification type is the same (e.g., LNA) or different. Modified nucleotides, such as LNA-modified nucleotides, do not have to be in a continuous row, but may be separated by one or more unmodified nucleotides. In the following table, exemplary modification patterns at the 5' and 3' ends of the ASOs of the invention are described, wherein unmodified nucleotides are indicated by "_", and the numbers on either side of the "_" refer to the number of modified nucleotides, such as LNA-modified nucleotides, in a column. One or more modified nucleotides are at any position at the 5' and / or 3' end of the ASO as shown in Table 3 below:

[0221] Table 3

[0222]

[0223]

[0224] Table 3 indicates the specific positions of LNA modifications at the 5' and 3' ends of each ASO in Table 4:

[0225] Table 4

[0226]

[0227]

[0228] In some embodiments, the oligonucleotides of the invention can reduce the expression of LRRK2 mRNA and / or LRRK2 protein by, for example, about 30%-100%, 35%-99%, 40%-98%, 45%-97%, 50%-96%, 55%-95%, 60%-90%, 65%-85%, 70%-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 reduction in the expression of LRRK2 mRNA and / or LRRK2 protein can be determined by comparing the expression of LRRK2 mRNA and / or LRRK2 protein in a sample treated with an oligonucleotide of the invention to a corresponding untreated control. An untreated control is, for example, LRRK2, LRRK2 mRNA, LRRK2 pre-mRNA expression or a combination thereof in a subject or untreated sample, such as cells, blood, urine, saliva, etc., prior to administration of an oligonucleotide of the invention. An untreated sample is, for example, obtained from a subject prior to administration of an oligonucleotide of the invention.

[0229] The oligonucleotides of the invention reduce the amount of LRRK2 mRNA and / or LRRK2 protein expression at nanomolar or micromolar concentrations, for example, at a concentration of 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 at a concentration of 1, 10 or 100 μM.

[0230] The oligonucleotides of the invention are used, for example, at a concentration of about 1, 3, 5, 9, 10, 15, 27, 30, 40, 50, 75, 82, 100, 250, 300, 500 or 740 nM, or about 1, 2.2, 3, 5, 6.6 or 10 μM.

[0231] The present invention also relates to a pharmaceutical composition comprising an oligonucleotide as described herein and a pharmaceutically acceptable carrier, excipient, stimulant, adjuvant and / or diluent. Optionally, the pharmaceutical composition further comprises another oligonucleotide, antibody and / or small molecule (such as a therapeutically active agent) different from the present invention for use in preventing and / or treating human diseases, such as PD (a prevalent and serious neurodegenerative disease).

[0232] The oligonucleotides or pharmaceutical compositions of the invention are for example used in methods for preventing and / or treating disorders such as neurodegenerative disorders. Neurodegenerative disorders are for example Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, multiple system atrophy, dementia with Lewy bodies (DLB), pure autonomic failure (PAF), frontotemporal dementia or prion disease.

[0233] The disorder is characterized, for example, by LRRK2 activity, such as overactivity, or LRRK2 mRNA and / or protein imbalance, i.e., LRRK2 mRNA and / or protein levels are abnormal (e.g., increased) compared to levels in normal healthy cells, tissues, organs, or subjects, or LRRK2 activity is abnormal. LRRK2 levels are increased, for example, by increased expression of LRRK2 mRNA and / or LRRK2 protein. Overactivity of LRRK2 function is caused, for example, by activating mutations or by unknown genetic, epigenetic, or environmental mechanisms.

[0234] LRRK2 mRNA and protein levels, respectively, can be measured by any standard method known to those skilled in the art, such as immunohistochemistry, Western blot, quantitative real-time PCR, or QUANTIGENE analysis known to those skilled in the art.

[0235] The oligonucleotides or pharmaceutical compositions described herein can be administered topically or systemically, e.g., orally, sublingually, nasally, subcutaneously, intravenously, intraperitoneally, intramuscularly, intratumorally, intrathecally, intracerebroventricularly, transdermally, and / or rectally. The oligonucleotides are administered, for example, alone or in combination with another oligonucleotide of the invention, and optionally in combination with another compound, such as another oligonucleotide, antibody, and / or small molecule different from the invention.

[0236] In addition, two or more oligonucleotides of the present invention are administered together, for example, at the same time point, for example, in the form of a pharmaceutical composition, or separately, or at staggered time intervals. Alternatively or in addition, one or more oligonucleotides described herein may be administered together, for example, at the same time point, for example, in the form of a pharmaceutical composition, or separately, or at staggered time intervals with another compound, such as another oligonucleotide (i.e., different from the present invention), an antibody and / or a small molecule. In some of these combinations, the oligonucleotides of the present invention reduce, for example, LRRK2 mRNA and / or LRRK2 protein expression, and another oligonucleotide (i.e., different from the present invention), an antibody and / or a small molecule inhibits (antagonist) or stimulates (agonist) another factor involved in the formation or progression of a neurodegenerative disease. The factor is, for example, α-synuclein, Tau, TDO, NLRP3, PFKB3, or a combination thereof. The factor may be a factor whose level or activity in a cell, tissue, organ, or subject increases or decreases depending on the cell, tissue, organ, or subject and its individual pathology.

[0237] The oligonucleotides of the present invention are combined, for example, with another therapeutic agent. Suitable therapeutic agents include, but are not limited to, L-dopa, dopamine agonists, monoamine oxidase inhibitors, etc., which are known to those skilled in the art.

[0238] The antibody combined with the oligonucleotide or pharmaceutical composition of the invention is, for example, an anti-α-synuclein such as Prasinezumab (RO7046015 / PRX002), Cinpanemab, ABBV-805, MEDI1341 or LU AF82422 antibody.

[0239] The subject of the present invention is, for example, a mammal (eg, human, dog, cat, horse, cow, pig), bird or fish.

[0240] Example

[0241] The following examples illustrate non-limiting embodiments of the present invention, but the present invention is not limited to these examples. The following experiments were performed on cells expressing LRRK2 endogenously, i.e., the cells do not represent an artificial system comprising a transfected reporter construct. Compared to endogenous systems that are closer to treatment-related in vivo systems, such artificial systems generally show higher inhibition levels and lower IC50 values. In addition, no transfection agent was used in the following experiments, i.e., autonomous delivery (gymnotic delivery) was performed. It is known that transfection agents increase the activity of oligonucleotides, which artificially affect IC50 values ​​(see, e.g., Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, No. 5, 2012). Because the artificial system using a transfection agent is difficult or impossible to convert into a treatment method, and there is no transfection formulation approved for oligonucleotides so far, the following experiments were performed without any transfection agent.

[0242] Example 1: Screening of human LRRK2-specific ASOs for target gene attenuation efficacy in A549 and MDA-MB-231 cells

[0243] LRRK2 specific ASO and gene attenuation efficacy were tested in human A549 cells and MDA-MB-231 cells. The cells were treated with 5 μM concentrations of LRRK2 specific ASO or control oligonucleotides, respectively, without using a transfection reagent. After three days of treatment, the cells were lysed. LRRK2 and HPRT1 mRNA expression was analyzed using QUANTIGENE SINGLEPLEX analysis (THERMOFISHER) and LRRK2 expression values ​​were standardized for HPRT1 values. The results of ASO in A549 cells and MDA-MB-231 cells are shown in Figures 3A to 3D, Table 5, and Figures 4A to 4D as residual LRRK2 mRNA expression relative to cells treated with a mock (set to 1). Use ASOA57162Hi (SEQ ID NO.164), A57147Hi (SEQ ID NO.149), A57208Hi (SEQ ID NO.210), A57187Hi (SEQ ID NO.189), A57146Hi (SEQ ID NO.148), A575302Hi (SEQ ID NO.69), A575307Hi (SEQ ID NO.69), A575308Hi (SEQ ID NO.69), A575309Hi (SEQ ID NO.69), A575310Hi (SEQ ID NO.69), A575313Hi (SEQ ID NO.69), A575321Hi (SEQ ID NO.142), A575323Hi (SEQ ID NO.142), A575326Hi (SEQ ID NO.142), A575327Hi (SEQ ID NO.142), A575335Hi (SEQ ID NO.209), A575337Hi (SEQ ID NO.209), A575338Hi (SEQ ID NO.209), A575341Hi (SEQ ID NO.209), A575349Hi, A575351Hi (SEQ ID NO.210), A575354Hi (SEQ ID NO.210) and A575355Hi (SEQ ID NO.209) respectively treated A549 cells to produce >85% target inhibition (represented by residual LRRK2 mRNA expression of <0.15 compared to cells treated with mock) (Figures 3A to 3D).As shown in Figures 4A to 4D, treatment with ASO A57252H (SEQ ID NO. 254), A57253H (SEQ ID NO. 255), and A57024Hi (SEQ ID NO. 26) resulted in ≥ 75% target inhibition (indicated by residual LRRK2 mRNA expression of ≤ 0.25 compared to mock-treated cells). Table 5 shows the average residual expression of human LRRK2 in A549 cells after ASO treatment (normalized to HPRT1 and compared to mock-treated cells):.

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252] Example 2: Concentration-response curves and IC50 value determination of selected human LRRK2-specific ASOs in A549 cells

[0253] The gene attenuation efficacy of the selected ASOs was tested at different concentrations in human A549 cells. Therefore, the cells were treated with the following concentrations of LRRK2 ASOs without transfection reagents: 10000nM, 3333nM, 1111nM, 370nM, 123nM, 41nM, 14nM. After three days of treatment, the cells were lysed. LRRK2 and HPRT1 mRNA expression was analyzed using QUANTIGENE SINGLEPLEX analysis (THERMOFISHER) and LRRK2 expression values ​​were normalized to HPRT1 values. The concentration-dependent attenuation of LRRK2 mRNA expression is shown in Table 6 below as residual LRRK2 mRNA expression normalized to HPRT1 mRNA expression relative to mock-treated cells (set to 1):

[0254]

[0255]

[0256] Example 3: Attenuation of human LRRK2 mRNA in transgenic mice:

[0257] The biological activity of ASOs targeting human LRRK2 was tested in mice expressing human LRRK2 (The Jackson Laboratory: C57BL / 6J-Tg(LRRK2*G2019S)2AMjff / J, strain number: 018785). These mice express endogenous mouse LRRK2 as well as human LRRK2 under the control of the human promoter / enhancer region within a bacterial artificial chromosome (BAC) transgene. Human LRRK2 expression can be detected in the central nervous system and periphery (Bieri et al., 2019; Sitzia et al., 2022).

[0258] Human LRRK2 transgenic mice were treated with a single intracerebroventricular (ICV, n=11 mice) administration of 100 μg of LRRK2 antisense oligonucleotide by pellet or received PBS as a vehicle control group (n=4 mice). Fourteen days after ICV injection, mice were sacrificed, tissues harvested, and total RNA extracted and processed using real-time PCR analysis to detect LRRK2 mRNA. Treatment with the selected ASOs resulted in a statistically significant reduction in human LRRK2 mRNA in the CNS region ranging from about 40% to about 90% compared to vehicle control.

[0259] Table 7 shows that treatment with ASO (SEQ ID NO. 209) caused a statistically significant decrease (approximately 45%-50%) in the striatum and cortical regions of the central nervous system compared to vehicle controls and normalized to the expression of HPRT1 (housekeeping gene) (Table 7). No statistically significant changes in human LRRK2 mRNA levels were detected in the peripheral tissues lung, liver, blood and kidney in the study group.

[0260] Table 7 shows attenuation of human LRRK2 mRNA in transgenic mice treated with SEQ ID NO. 209 ASO:

[0261]

[0262]

[0263] Table 7 shows the reduction in human LRRK2 mRNA levels detected by real-time PCR in different regions of the central nervous system and peripheral tissues 14 days after ICV injection of 100 μg LRRK2 ASO1 (n=12-13) or PBS vehicle (n=4). Gene expression was normalized to HPRT1 (housekeeping gene) and compared to human LRRK2 mRNA levels detected in animals treated with PBS vehicle (n=4). Differences are shown as standard error of the mean (SEM). Statistical significance was determined using one-way analysis of variance.

[0264] Embodiments of the present invention

[0265] 1. An oligonucleotide comprising 10 to 25 nucleotides, wherein the nucleotide sequence of the oligonucleotide is at least 80% to 90% complementary to the corresponding region in the LRRK2 nucleic acid sequence, and wherein at least one nucleotide is modified and the modification is selected from a modified sugar and optionally a modified internucleoside linkage.

[0266] 2. The oligonucleotide according to embodiment 1, wherein the LRRK2 nucleic acid is the mRNA of leucine-rich repeat kinase 2 (LRRK2) of SEQ ID NO. 1 and / or the pre-mRNA of LRRK2 of SEQ ID NO. 2 and / or a fragment thereof.

[0267] 3. The oligonucleotide of embodiment 1 or 2, wherein the modification is selected from the group consisting of LNA, ENA, cET, 2' fluorine-modified nucleotides, 2' O-methyl-modified nucleotides, 2' O-methoxy-modified nucleotides, FANA, and combinations thereof.

[0268] 4. The oligonucleotide according to any one of embodiments 1 to 3, wherein the modification is located in a sequence of at least 3 nucleotides or at least 5 nucleotides at the 5′ end and / or the 3′ end of the oligonucleotide.

[0269] 5. The oligonucleotide according to any one of embodiments 1 to 4, wherein the modification is located in a sequence of 5 nucleotides at the 5′ end and / or the 3′ end of the oligonucleotide.

[0270] 6. The oligonucleotide of any one of embodiments 1 to 5, wherein the internucleoside linkage is selected from the group consisting of phosphodiester, phosphorothioate, methylphosphonate, methylphosphorothioate, phosphoramidate, phosphorodiamidate, thiophosphoramidate, methylsulfonylphosphoramidate, or a combination thereof.

[0271] 7. An oligonucleotide comprising 10 to 25 nucleotides, at least one nucleotide having a bridging nucleic acid modification selected from the group consisting of: LNA, ENA, cET, 2' fluorine-modified nucleotides, 2'O-methyl-modified nucleotides, 2'O-methoxy-modified nucleotides, FANA and combinations thereof, wherein the modification is located in a sequence of 5 nucleotides at the 5' end and / or 3' end of the oligonucleotide, and wherein the oligonucleotide hybridizes with a transcript or mRNA of leucine-rich repeat kinase 2 (LRRK2) of SEQ ID NO.1, and / or hybridizes with a pre-mRNA and / or a fragment thereof of LRRK2 of SEQ ID NO.2.

[0272] 8. The oligonucleotide of any one of embodiments 1 to 7, wherein the oligonucleotide hybridizes outside the hybridization active region in the following positions or within the hybridization active region in the following positions: position 27001 to 27500, position 102001 to 102500, position 80501 to 81000, position 24501 to 25000, position 114001 to 114500, position 92001 to 92500, position 99501 to 100000, position 101001 to 101000 Position 67001 to 67500, position 15001 to 15500, position 24001 to 24500, position 26501 to 27000, position 79501 ​​to 80000, position 87001 to 87500, position 120501 to 121000, position 142001 to 142500, position 135001 to 135500, position 71001 to 71500, position 140501 to 141000 or a combination thereof.

[0273] 9. The oligonucleotide of any one of embodiments 1 to 8, wherein the oligonucleotide comprises SEQ ID NO.69, SEQ ID NO.210, SEQ ID NO.164, SEQ ID NO.254, SEQ ID NO.222, SEQ ID NO.186, SEQ ID NO.204, SEQ ID NO.142, SEQ ID NO.209, SEQ ID NO.255, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.60, SEQ ID NO.66, SEQ ID NO.162, SEQ ID NO.171, SEQ ID NO.224, SEQ ID NO.241, SEQ ID NO.250, SEQ ID NO.149, SEQ ID NO.256, SEW ID NO.239 or a combination thereof.

[0274] 10. An oligonucleotide according to any one of Embodiments 1 to 9, wherein the oligonucleotide is selected from the group consisting of: +A*+A*+T*A*C*T*A*C*T*A*T*T*T*G*T*T*+T*+C*+C (A57067Hi; SEW ID NO. 69), +T*+A*+C*A*A*T*A*T*T*T*A*T*G*G*T*T*+C*+T*+C (A57208Hi; SEW ID NO. 210), +G*+T*+C*A*T*A*A*G*T*T*G*T*C*A*G*+T*+A*+T (A57162Hi; SEQ ID NO. 164), +G*+A*+C*T*T*C*G*T*T*T*A*A*T*A*C*+C*+A*+G (A57252H; SEQ ID NO. 254), +C*+T*+T*T*G*C*T*T*A*T*T*C*G*T*G*+A*+C*+T (A57220Hi; SEQ ID NO. 222), +T*+T*+T*A*T*T*G*C*A*G*T*C*A*T*A*G*+T*+G*+A (A57184Hi; SEQ ID NO. 186), +C*+T*+T*T*G*T*A*A*A*T*G*A*C*G*T*T*+T*+C*+C (A57202Hi; SEQ ID NO. 204), +T*+G*+A*A*A*T*G*T*C*T*A*T*T*A*G*C*+A*+C*+A (A57140Hi, SEQ ID NO. 142), +A*+C*+A*A*T*A*T*T*T*A*T*G*G*T*T*C*+T*+C*+A (A57207Hi; SEQ ID NO. 209), +G*+G*+A*C*T*T*C*G*T*T*T*A*A*T*A*C*+C*+A*+G (A57253H; SEQ ID NO. 255), +C*+G*+G*C*T*A*A*A*T*G*T*T*T*C*A*G*+T*+T*+C (A57043Hi; SEQ IDNO. 45), +T*+C*+G*G*C*T*A*A*A*T*G*T*T*T*C*+A*+G*+T (A57044Hi; SEQ ID NO. 46), +G*+T*+G*T*A*A*C*T*A*A*G*T*G*A*T*+C*+T*+C (A57058Hi; SEQ ID NO. 60), +G*+A*+T*A*T*T*G*A*C*A*A*C*C*T*G*+C*+T*+G (A57064Hi; SEQ ID NO.66), +T*+G*+T*G*T*T*A*C*C*A*T*A*T*C*C*A*+T*+T*+T(A57160Hi; SEQ ID NO.162), +C*+T*+G*T*G*A*C*T*T*T*G*C*A*T*C*A*+A*+C*+T(A57169Hi; SEQ ID NO.171), +C*+A*+A*T*G*T*A*T*A*C*C*A*T*G*C*+A*+G*+T(A57222Hi; SEQ ID NO.224), +T*+T*+C*C*T*G*A*T*T*A*T*A*C*C*A*T*+A*+T*+G(A57239Hi; SEQ ID NO.241), +A*+T*+G*C*T*C*T*T*A*T*C*C*A*C*A*A*+T*+C*+C(A57248Hi; SEQ ID NO.250), +T*+T*+T*G*A*T*C*T*G*T*A*G*T*A*C*A*+T*+A*+C(A57147Hi; SEQ ID NO.149), +A*+T*+G*G*A*C*T*T*C*G*T*T*T*A*A*T*+A*+C*+C(A57254H; SEQ ID NO.256), +G*+G*+A*C*A*T*G*T*A*T*C*T*T*A*A*T*+A*+G*+T(A57237Hi; SEQ IDNO.239) and combinations thereof, wherein + indicates LNA nucleotides and * indicates phosphorothioate (PTO) linkages between the nucleotides. .

[0275] 11. The oligonucleotide of any one of embodiments 1 to 10, wherein the oligonucleotide has 80% to 99%, 85% to 98%, 90% to 95% or 93% sequence identity to any one of the oligonucleotides of SEQ ID NO. 3 to SEQ ID NO. 5301.

[0276] 12. An oligonucleotide as described in any one of embodiments 1 to 11, 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 consecutive nucleotides of any one of the oligonucleotides of SEQ ID NO.3 to SEQ ID NO.5301.

[0277] 13. The oligonucleotide of any one of embodiments 1 to 12, wherein the oligonucleotide inhibits the expression of LRRK2, LRRK2 mRNA, LRRK2 pre-mRNA, or a combination thereof at nanomolar or micromolar concentrations.

[0278] 14. A pharmaceutical composition comprising the oligonucleotide of any one of embodiments 1 to 13 and a pharmaceutically acceptable carrier, excipient, adjuvant or a combination thereof.

[0279] 15. The pharmaceutical composition of embodiment 14, further comprising a therapeutically active agent.

[0280] 16. An oligonucleotide according to any one of embodiments 1 to 13, or a pharmaceutical composition according to embodiment 14 or 15, for use in a method for preventing and / or treating a human disease, such as a neurodegenerative disease.

[0281] 17. The oligonucleotide or pharmaceutical composition of embodiment 16, wherein the pharmaceutically acceptable carrier, excipient, adjuvant or a combination thereof or the therapeutically active agent is administered separately before or after the delivery of the oligonucleotide, or is administered simultaneously via different delivery methods.

[0282] 18. The oligonucleotide of embodiment 16 or 17, wherein the disease is selected from the group consisting of Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, multiple system atrophy, dementia with Lewy bodies (DLB), pure autonomic failure (PAF), frontotemporal dementia, and prion disease.

[0283] 19. The oligonucleotide or pharmaceutical composition according to any one of embodiments 16 to 18, wherein the oligonucleotide and / or the pharmaceutical composition is for local or systemic administration.

[0284] 20. A method for preventing and / or treating a human disease, comprising administering a therapeutically effective amount of an oligonucleotide according to any one of embodiments 1 to 13 or a pharmaceutical composition according to any one of embodiments 14 or 15.

[0285] 21. The method of embodiment 20, wherein the human disease is selected from the group consisting of Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, multiple system atrophy, dementia with Lewy bodies (DLB), pure autonomic failure (PAF), frontotemporal dementia, and prion disease.

[0286] 22. The method of embodiment 20 or 21, wherein the oligonucleotide or pharmaceutical composition is administered locally or systemically.

Claims

1. An oligonucleotide comprising 10 to 25 nucleotides, wherein the nucleotide sequence of the oligonucleotide is at least 80% to 90% complementary to the corresponding region of the LRRK2 nucleic acid sequence, and wherein at least one nucleotide is modified and the modification is selected from a modified sugar and optionally a modified internucleoside linkage.

2. The oligonucleotide according to claim 1, wherein the LRRK2 nucleic acid is the mRNA of leucine-rich repeat kinase 2 (LRRK2) of SEQ ID NO. 1 and / or the pre-mRNA of LRRK2 of SEQ ID NO. 2 and / or a fragment thereof.

3. The oligonucleotide according to claim 1 or 2, wherein the modification is selected from the group consisting of LNA, ENA, cET, 2' fluorine-modified nucleotides, 2' O-methyl-modified nucleotides, 2' O-methoxy-modified nucleotides, FANA, and combinations thereof. 4 . The oligonucleotide according to claim 1 , wherein the modification is located in a sequence of at least 3 nucleotides or at least 5 nucleotides at the 5′ end and / or the 3′ end of the oligonucleotide.

5. The oligonucleotide according to any one of claims 1 to 4, wherein the modification is located in a sequence of 5 nucleotides at the 5' end and / or the 3' end of the oligonucleotide.

6. An oligonucleotide comprising 10 to 25 nucleotides according to claim 1, at least one nucleotide having a bridging nucleic acid modification selected from the group consisting of: LNA, ENA, cET, 2' fluorine-modified nucleotides, 2' O-methyl-modified nucleotides, 2' O-methoxy-modified nucleotides, FANA and combinations thereof, wherein the modification is located in a sequence of 5 nucleotides at the 5' end and / or 3' end of the oligonucleotide, and wherein the oligonucleotide hybridizes with a transcript or mRNA of leucine-rich repeat kinase 2 (LRRK2) of SEQ ID NO.1, and / or hybridizes with a pre-mRNA and / or a fragment thereof of LRRK2 of SEQ ID NO.

2.

7. The oligonucleotide according to any one of claims 1 to 6, wherein the oligonucleotide hybridizes outside the hybridization active region in the following positions or within the hybridization active region in the following positions: position 27001 to 27500, position 102001 to 102500, position 80501 to 81000, position 24501 to 25000, position 114001 to 114500, position 92001 to 92500, position 99501 to 100000, Position 67001-67500, position 15001-15500, position 24001-24500, position 26501-27000, position 79501-80000, position 87001-87500, position 120501-121000, position 142001-142500, position 135001-135500, position 71001-71500, position 140501-141000 or a combination thereof.

8. The oligonucleotide according to any one of claims 1 to 7, wherein the oligonucleotide comprises SEQ D NO.69, SEQ ID NO.210, SEQ ID NO.164, SEQ ID NO.254, SEQ ID NO.222, SEQ ID NO.186, SEQ ID NO.204, SEQ ID NO.142, SEQ ID NO.209, SEQ ID NO.255, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.60, SEQ ID NO.66, SEQ ID NO.162, SEQ ID NO.171, SEQ ID NO.224, SEQ ID NO.241, SEQ ID NO.250, SEQ D NO.149, SEQ ID NO.256, SEQ ID NO.239 or a combination thereof.

9. The oligonucleotide according to any one of claims 1 to 8, wherein the oligonucleotide is selected from the group consisting of: +A*+A*+T*A*C*T*A*C*T*A*T*T*T*G*T*T*+T*+C*+C (A57067Hi; SEQ ID NO.69), +T*+A*+C*A*A*T*A*T*T*T*A*T*G*G*T*T*+C*+T*+C (A57208Hi; SEQ ID NO.210), +G*+T*+C*A*T*A*A*G*T*T*G*T*C*A*G*+T*+A*+T (A57162Hi; SEQ ID NO.164), +G*+A*+C*T*T*C*G*T*T*T*A*A*T*A*C*+C*+A*+G (A57252H; SEQ ID NO.254), +C*+T*+T*T*G*C*T*T*A*T*T*C*G*T*G*+A*+C*+T (A57220Hi; SEQ ID NO.222), +T*+T*+T*A*T*T*G*C*A*G*T*C*A*T*A*G*+T*+G*+A (A57184Hi; SEQ ID NO.186), +C*+T*+T*T*G*T*A*A*A*T*G*A*C*G*T*T*+T*+C*+C (A57202Hi; SEQ ID NO.204), +T*+G*+A*A*A*T*G*T*C*T*A*T*T*A*G*C*+A*+C*+A (A57140Hi, SEQ ID NO.142), +A*+C*+A*A*T*A*T*T*T*A*T*G*G*T*T*C*+T*+C*+A (A57207Hi; SEQ ID NO.209), +G*+G*+A*C*T*T*C*G*T*T*T*A*A*T*A*C*+C*+A*+G (A57253H; SEQ ID NO.255), +C*+G*+G*C*T*A*A*A*T*G*T*T*T*C*A*G*+T*+T*+C (A57043Hi; SEQ IDNO.45), +T*+C*+G*G*C*T*A*A*A*T*G*T*T*T*C*+A*+G*+T (A57044Hi; SEQ ID NO.46), +G*+T*+G*T*A*A*C*T*A*A*G*T*G*A*T*+C*+T*+C (A57058Hi; SEQ ID NO.60), +G*+A*+T*A*T*T*G*A*C*A*A*C*C*T*G*+C*+T*+G (A57064Hi; SEQ ID NO.66), +T*+G*+T*G*T*T*A*C*C*A*T*A*T*C*C*A*+T*+T*+T (A57160Hi; SEQ ID NO.162), +C*+T*+G*T*G*A*C*T*T*T*G*C*A*T*C*A*+A*+C*+T (A57169Hi; SEQ ID NO.171), +C*+A*+A*T*G*T*A*T*A*C*C*A*T*G*C*+A*+G*+T (A57222Hi; SEQ ID NO.224), +T*+T*+C*C*T*G*A*T*T*A*T*A*C*C*A*T*+A*+T*+G (A57239Hi; SEQ ID NO.241), +A*+T*+G*C*T*C*T*T*A*T*C*C*A*C*A*A*+T*+C*+C (A57248Hi; SEQ ID NO.250), +T*+T*+T*G*A*T*C*T*G*T*A*G*T*A*C*A*+T*+A*+C (A57147Hi; SEQ ID NO.149), +A*+T*+G*G*A*C*T*T*C*G*T*T*T*A*A*T*+A*+C*+C (A57254H; SEQ ID NO.256), +G*+G*+A*C*A*T*G*T*A*T*C*T*T*A*A*T*+A*+G*+T (A57237Hi; SEQ IDNO.239) and combinations thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) linkage between said nucleotides.

10. The oligonucleotide according to any one of claims 1 to 9, wherein the oligonucleotide has 80% to 99%, 85% to 98%, 90% to 95% or 93% sequence identity to any one of the oligonucleotides of SEQ ID NO. 3 to SEQ ID NO. 5301.

11. An oligonucleotide according to any one of claims 1 to 10, 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 consecutive nucleotides of any one of the oligonucleotides of SEQ ID NO. 3 to SEQ ID NO. 5301.

12. The oligonucleotide of any one of claims 1 to 11, wherein the oligonucleotide inhibits expression of LRRK2, LRRK2 mRNA, LRRK2 pre-mRNA, or a combination thereof at nanomolar or micromolar concentrations.

13. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier, excipient, adjuvant or a combination thereof.

14. The pharmaceutical composition according to claim 13, further comprising a therapeutically active agent.

15. An oligonucleotide according to any one of claims 1 to 12, or a pharmaceutical composition according to claim 13 or 14, for use in a method for the prevention and / or treatment of a human disease such as a neurodegenerative disease.

16. The oligonucleotide or pharmaceutical composition according to claim 15, wherein the pharmaceutically acceptable carrier, excipient, adjuvant or combination thereof or the therapeutically active agent is administered separately before or after delivery of the oligonucleotide, or is administered simultaneously via different delivery methods.

17. The oligonucleotide according to claim 15 or 16, wherein the disease is selected from the group consisting of: Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, multiple system atrophy, dementia with Lewy bodies (DLB), pure autonomic failure (PAF), frontotemporal dementia and prion disease.

18. The oligonucleotide or pharmaceutical composition according to any one of claims 15 to 17, wherein the oligonucleotide and / or the pharmaceutical composition is for local or systemic administration.

19. A method for preventing and / or treating a human disease, comprising administering a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 12, or a pharmaceutical composition according to any one of claims 13 or 14.

20. The method of claim 19, wherein the human disease is selected from the group consisting of Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, multiple system atrophy, dementia with Lewy bodies (DLB), pure autonomic failure (PAF), frontotemporal dementia, and prion disease.

21. The method of claim 19 or 20, wherein the oligonucleotide or pharmaceutical composition is administered topically or systemically.

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