Oligonucleotides for reducing expression of leucine-rich repeat kinase 2 (LRRK2) and their use for the prevention and / or treatment of human diseases - Patents.com

Abstract

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

Description

[Technical field]

[0001] The present invention refers to oligonucleotides, particularly antisense oligonucleotides, that contain one or more modified nucleotides and hybridize with 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 technology]

[0002] Neurodegenerative diseases are caused by the progressive loss of neuronal structure or function in a process known as neurodegeneration. Such neuronal damage may ultimately result in 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 in the brain at many different levels of neural circuits.

[0003] Parkinson's disease (PD) is the second most common neurodegenerative disorder, affecting approximately 1 in 100 people over the age of 60. As current standard treatments are limited to symptomatic treatments, disease-modifying or neuroprotective therapies are highly desirable (Lees, Hardy, & Revesz, 2009). Pathological hallmarks of PD include the accumulation of α-synuclein (α-syn) in Lewy bodies (LBs) and Lewy neurites (LNs), as well as the progressive loss of dopaminergic (DA) neurons in the substantia nigra 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 indicates that LRRK2 function is directly involved in the risk of developing PD, underlying disease pathology, and PD progression, thus validating LRRK2 as an attractive therapeutic target. LRRK2 is a large protein of 2527 amino acids that is widely expressed throughout the body, with the 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 carboxyl-terminal ROC (COR) domain, and a serine / threonine protein kinase domain (Cookson, 2017). Taken together, LRRK2 appears to regulate aspects of intracellular vesicular trafficking and maintenance of organelles including the Golgi, endosomes and lysosomes.

[0006] Alterations in LRRK2 function due to genetic, epigenetic or environmental factors, as well as mutations in or around the LRRK2 gene, are directly involved in the development and progression of diseases, including synucleopathies such as PD. Therefore, suppression of its expression can provide an effective therapeutic opportunity for these diseases. Suppression of LRRK2 expression can be achieved, for example, by targeted degradation of the mRNA encoding LRRK2 or by blocking its translation.

[0007] Two competing technologies have been described primarily for specific suppression of mRNA expression: siRNA and antisense oligonucleotides (ASO). siRNA targets mRNA in the cytoplasm. In contrast, RNase H-dependent ASO targets mRNA and / or pre-mRNA in the nucleus. Thereby, intron regions can also be targeted by ASO that are inaccessible to siRNA, resulting in a significantly larger target space for ASO.

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

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

[0010] The mode of action of oligonucleotides differs from that of antibodies or small molecules and oligonucleotides offer significant advantages, for example with regard to: (i) their penetration into tissues, such as brain tissue, which is superior 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); (ii) long-term retention and duration of effect in the brain after direct administration; (iii) blocking each of multiple target functions and activities; (iv) combinations of oligonucleotides with each other or with antibodies or small molecules, and (v) Inhibition of intracellular effects that are inaccessible to antibodies or cannot be inhibited via small molecules. Summary of the Invention [Problem to be solved by the invention]

[0011] Thus, there is a need for new and improved therapies for the treatment of diseases (eg, PD) that are capable of reducing the amount of LRRK2 mRNA and protein with minimal toxic side effects. [Means for solving the problem]

[0012] The present invention refers to an oligonucleotide comprising 10-25 nucleotides, in which at least one nucleotide has a modification selected from the group consisting of bridged nucleic acids such as LNA, ENA, cET, 2'fluoro modified nucleotides, 2'O-methyl modified nucleotides, 2'O-methoxy modified nucleotides, FANA, and combinations thereof, the modification being located in a sequence of 5 nucleotides at the 5' and / or 3' end of the oligonucleotide, and the oligonucleotide hybridizes to a leucine-rich repeat kinase 2 (LRRK2) transcript or mRNA of SEQ ID NO: 1 and / or a pre-mRNA of LRRK2 of SEQ ID NO: 2 and / or a fragment thereof. The fragment results from a biological process including, but not limited to, alternative splicing. The oligonucleotides may be, for example, at positions 102001 to 102500, 27001 to 27500, 80501 to 81000, 24501 to 25000, 114001 to 114500, 92001 to 92500, 99501 to 100000, 67001 to 67500, 15001 to 15500, 24001 to 24500, and 26501 to 27000. , positions 79501 ​​to 80000, positions 87001 to 87500, positions 120501 to 121000, positions 142001 to 142500, positions 135001 to 135500, positions 71001 to 71500, positions 140501 to 141000, or combinations thereof, outside or inside the hybridization active region.

[0013] 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.

[0014] For example, the oligonucleotides can be +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, Sequence number 164), +G*+A*+C*T*T*C*G*T*T*T*A*A*T*A*C*+C* +A*+G(A57252H, Sequence number 254), +C*+T*+T*T*G*C*T*T*A*T*T*C*G*T*G*+A*+C*+T(A57220Hi, Sequence number 2 22), +T*+T*+T*A*T*T*G*C*A*G*T*C*A*T*A*G*+T*+G*+A(A57184Hi, Sequence number 186), +C*+T*+T*T*G *T*A*A*A*T*G*A*C*G*T*T*+T*+C*+C(A57202Hi, Sequence number 204), +T*+G*+A*A*A*T*G*T*C*T*A*T*T *A*G*C*+A*+C*+A(A57140Hi, array number 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, Sequence number 45), +T*+C*+G*G*C*T*A*A*A*T*G*T*T*T*C*+A*+G*+T(A57044Hi, Sequence number 46), +G*+T*+G*T*A*A*C*T*A*A*G*T*G*A*T*+C*+T*+C(A57058Hi, Sequence number 60), +G*+A*+T*A*T*T*G*A*C*A*A*C*C*T*G*+C*+T*+G(A57064Hi, Sequence number 66), +T*+G*+T*G*T*T*A*C*C*A*T*A*T*C*C*A*+T*+T*+T(A57160Hi, Sequence number 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, Sequence number 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, Sequence number 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, Sequence number 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, where + represents an LNA nucleotide and * represents an internucleotide phosphorothioate (PTO) bond. Alternatively, the oligonucleotide has 80-99%, 85-98%, 90-95, or 93% sequence identity to any one of the oligonucleotides of SEQ ID NO: 3 to SEQ ID NO: 5301 of the present invention.

[0015] Furthermore, 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 the present invention set forth in SEQ ID NO: 3 to SEQ ID NO: 5301. The nucleotides are, for example, consecutive nucleotides.

[0016] The oligonucleotides of the invention, for example, inhibit expression of LRRK2, LRRK2 mRNA, LRRK2 pre-mRNA, or a combination thereof, at nanomolar or micromolar concentrations.

[0017] The present invention further relates to a pharmaceutical composition comprising or consisting of the oligonucleotide of the present invention and a stimulating agent such as a pharma- ceutically acceptable carrier, excipient, diluent, adjuvant, or a combination thereof. The pharmaceutical composition may comprise an additional therapeutically active agent. The oligonucleotide of the present invention and the pharmaceutical composition are each for use in a method for the prophylaxis and / or treatment of a human disease, such as, for example, a neurodegenerative disease. The neurodegenerative disease is, for example, 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. The pharma- ceutically acceptable carrier, excipient, adjuvant, or a combination thereof, or the therapeutically active agent may be administered separately, for example, before or after delivery of the oligonucleotide, or may be administered simultaneously, for example, via different delivery methods. Delivery methods may be, for example, oral, sublingual, nasal, subcutaneous, intravenous, intraperitoneal, intramuscular, intratumor, intrathecal, intraventricular, transdermal and / or rectal.

[0018] The oligonucleotides and pharmaceutical compositions of the invention, respectively, are for example intended for local or systemic administration.

[0019] The present invention relates to a method for the prevention and / or treatment of a human disease, comprising administering a therapeutically effective amount of an oligonucleotide of the present invention or a pharmaceutical composition comprising such an oligonucleotide. The human disease, such as a neurological disease, may be 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 diseases. The oligonucleotide or pharmaceutical composition may be administered locally or systemically.

[0020] All documents cited or referenced herein ("documents cited herein"), and all documents cited or referenced in the documents cited herein, as well as any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein, are incorporated by reference herein and may be employed in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0021] [Figure 1-1] The RefSeq ID NM_198578 of LRRK2 mRNA (sequence number 1) is shown. [Figure 1-2] The RefSeq ID NM_198578 of LRRK2 mRNA (sequence number 1) is shown. [Figure 2-1] GRCh38:12:40224997:40369285, in particular LRRK2_GRCh38:12:40224997:40369285:1_seqGenomicENST00000298910.12 (LRRK2 pre-mRNA of sequence number 2). [Figure 2-2] This is a continuation of Figure 2-1. [Figure 2-3] This is a continuation of Figure 2-2. [Figure 2-4]This is a continuation of Figure 2-3. [Figure 2-5] This is a continuation of Figure 2-4. [Figure 2-6] This is a continuation of Figure 2-5. [Diagram 2-7] This is a continuation of Figure 2-6. [Figure 2-8] This is a continuation of Figure 2-7. [Figure 2-9] This is a continuation of Figure 2-8. [Figure 2-10] This is a continuation of Figure 2-9. [Figure 2-11] This is a continuation of Figure 2-10. [Figure 2-12] This is a continuation of Figure 2-11. [Figure 2-13] This is a continuation of Figure 2-12. [Figure 2-14] This is a continuation of Figure 2-13. [Figure 2-15] This is a continuation of Figure 2-14. [Figure 2-16] This is a continuation of Figure 2-15. [Figure 2-17] This is a continuation of Figure 2-16. [Figure 2-18] This is a continuation of Figure 2-17. [Figure 2-19] This is a continuation of Figure 2-18. [Figure 2-20] This is a continuation of Figure 2-19. [Figure 2-21] This is a continuation of Figure 2-20. [Figure 2-22] This is a continuation of Figure 2-21. [Figure 2-23] Continuation of Figure 2-22. [Figure 2-24] This is a continuation of Figure 2-23. [Figure 2-25] This is a continuation of Figure 2-24. [Figure 2-26] Continuation of Figure 2-25. [Figure 2-27] This is a continuation of Figure 2-26. [Figure 2-28] This is a continuation of Figure 2-27. [Figure 3A] Shows screening of human LRRK2-specific ASOs in A549 cells (human lung cancer). [Figure 3B] Shows screening of human LRRK2-specific ASOs in A549 cells (human lung cancer). [Figure 3C] Shows screening of human LRRK2-specific ASOs in A549 cells (human lung cancer). [Figure 3D] Shows screening of human LRRK2-specific ASOs in A549 cells (human lung cancer). [Figure 4A] Shows screening of human LRRK2-specific ASOs in MDA-MB-231 cells (human breast adenocarcinoma). [Figure 4B] Shows screening of human LRRK2-specific ASOs in MDA-MB-231 cells (human breast adenocarcinoma). [Figure 4C] Shows screening of human LRRK2-specific ASOs in MDA-MB-231 cells (human breast adenocarcinoma). [Figure 4D] Shows screening of human LRRK2-specific ASOs in MDA-MB-231 cells (human breast adenocarcinoma). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention provides oligonucleotides that hybridize with the mRNA and / or pre-mRNA sequence of, for example, leucine-rich repeat kinase 2 (LRRK2) from humans. These oligonucleotides hybridize with introns and / or exons and / or exon-exon junctions and / or exon-intron junctions of LRRK2 pre-mRNA, for example, in neuronal or tumor cells or other mammalian cells. Recruitment of enzymes such as RNase H degrades the pre-mRNA and reduces the level of LRRK2 mRNA. As a result, the production of LRRK2 protein is prevented and the level of LRRK2 protein is reduced, for example, in neuronal or tumor cells or other mammalian cells. As a result, the level of LRRK2 protein and its downstream effects are reduced. The oligonucleotides of the present invention represent promising and highly efficient tools for use in methods of prevention and / or treatment of disorders (e.g., neurodegenerative disorders) in which the expression, function, and / or activity of LRRK2 protein, respectively, are involved in the development and progression of the disease.

[0023] The oligonucleotides of the invention hybridize, for example, to the LRRK2 mRNA of SEQ ID NO: 1 (RefSeqIDNM_198578) and / or the LRRK2 pre-mRNA of SEQ ID NO: 2 (GRCh38:12:40224997:40369285).

[0024] The elements of the present invention are described in more detail below. Although these elements are listed in certain embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The examples and embodiments provided herein are not intended to limit the present invention to only the embodiments explicitly described. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutation or combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context dictates otherwise.

[0025] Throughout this specification and the claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated member, integer or step, or group of members, integers or steps, but not the exclusion of any other member, integer or step, or group of members, integers or steps. The terms "a" and "an" and "the" and similar references used in the context of describing the invention (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if set forth individually herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as," "for example") provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0026] The oligonucleotide of the present invention is, for example, an antisense oligonucleotide consisting of 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, or includes them. The oligonucleotide consists of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, or includes them. The oligonucleotide of the present invention includes at least one nucleotide that is modified. The modified nucleotide can be, for example, a bridged nucleotide such as a locked nucleic acid (LNA, for example, 2',4'-LNA), cET, ENA, a 2'fluoro-modified nucleotide, a 2'O-methyl-modified nucleotide, or a combination thereof. The oligonucleotide of the present invention includes, for example, nucleotides having one or more, two or more, three or more, or four or more of the same or different modifications. Additionally, the oligonucleotides of the present invention optionally comprise a modified phosphate backbone, where the phosphate is, for example, a phosphodiester, phosphorothioate, methylphosphonate, methyl phosphorothioate, phosphoramidate, phosphorodiamidate, thiophosphoramidate, mesyl phosphoramidate, or combinations thereof.

[0027] "Reducing" according to the present invention includes inhibiting an effect such as expression in different percentages and amounts.

[0028] The present invention refers to the provision of oligonucleotides, such as antisense oligonucleotides, that mediate the restriction of available LRKK2 mRNA for protein expression. To restrict protein expression, the oligonucleotide requires the presence of a complementary nucleic acid sequence that represents a hybridization target that allows the formation of a heteroduplex. The oligonucleotides of the present invention hybridize 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 leads to enzymes such as RNaseH-mediated degradation or inactivation of the target RNA, or termination of transcription, thus restricting the amount of available LRKK2 mRNA for protein expression.

[0029] Furthermore, the present invention relates to oligonucleotides of the present invention that contain one or more, two or more, three or more, or four or more modified nucleotides, for example at the 3' and / or 5' end of the oligonucleotide and / or at any position within the oligonucleotide, where the modified nucleotides are followed by a line 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 embodiments of oligonucleotides that contain modified nucleotides, for example, LNA, indicated by (+), and phosphorothioate (PTO), indicated by (*). Oligonucleotides that consist of or contain the sequences of Table 1 may also contain any other modified nucleotides, or any other combination of modified and unmodified nucleotides. The oligonucleotides of Table 1 hybridize to human LRKK2 mRNA. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

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Table 1-156

[0030] Table 1: List of human LRRK2-specific ASOs and control oligonucleotides. "H" after the ASO name indicates a human LRRK2-specific sequence that binds to the exon region of pre-mRNA, and "Hi" after the ASO name indicates a human LRRK2-specific sequence that binds to the intron region of pre-mRNA. 16-, 17-, 18- and 19-mers were designed according to in-house standards, and Neg1 (described in WO2014154843A1), R01002, R01011 and R01025 were used as non-targeting control oligonucleotides in some experiments.

[0031] The ASO of the present invention hybridizes with, for example, a hybridization active region. Such a region may be, for example, a region located at positions 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, 125 01~13000, 13001~13500, 13501~1400, 14501~15000, 15001~15500, 15501~16000, 16001~16500, 16501~17000, 17001~17500, 17501~18000, 18001~18500, 18501~19000, 19001~19500, 20001~20500, 20501~21000, 21001~21500, 21501~22000, 22001~22500, 23001~23500, 23501~24000, 240 01~24500, 24501~25000, 25001~25500, 26001~26500, 26501~27000, 27001~27500, 27501~28000, 28001~28500, 29001~29500, 29501~30000, 30001~30500, 30501~31000, 31001~31500, 32001~32500, 33001~33500, 33501~34000, 34001~34500, 34501~35000, 35001~35500, 35501~36000, 3 6001~36500, 36501~37000, 37001~37500, 37501~38000, 38001~38500, 38501~39000, 39001~39500, 39501~40000, 40001~40500, 40501~41000, 42001~42500, 42501~43000, 43001~43500, 43501~44000, 44001~44500, 44501~45000, 45001~45500, 45501~46000, 46001~46500, 46501~47000,47001~47500、47501~48000、48001~48500、48501~49000、49001~49500、49501~50000、50001~50500、50501~51000、51001~51500、51501~52000、52001~52500、52501~53000、53001~53500、53501~54000、54001~54500、54501~55000、55001~55500、56001~56500、56501~57000、57001~57500、57501~58000、58001~58500、58501~59000、59001~59500、59501~60000、60001~60500、60501~61000、61001~61500、61501~62000、62001~62500、62501~63000、63001~63500、63501~64000、64001~64500、64501~65000、65001~65500、65501~66000、66001~66500、66501~67000、67001~67500、67501~68000、68001~68500、68501~69000、69001~69500、69501~70000、70001~70500、70501~71000、71001~71500、71501~72000、72001~72500、72501~73000、73001~73500、73501~74000、74001~74500、74501~75000、75001~75500、75501~76000、76001~76500、76501~77000、77001~77500、77501~78000、78001~78500、78501~79000、79001~79500、79501~80000、80001~80500、80501~81000、81001~81500、81501~82000、82001~82500、82501~83000、83001~83500、83501~84000、84001~84500、84501~85000、85001~85500、85501~86000、86001~86500、86501~87000、87001~87500、87501~88000、88001~88500、88501~89000、89001~89500、89501~90000、90001~90500、90501~91000、91001~91500、91501~92000、92001~92500、92501~93000、93001~93500、93501~94000、94001~94500、94501~95000、95001~95500、95501~96000、96001~96500、96501~97000、97001~97500、97501~98000、98001~98500、98501~99000、99001~99500、99501~100000、100001~100500、100501~101000、101001~101500、101501~102000、102001~102500、102501~103000、103001~103500、103501~104000、104001~104500、105001~105500、105501~106000、106001~106500、106501~107000、107001~107500、108001~108500、108501~109000、109001~109500、109501~110000、110001~110500、110501~111000、111001~111500、111501~112000、112001~112500、112501~113000、113001~113500、113501~114000、114001~114500、114501~115000、115001~115500、115501~116000、116001~116500、116501~117000、117001~117500、117501~118000、118001~118500、118501~119000、119001~119500、119501~120000、120001~120500、120501~121000、121001~121500、122001~122500、122501~123000、123001~123500、124001~124500、124501~125000、125001~126000、126001~126500、127001~127500、129001~129500、129501~130000、130001~130500、131001~131500, 131501~132000, 132501~133000, 134001~134500, 134501~135000, 135001~135500, 135501~136000, 136001~136500, 136501~137000, 137001~137500, 137501~138000, 138001~138500, 138501~1 39000, 139001-139500, 139501-140000, 140001-140500, 140501-141000, 141001-141500, 141501-142000, 142001-142500, 142501-143000, 143001-143500, 143501-144000, or 144001-144173. 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

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

Table 2-38

Table 2-39

Table 2-40

Table 2-41

Table 2-42

Table 2-43

Table 2-44

Table 2-45

Table 2-46

Table 2-47

Table 2-48

Table 2-49

Table 2-50

Table 2-51

Table 2-52

Table 2-53

Table 2-54

Table 2-55

Table 2-56

Table 2-57

Table 2-58

Table 2-59

Table 2-60

Table 2-61

Table 2-62

Table 2-63

Table 2-64

Table 2-65

Table 2-66

Table 2-67

Table 2-68

Table 2-69

Table 2-70

Table 2-71

Table 2-72

Table 2-73

Table 2-74

Table 2-75

Table 2-76

Table 2-77

Table 2-78

Table 2-79

Table 2-80

Table 2-81

Table 2-82

Table 2-83

Table 2-84

Table 2-85

Table 2-86

Table 2-87

Table 2-88

Table 2-89

Table 2-90

Table 2-91

Table 2-92

Table 2-93

Table 2-94

Table 2-95

Table 2-96

Table 2-97

Table 2-98

Table 2-99

Table 2-100

Table 2-101

Table 2-102

Table 2-103

Table 2-104

Table 2-105

Table 2-106

Table 2-107

Table 2-108

Table 2-109

Table 2-110

Table 2-111

Table 2-112

[0032] The oligonucleotide of the present invention hybridizes with, for example, human LRRK2 mRNA and / or pre-mRNA of SEQ ID NO: 1 and / or SEQ ID NO: 2. Such oligonucleotides are called LRRK2 antisense oligonucleotides. The oligonucleotide of the present invention is, for example, an antisense oligonucleotide, and is shown in Table 1. The present invention further refers 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 to the oligonucleotides in Table 1.

[0033] Furthermore, the present invention refers 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 set forth in SEQ ID NO: 3 to SEQ ID NO: 5301.

[0034] Each nucleotide of the sequence may be modified, and the ASO of the invention preferably comprises a core of 6 to 8 unmodified nucleotides. The ASO of the invention comprises one or more modified nucleotides, for example 1, 2, 3, 4 or 5 nucleotides, for example at the 5' and / or 3' end of the oligonucleotide, i.e. at the 5' and / or 3' side of the core. The 5' and 3' ends can be identically modified or differently modified. When the 5' and 3' ends are identically modified, the nucleotides are modified at the same position counted from the 5' and 3' ends, respectively (in both cases starting counting at 1 from the ends), with the same modification, for example an LNA modification. When the 5' and 3' ends are differently modified, the position of the modified nucleotide and / or the type of modification at the 5' and 3' ends are different, the type of nucleotide modification being the same (for example LNA) or different. The modified nucleotides, such as LNA modified nucleotides, do not have to follow each other contiguously, but can be separated by one or more unmodified nucleotides. Exemplary modification patterns for the 5' and 3' ends of ASOs of the invention are set forth where unmodified nucleotides are indicated with an "_" and the number on either side of the "_" refers to the number of modified nucleotides, such as LNA modified nucleotides, in the row. The modified nucleotides may be located anywhere at the 5' and / or 3' ends of the ASO, as shown in Table 3 below. [Table 3]

[0035] The specific positions of the LNA modifications at the 5' and 3' ends of each ASO are shown in Table 4. [Table 4]

[0036] In some embodiments, the oligonucleotides of the present invention can reduce the amount of LRRK2 mRNA and / or LRRK2 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%. Reduction in the amount of LRRK2 mRNA and / or LRRK2 protein expression can be determined by comparing the amount of LRRK2 mRNA and / or LRRK2 protein expression in a sample treated with the oligonucleotide of the present invention to a corresponding untreated control. An untreated control is, for example, LRRK2, LRRK2 mRNA, LRRK2 pre-mRNA expression in a subject prior to administration of an oligonucleotide of the invention, or a combination thereof, or is an untreated sample of cells, blood, urine, saliva, etc. An untreated sample is, for example, taken from a subject prior to administration of an oligonucleotide of the invention.

[0037] The oligonucleotides of the invention reduce the amount of LRRK2 mRNA and / or expression of 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 1, 10 or 100 μM.

[0038] 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.

[0039] The present invention also refers to a pharmaceutical composition comprising the oligonucleotides described herein and pharma- ceutically acceptable carriers, excipients, stimulants, adjuvants, and / or diluents. Optionally, the pharmaceutical composition further comprises another oligonucleotide different from the present invention, a small molecule such as an antibody and / or a therapeutic active agent for use in the prophylaxis and / or treatment of human diseases, e.g., PD-prevalent and severe neurodegenerative diseases.

[0040] The oligonucleotide or pharmaceutical composition of the invention is for use in a method for the prevention and / or treatment of a disorder, such as, for example, a neurodegenerative disorder, such as, 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.

[0041] The disorder is characterized by LRRK2 activity, such as, for example, 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 amounts of LRRK2 mRNA and / or LRRK2 protein expression. Overactivity of LRRK2 function is caused, for example, by activating mutations, or by unknown genetic, epigenetic, or environmental mechanisms.

[0042] LRRK2 mRNA and protein levels, respectively, can be measured by any standard method known to those of skill in the art, such as immunohistochemistry, Western blot, quantitative real-time PCR, or the QUANTIGENE assay, as known to those of skill in the art.

[0043] The oligonucleotides or pharmaceutical compositions described herein can be administered locally or systemically, for example, 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, optionally in combination with another compound, such as another oligonucleotide different from the invention, an antibody, and / or a small molecule.

[0044] Furthermore, two or more oligonucleotides of the present invention may be administered, for example, simultaneously, for example, in a pharmaceutical composition, or separately, or at staggered intervals. Alternatively or additionally, one or more oligonucleotides described herein may be administered together with another compound, such as another oligonucleotide (i.e., different from the present invention), an antibody, and / or a small molecule, for example, at the same time in a pharmaceutical composition, or separately, or at staggered intervals. In some of these combinations, the oligonucleotide of the present invention, for example, reduces the amount of LRRK2 mRNA and / or LRRK2 protein expression, and the other oligonucleotide (i.e., different from the present invention), an antibody, and / or a small molecule inhibits (antagonist) or stimulates (agonist) a further factor involved in the formation or progression of a neurodegenerative disease. The factor may be, for example, α-synuclein, Tau, TDO, NLRP3, PFKB3, or a combination thereof. The factor may be a factor whose level or activity is increased or decreased in a cell, tissue, organ, or subject, depending on the cell, tissue, organ, or subject, and its individual condition.

[0045] The oligonucleotides of the invention may, for example, be combined with another therapeutically active agent. Suitable therapeutically active agents include, but are not limited to, L-dopa, dopamine agonists, monoamine oxidase inhibitors, and the like.

[0046] The antibody combined with the oligonucleotide or pharmaceutical composition of the invention is, for example, an anti-alpha-synuclein antibody, such as prasinezumab (RO7046015 / PRX002), simpanemab, ABBV-805, MEDI1341 or LU AF82422 antibody.

[0047] The subject of the present invention is, for example, a mammal (eg, human, dog, cat, horse, cow, pig), bird, or fish. EXAMPLES

[0048] 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 that endogenously express LRRK2, i.e., the cells do not represent an artificial system that contains a transfected reporter construct. Such artificial systems generally exhibit higher inhibition and lower IC than endogenous systems that are closer to the therapeutically relevant in vivo system. 50 Furthermore, in the following experiments, no transfection agent was used, i.e., gymnotic delivery was performed. The transfection agent was IC 50 It is known that transfection agents increase the activity of oligonucleotides that artificially affect the expression level of the target gene (see, for example, Zhang et al., Gene Therapy, 2011, 18, 326-333; Stanton et al., Nucleic Acid Therapeutics, Vol. 22, No. 5, 2012). Artificial systems using transfection agents are difficult or impossible to translate into therapeutic approaches, and no transfection agents have been approved for oligonucleotides so far, so the following experiments were performed without transfection agents.

[0049] Example 1: Targeted knockdown efficacy screening of human LRRK2-specific ASOs in A549 and MDA-MB-231 cells The knockdown efficacy of LRRK2-specific ASO was tested in human A549 and MDA-MB-231 cells. Cells were treated with each LRRK2-specific ASO or control oligonucleotide at a concentration of 5 μM without the use of transfection reagent. After 3 days of treatment, cells were lysed. LRRK2 and HPRT1 mRNA expression was analyzed using the QUANTIGENE SINGLEPLEX assay (THERMOFISHER), and LRRK2 expression values ​​were normalized to HPRT1 values. The results of ASO in A549 and MDA-MB-231 cells are shown as the remaining LRRK2 mRNA expression compared to mock-treated cells (set as 1) in Figures 3A-3D, Table 5, and Figures 4A-4D. ASO A57162Hi (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), A57 Treatment of A549 cells with 5326Hi (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) each resulted in >85% target inhibition (represented by residual LRRK2 mRNA expression of <0.15 compared to mock-treated cells) (Figures 3A-3D). As shown in Figures 4A-4D, treatment with ASOs A57252H (sequence number 254), A57253H (sequence number 255) and A57024Hi (sequence number 26) resulted in ≧75% target inhibition (represented by residual LRRK2 mRNA expression of ≦0.25 compared to mock-treated cells).Table 5 shows the mean remaining expression of human LRRK2 (normalized to HPRT1 and compared to mock-treated cells) in A549 cells following ASO treatment. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7]

[0050] Example 2: Concentration-response curves and IC of selected human LRRK2-specific ASOs in A549 cells 50 Determining Values The knockdown efficacy of selected ASOs was tested in human A549 cells at different concentrations. Thus, cells were treated with each LRRK2 ASO at the following concentrations: 10000nM, 3333nM, 1111nM, 370nM, 123nM, 41nM, 14nM without using transfection reagent. After 3 days of treatment, cells were lysed. LRRK2 and HPRT1 mRNA expression was analyzed using QUANTIGENE SINGLEPLEX assay (THERMOFISHER), and LRRK2 expression values ​​were normalized to HPRT1 values. The concentration-dependent knockdown of LRRK2 mRNA expression is shown in Table 6 below as the remaining LRRK2 mRNA expression normalized to HPRT1 mRNA expression compared to mock-treated cells (set as 1). [Table 6]

[0051] Example 3: Knockdown of human LRRK2 mRNA in transgenic mice: The bioactivity of ASOs targeting human LRRK2 was tested in mice expressing human LRRK2 (The Jackson Laboratory: C57BL / 6J-Tg(LRRK2*G2019S)2AMjff / J, stain number: 018785). These mice express endogenous mouse LRRK2 as well as human LRRK2 under the control of a 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).

[0052] Human LRRK2 transgenic mice were treated with a single intracerebroventricular (ICV, n=11 mice) bolus dose of 100 μg of LRRK2 antisense oligonucleotide or received PBS as a vehicle control group (n=4 mice). 14 days after ICV injection, mice were sacrificed, tissues were harvested, total RNA was extracted, and processed to detect LRRK2 mRNA using real-time PCR analysis. Treatment with selected ASOs resulted in a statistically significant reduction of human LRRK2 mRNA in CNS regions, ranging from about 40% to about 90%, compared to vehicle controls.

[0053] Table 7 shows that treatment with ASO (SEQ ID NO: 209) resulted in a statistically significant reduction (approximately 45-50%) of human LRRK2 mRNA in the striatal and cortical regions of the central nervous system compared to vehicle controls, normalized to expression of the housekeeping gene, HPRT1 (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 cohort.

[0054] Table 7 shows human LRRK2 mRNA knockdown in transgenic mice treated with sequence number 209 ASO. [Table 7]

[0055] Table 7 shows the reduction in human LRRK2 mRNA levels in different regions of the central nervous system and peripheral tissues as detected by real-time PCR 14 days after ICV injection of either 100 μg LRRK2 ASO1 (n=12-13) or PBS vehicle (n=4).

[0056] Gene expression was normalized to the housekeeping gene, HPRT1, and compared to human LRRK2 mRNA levels detected in PBS vehicle-treated animals (n=4). Variance is displayed as standard error of the mean (SEM). One-way ANOVA was used to determine statistical significance.

[0057] EMBODIMENTS OF THE PRESENT DISCLOSURE 1. An oligonucleotide comprising 10-25 nucleotides, wherein the sequence of nucleotides of the oligonucleotide is at least 80-90% complementary to the corresponding region of the LRRK2 nucleic acid sequence, and wherein at least one nucleotide is modified, the modification being selected from a modified sugar and an optionally modified internucleoside linkage.

[0058] 2. The oligonucleotide of embodiment 1, wherein the LRRK2 nucleic acid is the leucine-rich repeat kinase 2 (LRRK2) mRNA of SEQ ID NO: 1, and / or the LRRK2 pre-mRNA of SEQ ID NO: 2, and / or fragments thereof.

[0059] 3. The oligonucleotide of embodiment 1 or 2, wherein the modification is selected from the group consisting of LNA, ENA, cET, 2'fluoro modified nucleotides, 2'O-methyl modified nucleotides, 2'O-methoxy modified nucleotides, FANA, and combinations thereof.

[0060] 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.

[0061] 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.

[0062] 6. The oligonucleotide according to any one of embodiments 1 to 5, wherein the internucleoside linkages are selected from the group consisting of phosphodiester, phosphorothioate, methylphosphonate, methyl phosphorothioate, phosphoramidate, phosphorodiamidate, thiophosphoramidate, mesyl phosphoramidate, or combinations thereof.

[0063] 7. An oligonucleotide comprising 10 to 25 nucleotides, wherein at least one nucleotide has a bridged nucleic acid modification selected from the group consisting of LNA, ENA, cET, 2'fluoro modified nucleotides, 2'O-methyl modified nucleotides, 2'O-methoxy modified nucleotides, FANA, and combinations thereof, said modification being located in a sequence of 5 nucleotides at the 5' end and / or 3' end of the oligonucleotide, and wherein the oligonucleotide hybridizes to a leucine-rich repeat kinase 2 (LRRK2) transcript or mRNA of SEQ ID NO: 1 and / or a LRRK2 pre-mRNA of SEQ ID NO: 2 and / or a fragment thereof.

[0064] 8. The oligonucleotide is selected from the group consisting of positions 27001 to 27500, 102001 to 102500, 80501 to 81000, 24501 to 25000, 114001 to 114500, 92001 to 92500, 99501 to 100000, 67001 to 67500, 15001 to 15500, 24001 to 24500, 26501 to 27000, and 79501 ​​to 80000; The oligonucleotide according to any one of embodiments 1 to 7, which hybridizes outside or inside the hybridization active region of positions 87001 to 87500, positions 120501 to 121000, positions 142001 to 142500, positions 135001 to 135500, positions 71001 to 71500, positions 140501 to 141000, or combinations thereof.

[0065] 9. The oligonucleotide of any one of embodiments 1-8, 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, SEQ ID NO:239, or a combination thereof.

[0066] 10. The oligonucleotide is +A*+A*+T*A*C*T*A*C*T*A*T*T*T*G*T*T*+T*+C*+C(A57067Hi, Sequence number 69), +T*+A*+C*A*A*T*A*T*T*T*A*T*G*G*T*T*+C*+T*+C(A57208Hi, serial number 210), +G*+T*+C*A*T*A*A*G*T*T*G*T*C*A*G*+T*+A*+T(A57162Hi, serial number 164), +G*+A*+C*T*T*C* G*T*T*T*A*A*T*A*C*+C*+A*+G(A57252H, configuration number 254), +C*+T*+T*T*G*C*T*T*A*T*T* C*G*T*G*+A*+C*+T (A57220Hi, configuration number 222), +T*+T*+T*A*T*T*G*C*A*G*T*C*A*T*A*G *+T*+G*+A(A57184Hi, serial number 186), +C*+T*+T*T*G*T*A*A*A*T*G*A*C*G*T*T*+T*+C*+C(A57202Hi, serial number 204)), +T*+G*+A*A*A*T*G*T*C*T*A*T*T*A*G*C*+A*+C*+A(A57 140Hi, serial number 142), +A*+C*+A*A*T*A*T*T*T*A*T*G*G*T*T*C*+T*+C*+A(A57207Hi, serial number 209), +G*+G*+A*C*T*T*C*G*T*T*T*A*A*T*A*C*+C*+A*+G(A57253H, serial number 255), +C*+G*+G*C*T*A*A*A*T*G*T*T*T*C*A*G*+T*+T*+C(A57043Hi, serial number 45), +T*+C*+G*G*C*T*A*A*A*T*G*T*T*T*C*+A*+G*+T(A57044Hi, serial number 46), +G*+T*+G*T*A*A*C*T*A*A*G*T*G*A*T*+C*+T*+C(A57058Hi, serial number 60), +G*+A*+T*A*T*T*G*A*C*A*A*C*C*T*G*+C*+T*+G(A57064Hi, serial number 66), +T*+G*+T*G*T*T*A*C*C*A*T*A*T*C*C*A*+T*+T*+T(A57160Hi, serial number 162), +C*+T*+G*T*G*A*C*T*T*T*G*C*A*T*C*A*+A*+C*+T(A57169Hi, serial number 171), +C*+A*+A*T*G*T*A*T*A*C*C*A*T*G*C*+A*+G*+T(A57222Hi, Sequence number 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, Sequence number 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, Sequence number 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 an internucleotide phosphorothioate (PTO) bond.

[0067] 11. The oligonucleotide according to 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.

[0068] 12. The oligonucleotide according to 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 in sequence number 3 to SEQ ID NO:5301.

[0069] 13. The oligonucleotide of any one of embodiments 1-12, wherein the oligonucleotide inhibits expression of LRRK2, LRRK2 mRNA, LRRK2 pre-mRNA, or a combination thereof at nanomolar or micromolar concentrations.

[0070] 14. A pharmaceutical composition comprising an oligonucleotide according to any one of embodiments 1 to 13, and a pharma- ceutically acceptable carrier, excipient, adjuvant, or combination thereof.

[0071] 15. The pharmaceutical composition according to embodiment 14, further comprising a therapeutically active agent.

[0072] 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 the prevention and / or treatment of a human disease, such as a neurodegenerative disease.

[0073] 17. The oligonucleotide or pharmaceutical composition according to embodiment 16, wherein a pharma- ceutically acceptable carrier, excipient, adjuvant, or combination thereof, or a therapeutically active agent is administered separately before or after delivery of the oligonucleotide, or simultaneously via a different delivery method.

[0074] 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 diseases.

[0075] 19. The oligonucleotide or pharmaceutical composition for use according to any one of embodiments 16 to 18, wherein the oligonucleotide and / or pharmaceutical composition is for local or systemic administration.

[0076] 20. A method for the prevention and / or treatment of a human disease, comprising administering a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 13, or a pharmaceutical composition according to any one of embodiments 14 or 15.

[0077] 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 paradementia, and prion diseases.

[0078] 22. The method of embodiment 20 or 21, wherein the oligonucleotide or pharmaceutical composition is administered locally or systemically.

Claims

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

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

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

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

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

7. The oligonucleotide is at positions 27001-27500, 102001-102500, 80501-81000, 24501-25000, 114001-114500, 92001-92500, 99501-100000, 67001-67500, 15001-15500, 24001-24500, 26501-27000, 79501-80000 7. The oligonucleotide according to claim 1 , which hybridizes outside or inside the hybridization active region of positions 87001 to 87500, 120501 to 121000, 142001 to 142500, 135001 to 135500, 71001 to 71500, 140501 to 141000, or a combination thereof.

8. 8. The oligonucleotide of any one of claims 1 to 7, 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, SEQ ID NO:239, or a combination thereof.

9. The oligonucleotide is +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*A*G*T*T*T*G*T*C*A*G*+T*A*+T (A57162Hi, SEQ ID NO: 164), +G*+A*+C*T*C*T*T*T*A*A*+C*+A*+G (A57252H, SEQ ID NO: 254), +C*+T*+T*T*G*C*T*A*T*T*C*G*T*G*G*+A*+C*+T (A57220Hi, SEQ ID NO: 222), +T*+T*+T*+T*A*T*T*G*C*A*G*G*T*+G*+A (A57184Hi, SEQ ID NO: 186), +C*+T*+T*T*T*G*A*A*A*T*A*T*T*T*T*+C*+C*+C (A57202Hi, SEQ ID NO: 204)), +T*+G*+A*A*A*T*G*T*C*T*A*T*T*A*G*C*+A*+A (A57140Hi, SEQ ID NO: 142), +A*+C*+A*A*T*A*T*T*T*A*T*A*T*T*G*T*T*C*+C*+A (A57207Hi, SEQ ID NO: 209), +G*+G*+A*C*T*T*T*A*A*T*A*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 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 ID NO: 239), and combinations thereof, wherein + indicates an LNA nucleotide and * indicates a phosphorothioate (PTO) bond between said nucleotides.

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

11. The oligonucleotide of 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 of any one of claims 1 to 12 and a pharma- ceutically acceptable carrier, excipient, adjuvant, or combination thereof.

14. 14. The pharmaceutical composition of 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 of claim 15, wherein the pharma- ceutically acceptable carrier, excipient, adjuvant, or combination thereof, or the therapeutically active agent is administered separately before or after delivery of the oligonucleotide, or administered simultaneously via different delivery methods.

17. 17. The oligonucleotide of 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 lobe dementia, and prion disease.

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

19. A method for the prevention and / or treatment of 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 claim 13 or 14.

20. 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 paradementia, and prion diseases.

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

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