Antimir-27b for treatment of parkinson's disease

Abstract

The present invention relates to the use of antimiR-27b compounds for treatment or alleviation of Parkinsons Disease. The compounds of the invention have been found to be capable of preventing dopaminergic neuronal degradation in PD models, as well as influencing important pathways relevant for development of Parkinsons Disease.

Description

[0001] P477937PC00 AntimiR-27b for treatment of Parkinson’s diseaseField of the invention The present invention relates to compounds and compositions capable of inhibiting the activity ofmicroRNA-27b (miR-27b) in mammals such as humans. In particular, the invention providesantisense oligonucleotide compounds capable of modulating the activity of miR-27b in a humanin vivo useful for treating CNS disorders such as Parkinson’s disease.BackgroundParkinson’s disease (PD) is a serious, chronic, progressive neurological disorder that affects thenervous system and causes unintended or uncontrollable movements, such as shaking, stiffness,and difficulty with balance and coordination. Common symptoms may include tremor and stiffnessor slowing of movement (bradykinesia), rigid muscles, impaired posture and balance, loss of automatic movements, and speech changes.The hallmarks of PD include the progressive loss of dopamine producing cells in the substantianigra. Further, during progress of PD, loss of motor neurons in the Thalamus area is an importantfeature.The standard of care for PD is dopamine replacement, which often is an effective treatment ofsymptoms, but as the disease progresses, dopamine therapy becomes less effective.There is a great need for new treatments of PD, for use in the early stages before the neuronal loss is too significant, as well as for treating later stages where present dopamine therapy does not work.The present invention uses RNA therapeutics to modulate microRNAs (miRNAs). miRNAs aresmall non-coding RNAs that negatively regulate their target mRNAs by imperfect base pairingwith complementary target sites of the 3’ untranslated regions leading to translational repression and / or target mRNA degradation. Each miRNA regulates the expression of multiple mRNAs and can influence several disease-associated pathways simultaneously. The miR-27 family is comprised of miR-27a and miR-27b, which are highly expressed in thesubstantia nigra and thalamus [1, 2]; brain areas affected in the pathogenesis of PD. Notably, theexpression of miR-27 is dysregulated in peripheral blood mononuclear cells [3] and in plasma [4]of sporadic PD patients [5], linking miR-27 dysregulation to PD. In addition, miR-27 has beenshown to be upregulated at the early stages of PD [3].Mitochondria constitute the core structure of cellular metabolism, and mitophagy, the selective degradation of mitochondria, is key to maintaining mitochondrial function and neuronal P477937PC00homeostasis, and thereby cellular health [7]. Dysfunction of mitophagy is a common feature ofmany neurodegenerative diseases and leads to accumulation of damaged mitochondria andsubsequent neurodegeneration [8]. Importantly, mitophagy has been shown to play a crucial rolein PD pathogenesis, and to be impaired in both idiopathic and familial PD patients, where severalmutations in mitophagy-related genes have been identified [9]. Furthermore, mitophagy inductionhas shown beneficial properties in several preclinical PD models [7, 10, 11]. PTEN-inducedputative kinase 1 (PINK1) is a sensor of mitochondrial damage and master regulator of mitophagy, which functions by recruiting Parkin and thereby initiating mitochondrial breakdown. PINK1 is a direct target of miR-27b, and knockdown of miR-27b has been found to increase the expressionof PINK1 and induce mitophagy [12, 13]. At the same time, knockdown of miR-27a leads toimproved mitochondrial function measured through metabolic reprogramming via PGC1-a

[0014] .In addition to modulating mitophagy, miR-27b has also been shown to regulate immune responses, which are strongly linked to the aetiology and progression of PD. Microglia exert manyfunctions in the CNS and are the brain's resident immune cells

[0015] . Once activated, microgliamigrate to the site of injury (microgliosis) where they phagocytize cellular debris and secrete pro- and anti-inflammatory factors, which during a pathological chronic activation state result in a feed-forward cycle promoting further inflammation and neurodegeneration

[0016] . Increased CNSinflammation is strongly linked to PD though the exact pathogenesis is not determined

[0017] . Interestingly, miR-27b has been shown to promote an activated microglial phenotype, whileinhibition of miR-27b has been found to dampen inflammatory responses, including cytokinerelease

[0018] . Similarly, levels of miR-27 are increased in T cells of multiple sclerosis patients, andtreatment of patient-derived cells with miR-27 inhibitors has led to decreased proinflammatorycytokine production

[0019] .Oxidative stress has also been indicated in the pathogenesis of PD. Dopaminergic signalling is known to be oxidative stress-generating, and the dopaminergic system has been found to beselectively vulnerable to increased oxidative stress

[0020] . Furthermore, several of the preclinicalPD models are induced by chemical compounds known to cause oxidative stress [21, 22], furtherhighlighting its correlation with PD. In preclinical models, targeted downregulation of miR-27b using ASOs has provided therapeutic benefit in haemorrhage-induced brain injury by inducing theAntioxidant Response Element (ARE) pathway

[0023] . The ARE pathway is a powerful antioxidativeand detoxifying pathway controlling more than 250 genes involved in oxidant defences and cellular homeostasis. The pathogenesis of PD is an extremely complex process where not all elements are known. However, there is a great need in the market for treatments that can prevent, alleviate, ameliorate or delay the progression of the disease.The present invention provides compounds and methods useful for such treatment. P477937PC00 Summary of the inventionThe present invention provides novel treatments for Parkinson’s Disease. The treatmentsprovided in the invention are antisense oligonucleotides targeting miR-27b, and capable ofdownregulating expression of miR-27b in deep brain areas and tissues relevant for progressionof PD. The inventors have shown that the compounds according to the invention are capable ofdownregulating miR-27b in the posterior cortex, substantia nigra and striatum. These datatogether with the data showing effects on different molecular mechanisms relevant for treatment of Parkinson’s disease show that the compounds according to the invention are indeed relevant for treatment of such diseases. In particular, the compounds of the invention, such as the antisense oligonucleotides comprisingLNA-modified ASOs of the invention are capable of rescuing viability and mitochondrial functionin an in vitro model of PD. In further in vivo experiments using an in vivo PD model, the compoundsof the invention can rescue dopaminergic neuron loss. Of the relevant pathways identified asimplicated in PD progression, the compounds according to the invention are capable ofinfluencing inflammation, mitophagy, oxidative stress and synuclein degradation in an in vivo PDmodel. The data support that the compounds according to the invention are useful in treatmentand alleviation of age-related neuro-degenerative diseases such as Parkinson’s disease.Based upon the effects of the compounds according to the invention, combined with the finding that miR-27b is upregulated in early-stage idiopathic PD patients, our target population comprises early-stage idiopathic PD patients.Mechanistically, the inventors show that miR-27 regulates several disease pathways implicatedin PD etiopathogenesis, including mitochondrial function, inflammation, and oxidative stress. This opens for the therapeutic possibility of targeting multiple, separate PD-associated molecular pathways by modulating a single target; an approach that significantly differentiates our drug candidates from others in the clinic and in development. Furthermore, by modulating these common pathological pathways and not focusing on genetic components, this therapeuticapproach targets sporadic PD, which accounts for over 90% of PD patients [6].In the present invention, the inventors have shown that ICV administration of an ASO targeting miR-27b in mice leads to efficient knockdown in posterior cortex and substantia nigra, and also detectable levels of the ASO in the tissues.In conclusion, inhibition of miR-27 can lead to disease-modification via three independentpathways, all proposed to be crucial for PD pathogenesis: mitochondrial function, oxidative stress, and inflammation. This unique approach of one-target-multiple pathways offers the potential fordisease-modification in PD by halting the progression of the disease, thereby benefitting patients,their families and healthcare providers. P477937PC00In some embodiments, the inventors has shown that a compound comprising an antisenseoligonucleotide capable of inhibiting microRNA-27b expression, wherein the oligonucleotidecomprise 8 to 21, such as 10 to 21, such as 12 to 21 or such as 14 to 21 consecutive nucleotidesthat are complementary to the miR-27b mature sequence (SEQ ID NO 1) 5'- uucacaguggcuaaguucugc-3', wherein the oligonucleotide comprise a sequence that is complementary to the seed sequence in position 2-7 of SEQ ID NO 1, and wherein at least onenucleotide is an LNA, wherein the compound is useful for the treatment or alleviation ofParkinson’s disease. In some embodiments, the compound according to the invention is deliveredto the brain. The compounds of the invention comprise inhibitors of miR-27b, such as in some embodimentsthe inhibitor is LNA-modified antisense oligonucleotide compounds that are complementary to themature miR-27b sequence, and which are capable of inhibiting miR-27b expression and / orfunction. Due to the ability of the compounds of the invention, to be distributed into deep areas ofthe brain, and exert their function in tissues such as the striatum, posterior cortex and substantia nigra, and the observed effects of the compounds in influencing important parameters relevant for progression of PD, the compounds according to the invention are for use in the treatment or alleviation of one or more symptoms of Parkinson’s disease, and for use in methods for preventing neuronal death, or dopaminergic neuronal loss. Further, in vivo data generated by the inventorssupport use of the compounds according to the invention for inhibiting miR-27b expression in theposterior cortex, striatum or substantia nigra when e.g. administered to the brain such as to thecerebrospinal fluid, and thereby preventing or alleviating neuronal inflammation, such as dopaminergic neuronal inflammation. In some embodiments, the compounds according to the invention are for use in methods forinducing mitophagy, or alleviate oxidative stress in the brain, or for promoting or alleviation ofsynuclein degradation in the brain. Figure LegendsFigure 1A. antimiR-27b compound potency and IC50 curves. Potency of test compound (SEQID NO: 16 and SEQ ID NO:20) in the Renilla / Luciferase Assay. n(N)=2(2), least squaresregression with log(inhibitor) vs. a three-parameter response, luciferase assay in human U-87cells.Figure 1B. antimiR-27b compound shows viability rescue in a PD in vitro model; n(N)=3 (2),Viability assessed using the PrestoBlue HS assay in PC-12 cells with and without MPP+ (200 ^M) and antimiR-27b compound (1^M).Bars represent mean±SEM.Figure 2. antimiR-27b compounds improve mitochondrial function and mitophagy in vitro.A, Mitochondrial membrane potential evaluation by TMRE assay with or without MPP+ after P477937PC00 antimiR-27b treatment. B Membrane potential assessment by TMRE assay after treatment with antimiR-27b compounds (1^M, 48h). C Mitophagy rate assay upon AntimiR-27b compound treatment under normal or depolarizing conditions (10 ^M CCCP, 5 ^M FCCP), together with anactive comparator (MTK458 – PINK1 activator).Figure 3. AntimiR-27b compound induce the antioxidant response in vitro. Primary corticalneurons (DIV 11) treated with an antimiR-27b compound for 48h at 1 ^M dose. A. Expressionlevels of Nrf2, Nqo1 and Hmox1 assessed by RT-PCR. B Hmox1 protein levels assessed by immunoblotting. Bars are mean+SEM. N=3-6.Figure 4. Potency and biodistribution of antimiR-27b compound in the brain A: Targetengagement in the Posterior cortex, left panel depicts miR-27b expression by RT-PCR 7 days after ICV injection with antimiR-27b compound, and right panel indicates tissue concentrations ofantimiR27b compound measured by hELISA assay at 3 escalating doses. B: Target engagementin Substantia nigra, left panel depicts miR-27b expression by RT-PCR 7 days after ICV injectionwith antimiR-27b compound, and right panel indicates tissue concentrations of antimiR27bcompound measured by hELISA assay at 3 escalating doses.Figure 5. Selectivity of antimiR-27b compound in the brain. Panels depict miR-27a and miR-27b expression by RT-PCR from the posterior cortex (A) and substantia nigra (B) from micetreated for 7 days with 2 escalating doses of an antimiR-27b compound. Figure 6. Effect of antimiR-27b compound on dopaminergic neuron rescue in the MPTP mouse model of PD. Stereological TH+ counts in the Substantia Nigra of mice treated with antimiR-27b compound. NMT.002 is an antimiR-27b compound. Figure 7. Effect of an antimiR-27b compound on PD pathways in the MPTP mouse modelof PD. A. Assessment of inflammatory marker expression by RT-PCR on striatum samples fromMPTP mice (n=4-9 mice / group). i.e. Il1b, Tnf, Nfkb and Nlrp3.B. Effect in striatum of MPTP miceof antimiR-27b on mitophagy relevant gene Pink1 expression as measured by RT-PCR. C. Effectin striatum of MPTP mice of antimiR-27b on antioxidant response relevant gene Nrf2 expressionas measured by RT-PCR. D. Effect in striatum of MPTP mice of antimiR-27b on synucleindegradation relevant gene expression BMl-1 as measured by RT-PCR. Figure 8. Uptake, potency and translatability of an antimiR-27b compound in iPSC-derivedhuman neurons. Unassisted uptake in human iPSCs of FAM-labelled antimiR-27b compound,8B. RT-PCR detection with U6 and GAPDH as house-keeping controls of miR-27b, Hmox-1 andPink-1 in human iPSCs (DIV 11, n=2). P477937PC00Figure 9. Target engagement and selectivity in rats via intracisternal injection. miR-27b andmiR-27a inhibition in posterior cortex of rats using different antimiR-27b compounds at twoescalating doses, 30 days post-injection. Compounds were administered by intracisternalinjection. One-way ANOVA with multiple comparisons was used to analyse differences. N=4rats / group. Figure 10. Target engagement, duration of action and biodistribution in a rat PD model (AAV-hSNCA). miR-27b expression in the substantia nigra, CSF and plasma of AAV-hSNCA ratmodel or GFP control treated with antimiR-27b compounds, 8 weeks after injection (n=6-8rats / group). Bars represent mean+SEM. Two-way ANOVA with multiple comparisons.Figure 11. antimiR-27b compound rescues weight loss in a rat PD model (AAV-hSNCA). Rat weights in the AAV-hSNCA rat model or GFP control treated with an antimiR-27b compound,8 weeks after injection. Bars represent mean+SEM. N=6-8 rats / group. Graphs separated bygender. Figure 12. antimiR-27b compound rescues motor symptoms and gait defects in a rat PD model (AAV-hSNCA). Actimetry counts from the locomotion test (A,C) and number of adjustingsteps in the stepping test (B,D) in the AAV-hSNCA rat model or GFP control treated with antimiR-27b compounds, 8 weeks after injection. N=6-8 rats / group. Bars represent mean+SEM. Figure 13. antimiR-27b compounds rescue nigral degeneration and neuroinflammation(microgliosis) in a rat PD model (AAV-hSNCA). Histology of TH+ cell counts in the SubstantiaNigra (A), optical density of TH+ projections in the striatum (B), and microglial cell counts perhemisphere based on Iba1+ staining in the AAV-hSNCA rat model or GFP control treated withantimiR-27b compounds, 8 weeks after injection. N=6-8 rats / group. Bars represent mean+ SEM. Figure 14. Target engagement and weight rescue of an antimiR-27b compound in a mousePD model (MitoPark). A. miR-27b expression in the Substantia Nigra of the MitoPark mousemodel treated ICV with an antimiR-27b compound after 12 weeks with 2 doses every 6 weeks. BWeight timecourse in the MitoPark mouse model (left), and bar plot of weight for week 18. Bars represent mean+SEM.n=6-12 mice / group Figure 15. Rescue of learning and memory deficits of an antimiR-27b compound in a mouse PD model (MitoPark). Barnes maze test for memory and learning in female Mitoparkmice 5 weeks post antimiR-27b compound administration (17-week-old mice). A Latency to Goal P477937PC00 for trials 1,2 and 3 in MitoPark mouse. B Latency to goal and distance travelled on Probe Day. N=4-6 mice / group. Bars represent mean+SEM. Detailed description of the inventionIn describing the embodiments of the invention, specific terminology will be resorted to for thesake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is understood that each specific term includes all technical equivalents, which operate in a similar manner to accomplish a similar purpose. The term "therapeutically effective amount", or “effective amount” or effective dose”, refers to an amount of a therapeutic agent, which confers a desired therapeutic effect on an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, the method of administration, assessment of the individual's medical condition, and other relevant factors. The term "treatment" refers to any administration of a therapeutic medicament, herein comprising an antisense oligonucleotide that partially or completely cures or reduces one or more symptoms or features of a given disease. The term “compound” as used herein, refers to a compound comprising an miR-27b inhibitor, such as an antisense oligonucleotide targeting miR-27b according to the invention. In some embodiments, a compound may comprise other elements apart from the oligonucleotide of the invention. Such other elements may in non-limiting example be a delivery vehicle which isconjugated or in other way bound to the oligonucleotide. In some embodiments, in its broadestsense, the inhibitor of miR-27b is an other type of molecule which is capable of effectively inhibiting the function or expression of miR-27b. Examples of such other types of inhibitors include but are not limited to siRNA’s. “antimiR” means a compound comprising an oligonucleotide that is complementary to a microRNA, i.e. an antimiR-27b compound comprise an oligonucleotides complementary tomicroRNA 27b, such as the antimiR-27b compounds according to the present invention. In someembodiments, the antimiR compounds are LNA / DNA mixmers, in some embodiments, theantimiR compounds are or comprise any of SEQ ID NO: 5 – 22.“ASO” means antisense oligonucleotide. P477937PC00 "Antisense oligonucleotide" means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid. The antisense oligonucleotide of the present invention is preferably a “mixmer”.A “mixmer” is an antisense oligonucleotide, comprising a mix of nucleoside analogues such asLNA and DNA nucleosides (LNA / DNA mixmer), and wherein the antisense oligonucleotide doesnot comprise an internal region having a plurality of nucleosides (such as a region of at least 6 or7 DNA nucleotides), capable of recruiting an RNase, such as RNase H, wherein the nucleosidescomprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external wings. “Nucleoside analogues” are described by e.g. Freier & Altmann; Nucl. Acid. Res., 1997, 25, 4429– 4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and examples ofsuitable and preferred nucleoside analogues are provided by WO2007031091, which are hereby incorporated by reference. "5-methylcytosine" means a cytosine modified with a methyl group attached to the 5’ position. A 5-methylcytosine is a modified nucleobase. "2'-O-methoxyethyl" (also 2'-MOE and 2'-O(CH~)~-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of a furanose ring. "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside) means a nucleoside comprising a 2'- MOE modified sugar moiety. A “locked nucleic acid” or “LNA” is often referred to as inaccessible RNA, and is a modified RNA nucleobase. The ribose moiety of an LNA nucleobase is modified with an extra bridge connecting the 2' oxygen and 4' carbon. An LNA oligonucleotide offers substantially increased affinity for its complementary strand, compared to traditional DNA or RNA oligonucleotides. In some aspects bicyclic nucleoside analogues are LNA nucleotides, and these terms may therefore be used interchangeably, and in such embodiments, both are characterized by the presence of a linker group (such as a bridge) between C2' and C4' of the ribose sugar ring. When used in the present context, the terms "LNA unit", "LNA monomer", "LNA residue", "locked nucleic acid unit", "locked nucleic acid monomer" or "locked nucleic acid residue", refer to a bicyclic nucleoside analogue. LNA units are described in inter alia WO 99 / 14226 , WO 00 / 56746 , WO 00 / 56748 , WO 01 / 25248 , WO 02 / 28875 , WO 03 / 006475, WO2015071388, and WO 03 / 095467. “Beta-D-Oxy LNA”, is a preferred LNA variant. P477937PC00 "Bicyclic nucleic acid" or "BNA" or "BNA nucleosides" mean nucleic acid monomers having a bridge connecting two carbon atoms between the 4' and 2' position of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugar include, but are not limited to A) pt-L-methyleneoxy (4'-CH2-0-2') LNA, (B) P-D-Methyleneoxy (4'-CH2-0-2') LNA, (C)Ethyleneoxy (4'- (CH2)2-0-2') LNA, (D) Aminooxy (4'-CH2-0-N(R)-2') LNA and (E) Oxyamino (4'-CH2-N(R)-0-2') LNA.As used herein, LNA nucleotides include, but are not limited to, nucleotides having at least onebridge between the 4' and the 2' position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from -[C(R~)(R2)]„-, - C(R~)=C(R2)-, -C(R~)=N, -C(=NREM)-, -C(=0)-, -C(=S)-, -0-, -Si(Ri)q-, -S(=0) —and -N(R&)-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R& and R2 is, independently, H, a protecting group, hydroxyl, C»C» alkyl, substituted C» (-CHz-) group connecting the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy (4'-CH&-0-2') LNA is used. Furthermore; in the case of the bicyclic sugar moiety having an ethylene bridging group in thisposition, the ethyleneoxy (4'-CH&CH&-0-2') LNA is used. n -L- methyleneoxy (4'-CH&-0-2'), anisomer of methyleneoxy (4'-CH&-0-2') LNA is also encompassed within the definition of LNA, asused herein. In some embodiments, the nucleoside unit is an LNA unit selected from the list of beta-D-oxy- LNA, alpha-Loxy-LNA, beta-D-amino-LNA, alpha-L-amino-LNA, beta-D-thio-LNA, alpha-L-thio- LNA, 5'-methyl-LNA, beta-D-ENA and alpha-L-ENA. "cEt" or "constrained ethyl" means a bicyclic sugar moiety comprising a bridge connecting the 4'- carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CHq)-0-2'. "Constrained ethyl nucleoside" (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge. cEt and some of its properties are described in Pallan et al. Chem Commun (Camb).2012, August 25; 48(66): 8195–8197. “Tricyclo (tc)-DNA” belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. Structure and method of production may be seen in Renneberg et al. Nucleic Acids Res.2002 Jul 1; 30(13): 2751–2757. “2’-fluoro”, as referred to herein is a nucleoside comprising a fluoro group at the 2’ position of the sugar ring.2’-fluorinated nucleotides are described in Peng et al. J Fluor Chem.2008 September; 129(9): 743–766. P477937PC00 “2’-O-methyl”, as referred to herein, is a nucleoside comprising a sugar comprising an –OCH3 group at the 2’ position of the sugar ring. “Conformationally Restricted Nucleosides (CRN)” and methods for their synthesis, as referred to herein, are described in WO2013036868, which is hereby incorporated by reference. CRN are sugar-modified nucleosides, in which, similar to LNA, a chemical bridge connects the C2' and C4'carbons of the ribose. However, in a CRN, the C2’ – C4’ bridge is one carbon longer than in anLNA molecule. The chemical bridge in the ribose of a CRN locks the ribose in a fixed position,which in turn restricts the flexibility of the nucleobase and phosphate group. CRN substitutionwithin an RNA- or DNA-based oligonucleotide has the advantages of increased hybridizationaffinity and enhanced resistance to nuclease degradation. “Unlocked Nucleic Acid” or “UNA”, is as referred to herein unlocked nucleic acid typically wherethe C2 — C3 C-C bond of the ribose has been removed, forming an unlocked "sugar" residue(see Fluiter et al., Mol. Biosyst., 2009, 10, 1039, hereby incorporated by reference, and Snead et al. Molecular Therapy—Nucleic Acids (2013) 2, e103;). "Target region" means a portion of a target nucleic acid to which one or more antisense compounds is targeted. “Targeted delivery” as used herein means delivery, wherein the antisense oligonucleotide has either been formulated in a way that will facilitate efficient delivery in specific tissues or cells, or wherein the antisense oligonucleotide in other ways has been for example modified to comprise a targeting moiety, or in other way has been modified in order to facilitate uptake in specific target cells. An “effective dosage or dose” means the dose of a drug that will achieve the desired effect. In the context of the present invention, an effective dosage or dose of one of the compounds of theinvention, is a dose that will lead to sufficient inhibition of miR-27b in the tissue desired, and leadto the desired clinical effect.Compounds and useIn its broadest sense, the inhibitor of miR-27b comprised in the compounds according to theinvention is other than an oligonucleotide. In some instances, the inhibitor may be an siRNA. Inany case, the compounds are for use in an effective dosage that will inhibit the function or expression of miR-27b sufficiently to achieve the desired effect. The antisense oligonucleotidesof the invention are for use in treating Parkinson’s disease or related conditions as describedbelow and are designed to target microRNA-27b (miR-27b). P477937PC00 Specific antisense oligonucleotides have been designed to target regions of miR-27b having the mature sequence 5’-uucacaguggcuaaguucugc-3’ (SEQ ID NO: 1) (miRBase acc # MIMAT0000419).The above reference to “miRBase” is according to miRBase release 22.1.In some embodiments, the invention provides a compound comprising an antisenseoligonucleotide capable of inhibiting miRNA-27b expression, wherein the oligonucleotidecomprise 8 to 21, such as 10 to 21, such as 12 to 21, such as 14 to 21 consecutive nucleotidesthat are complementary to the miR-27b mature sequence (SEQ ID NO 1) 5'- uucacaguggcuaaguucugc-3', wherein the oligonucleotide comprise a sequence that is complementary to the seed sequence in position 2-7 of SEQ ID NO 1, and wherein at least one nucleotide is an LNA, and wherein the compound is for use in the treatment or alleviation ofParkinson’s disease. In some embodiments, the antisense oligonucleotide complementary tomiR-27b according to the invention is at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as 100% complementary to SEQ ID NO 1, 2 or 3 over the length of the oligonucleotide. In some embodiments, the invention provides antisense oligonucleotides designed to target part of or the whole of 5’-ucacaguggcuaaguucug-3’ (SEQ ID NO: 2). In some embodiments, the antisense oligonucleotides of the invention are designed to target at least 5’-ucacaguggcuaaguucu-3’ (SEQ ID NO: 3). In some embodiments, the antisense oligonucleotides comprise the sequence 5’- agaacttagccactgtga-3’ (SEQ ID NO: 4). In some embodiments, the antisense oligonucleotide is 18 or 19 nucleotides in length, and comprises the sequence 5’ -agaacttagccactgtga-3’ (SEQ ID NO: 4). In some embodiments, the antisense oligonucleotide is 18 or 19 nucleotides in length, comprises the sequence 5’-agaacttagccactgtga-3’ (SEQ ID NO: 4) and is a mixmer.In some embodiments, the antisense oligonucleotide targeting miR-27b is 18 or 19 nucleotides inlength, comprises the sequence 5’-agaacttagccactgtga-3’ (SEQ ID NO: 4) and is an LNA / DNAmixmer. It has surprisingly been found that antisense oligonucleotides which are LNA / DNA mixmers, 18 or 19 nucleotides in length, and comprise SEQ ID NO: 4 are particularly potent in downregulating miR-27b activity. Such antisense oligonucleotides complementary to miR-27b show superior efficiency in downregulating their target miR-27b when they are 18 or 19 nucleotides in length, LNA / DNA mixmers, comprising from 50-70 % LNA and has no more than three consecutive DNA nucleotides. In some embodiments, the invention provides an antisense oligonucleotide complementary tomiR-27b consisting of a sequence of 18-19 nucleobases in length that is a mixmer which doesnot comprise a region of more than three consecutive DNA nucleotides, and which comprisesbetween seven and 14 affinity-enhancing nucleotide analogues, and wherein the antisense P477937PC00 oligonucleotide comprises between 1 and 18 phosphorothioate internucleotide linkages, andwherein the oligonucleotide is complementary to any of SEQ ID NO: 2 - 3, or which comprisesSEQ ID NO: 4. In some embodiments, the antisense oligonucleotide complementary to miR-27b, is 18 or 19nucleotides in length, comprises SEQ ID NO: 4 and is an LNA / DNA mixmer having between 50and 70 % LNA, such as between 52 and 68 % LNA, such as at least 50% LNA, such as at least 52% LNA.In some embodiments, the antisense oligonucleotides complementary to miR-27b according toany one of the above embodiments, have two terminal LNA nucleotides in each end. Further, in preferred embodiments, the LNA used in the antisense oligonucleotides of the invention are Beta- D-Oxy LNA. In some preferred embodiments, all LNA cytosines are 5-methylcytosine, i.e. in sequence listings, all capital C’s are methyl C’s.For in vivo use, stability of the antisense oligonucleotide will benefit from having one or morephosphorothioate linkages. In some embodiments, the antisense oligonucleotides complementary to miR-27b comprise phosphorothioate internucleoside bonds, such as at least one bond is phosphorothioate, or in some instances, the oligonucleotides have a complete phosphorothioate backbone, i.e. all internucleoside linkages are phosphorothioate linkages. The inventors have previously identified a series of highly potent antisense oligonucleotides complementary to miR-27b that all have the features listed in the above embodiments. Thesecompounds are listed in Table 1 as SEQ ID NO’s: 5 - 22. All of these compounds are preferred.In some embodiments, the compounds having any one of SEQ ID NO’s: 8, 12, 16, 19, 20 and 22 are especially preferred.Table 1 lists SEQ ID NO: 5 - 22 that are LNA / DNA mixmer antisense oligonucleotidescomplementary to miR-27b. Table 1 AntimiR-27b compounds SEQ ID NO Length # LNA % LNA5 AGaaCTtAgCCaCtGtGA 18 11 616 AGaAcTTaGcCACtGtGA 18 12 677 AGaaCTTaGCcaCtGtGA 18 11 618 AGaActTAgcCaCTGtGA 18 11 619 AGaaCTtAGcCaCTgTGA 18 12 6710 AGaaCTtAgCCAcTgTGA 18 12 6711 AGaAcTTaGcCACtgTGA 18 12 67 P477937PC00 12 AGaaCTtAGcCaCtgTGA 18 11 6113 AGAacTTagCcACTgtGA 18 11 6114 AGaaCTtaGccAcTgTGA 18 10 5515 AGaActTaGccACTgTGA 18 11 6116 AGaActTagCcaCTgTGA 18 10 5517 CAgaAcTTagCcaCTgTGA 19 11 5818 CAGaACtTAgcCaCTGtGA 19 13 6819 CAgaaCTtaGccACtgTGA 19 10 5220 AGAacTTagCcACTgtGA 18 11 6121 CAGaACtTAgcCaCTGtGA 19 13 6822 AGAacTTaiCcACTgtGA 18 11 61In table 1, uppercase letters denote LNA nucleotides and lowercase letters DNA nucleotides. Theletter “i” denotes inosine and capital C denotes LNA 5-methylcytocine. All internucleoside bondsare phosphorothioate linkages. In some embodiments, DNA cytosine may be methylated.In some instances, it will add to the potency or other characteristics of a compound to substitute one or more of the DNA nucleotides in an LNA / DNA mixmer ASO with other high-affinity nucleotides than LNA.In some instances, the antisense oligonucleotides complementary to miR-27b of the invention areLNA / DNA mixmer ASOs wherein one or more of the DNA nucleotides have been replaced withone or more nucleosides that are anyone of tricyclo-DNA, 2'-Fluoro, 2'-0-methyl, 2'methoxyethyl(2'MOE), 2' cyclic ethyl (cET), UNA,, 2’fluoro and Conformationally Restricted Nucleoside (CRN).In some embodiments, the inventors has shown that an effective dosage of a compoundcomprising an antisense oligonucleotide capable of inhibiting microRNA-27b expression, wherein the oligonucleotide comprise 10 to 21 consecutive nucleotides that are complementary to the miR-27b mature sequence (SEQ ID NO 1) 5'-uucacaguggcuaaguucugc-3', wherein the oligonucleotide comprise a sequence that is complementary to the seed sequence in position 2-7 of SEQ ID NO 1, and wherein at least one nucleotide is an LNA, is useful for the treatment oralleviation of Parkinson’s disease. The inventors have found that the compounds according to theinvention can rescue mitochondrial function and viability in an in vitro Parkinson’s disease model.Mitochondrial function and viability are known to be essential for neuronal health in Parkinsonsdisease. The inventors have previously made very potent antisense oligonucleotides targeting miR-27bthat are 18 or 19 nucleotides long, such as anyone of SEQ ID NO: 5-21. Thus, in someembodiments, the compound according to the invention is a compound comprising an antisenseoligonucleotide capable of inhibiting microRNA-27b expression, wherein the oligonucleotideconsists of 18 or 19 consecutive nucleotides comprising (SEQ ID NO 2) 5'-agaacttagccactgtga-3’and wherein at least one nucleotide is an LNA, and wherein the compound is for use in the treatment or alleviation of Parkinson’s disease, or related conditions or symptoms, such as other P477937PC00synucleinopathies. In some such embodiments, the compound according to the invention isselected from SEQ ID NO: 5-21.In some embodiments, the compounds according to the invention is for use in treatment of early-stage idiopathic Parkinsons’ disease.The inventors have tested the compounds according to the invention in a rat AAV synuclein modeland found that the compounds according to the invention are useful for treatment, prevention,reduction or alleviation of gait disturbances associated with Parkinsons disease. In someembodiments, the compounds according to the invention are for use in treatment, prevention,reduction or alleviation of gait disturbances associated with Parkinsons disease. Further, it wasfound that the compounds according to the invention are useful for treatment, prevention, reduction or alleviation of motor symptoms associated with Parkinsons disease. Therefore, in some embodiments, the compounds according to the invention are for use in for treatment, prevention, reduction or alleviation of motor symptoms associated with Parkinsons disease. The inventors of the present invention further found that the compounds according to the invention are capable of rescuing learning and memory ability in a mouse model of Parkinsons disease. Therefore, in some embodiments, the compounds according to the invention are for treatment,rescuing or alleviation of learning and memory loss associated with Parkinsons Disease.The compounds according to the invention such as antisense oligonucleotides targeting miR-27b,are effective in influencing important pathogenic pathways relevant for Parkinson’s disease, andtherefore these compounds are for use in the treatment or alleviation of one or more symptomsof Parkinson’s disease. The inventors further have found that the compounds according to theinvention such as antisense oligonucleotides targeting miR-27b, are effective for use in treatment,reducing, preventing or alleviation of unintended weight loss associated with Parkinsons disease. The antimiR-27b compounds of the invention have been tested in a Parkinson’s disease in vitromodel and in a MPTP in vivo model of Parkinson’s disease and shown the ability to rescueneurons from death. Therefore, the compounds according to the invention, such as thosecomprising potent antimiR-27b antisense oligonucleotides are for use in preventing neuronaldeath, or dopaminergic neuronal loss. Furthermore, the inventors have shown that the compounds of the invention are capable of deep tissue penetration in the brain, shown by miR-27b knockdown in posterior cortex, striatum and substantia nigra in experimental rats. Therefore, in some embodiments, the compounds according to the invention are for inhibiting miR-27b expression in the posterior cortex, striatumor substantia nigra when administered to the brain. The inventors also demonstrated the abilityof the compounds according to the invention to prevent degeneration of the nigrostriatal systemin the rat AAV-synuclein model. In some embodiments, the compounds according to the inventionare for preventing degeneration of the nigrostriatal system in association with ParkinsonsDisease. In some embodiments, the compounds according to the invention are for use in P477937PC00preventing or alleviating CNS inflammation, such as microgliosis. Such use of the compounds issupported by the findings shown in figure 13.The compounds of the invention have further shown to potently inhibit pathways including Il1b,Tnf, Nfkb, and Nlrp3 which have been shown to be involved in Parkinson’s disease related neuroinflammation. Consequently, in some embodiments, the compounds according to theinvention, such as the antisense oligonucleotides targeting miR-27b are for preventing oralleviating neuronal inflammation, such as dopaminergic neuronal inflammation. The compounds according to the invention were shown to be involved in other pathogenic events involved in progression of Parkinson’s disease, including mitophagy and oxidative stress. The inventors have shown in the MPTP in vivo model, that Pink1 and Nrf2 are induced by an effective dosage of the compounds of the invention, and therefore the compounds according to theinvention, such as the antisense oligonucleotides of the invention are for inducing of mitophagy,or preventing or alleviating oxidative stress in the brain. Further, the compounds have been shownto be absorbed by human cells and effectively dose dependently downregulate miR-27b and upregulate target genes such as Pink-1. BMI-1 is also induced in the MPTP model by an effective dosage of the compounds of theinvention, and as BMI-1 is involved in the synuclein degradation pathway relevant for Parkinson’sdisease, the compounds according to the invention, such as the antisense oligonucleotidestargeting miR-27b of the invention are for promoting or alleviation of synuclein degradation in thebrain. In some embodiments, the compounds according to the invention, such as antisenseoligonucleotides inhibiting miR-27b, are for use in treating synucleinopathies, such as alpha-synucleinopathies. In some embodiments, the compounds such as the antisense oligonucleotidestargeting miR-27b according to the invention are for treating Parkinson’s disease.The compounds for use according to anyone of the above embodiments, are antimiR-27bantisense oligonucleotides, wherein the oligonucleotide is a LNA / DNA mixmer having not morethan 4 contiguous DNA nucleotides, such as not more than 3 contiguous DNA nucleotides, such as not more than two contiguous DNA nucleotides.The compound for use according to the previous embodiment has at least one internucleosidebond which is a phosphorothioate bond.In some embodiments, the compound for use according to any of the previous embodiments, isan oligonucleotide selected from anyone of the sequences:(SEQ ID NO 16) 5'-AGaActTagCcaCTgTGA-3’, (SEQ ID NO 20) 5'-AGAacTTagCcACTgtGA-3’, wherein capital letters depict LNA nucleotides and lowercase letters depict DNA nucleotides, and at least one internucleoside bond is a phosphorothioate linkage. In some embodiments, the compounds for use according to the above embodiments are those ofe.g. SEQ ID NO: 16 or 20, wherein capital C denotes LNA 5-methylcytosine. P477937PC00 In some embodiments, the LNA is beta-D-oxy LNA. In some embodiments, the compound for use according to the above embodiments are designed wherein all internucleoside bonds are phosphorothioate bonds. In some embodiments, the compound for use according to any of the above embodiments are made, wherein LNA is beta-D-oxy LNA and all internucleoside bonds are phosphorothioate bonds. In some embodiments, the compound for use according to any one of the above embodiments are made, wherein the oligonucleotide is selected from anyone of the sequences: (SEQ ID NO 16) 5'-AGaActTagCcaCTgTGA-3’, or (SEQ ID NO 20) 5'-AGAacTTagCcACTgtGA-3’, andwherein capital letters denote LNA nucleotides and lowercase letters denote DNA nucleotides,and LNA is beta-D-oxy LNA, and all internucleoside bonds are phosphorothioate linkages. In some embodiments according to the previous embodiment is made, wherein capital C is LNA 5-methylcytosine. In some embodiments according to the invention, the antisense oligonucleotide for use according to the invention is anyone of (SEQ ID NO 22) 5’-AGAacTTaiCcACTgtGA-3’ (SEQ ID NO 20) 5’-AGAacTTagCcACTgtGA-3’ (SEQ ID NO 19) 5’-CAgaaCTtaGccACtgTGA-3’ (SEQ ID NO 16) 5’-AGaActTagCcaCTgTGA-3’ (SEQ ID NO 12) 5’-AGaaCTtAGcCaCtgTGA-3’ (SEQ ID NO 8) 5’-AGaActTAgcCaCTGtGA-3’Wherein capital letters are LNA, small letters are DNA, capital C is 5-methylcytosine, LNA is beta-D-oxy LNA, “i” is inosine and all internucleoside bonds are phosphorothioate bonds.In some embodiments, the antisense oligonucleotide such as anyone of SEQ ID NO: 5-22, suchas any of SEQ ID NO: 8, 12, 16, 19, 20 or 22 are for use in treating or alleviating Parkinson’sdisease, such as symptoms of Parkinson’s disease and / or alleviating neuronal inflammation, suchas dopaminergic neuronal inflammation, such as for use in preventing neuronal death, ordopaminergic neuronal loss in PD.In some such embodiments, the compound for use according to anyone of the aboveembodiments, is made wherein the compound is a conjugate that comprises the antisenseoligonucleotide of any one of the above embodiments. In some such embodiments, the conjugatefurther comprises a means for improving the cellular uptake of compound. In some embodiments, the compound for use according to the above embodiments is a bioconjugate. In some embodiments, the compound, such as the antisense oligonucleotide targeting miR-27b for use in treating or alleviating Parkinson’s disease or related conditions are for use in P477937PC00 combination with other drugs relevant for treatment of the diseases and conditions described inthis application. In some embodiments, the compound according to the invention comprises anantisense oligonucleotide that is 80% complementary to SEQ ID NO:1, such as 90% complementary. In some embodiments, the antisense compound is 80% or 90% identical to any one of SEQ ID NO: 5-22. Methods Synthesis of LNA-modified antisense oligonucleotides Synthesis of LNA-modified antisense oligonucleotides is well known in the art and such ASOs are commercially available. Cell Culture PC-12 cells The PC-12 Adh rat cell line was purchased from ATCC® (CRL-1721.1™) and cells were routinely cultivated in Ham's F-12K (Kaighn's) Medium (Gibco, 21127022) supplemented with 2.5% Fetal Bovine Serum (FBS) (Sigma, F4135-500), 15% Horse Serum and 1% Penicillin / Streptomycin (Sigma, P4333-100). Primary cortical rat neurons Culture plates were coated 1-day prior to cell plating with 50µg / mL Poly-D-Lysine (Gibco, A3890401) diluted in sterile H2O for 1h at 37°C, washed 3x in sterile H2O, and incubated with 1180µg / mL mouse laminin (Gibco, 23017015) diluted in sterile PBS at 4°C overnight. The laminin was aspirated, and wells were washed with sterile H2O twice. Primary rat cortex neurons were purchased from Thermo Fisher (A1084002) and defrosted as well as cultivated in Neurobasal complete medium supplemented with 2% B27 (Gibco, A3582801), 1% Penicillin / Streptomycin, 0.5mM GlutaMAX (Gibco, A1286001), according to manufacturer’s instructions. On DIV2 and DIV4 after defrosting, half of the cell medium was aspirated and replaced by fresh and 37°C-warm Neurobasal complete medium. iPSC derived neurons Generation of iPSC and culture of stem cells. Induced pluripotent stem cells were generated from human skin biopsies. The skin biopsies were kept in DMEM-1885 media containing 10% Fetal bovine serum (FBS) and 1% Penicillin / streptomycin (P / S) for at least two weeks and dividing fibroblasts were split into new culture dishes and frozen for N2 freezer storage. Fibroblast cells were transformed into induced pluripotent stem cells (iPSC) by transfecting with the transcription factors Oct3 / 4, Klf-4, Sox-2, c- myc, also referred to as Yamanaka factors (Ref: Cell. 2006 Aug 25;126(4):663-76. doi: 10.1016 / j.cell.2006.07.024. Epub 2006 Aug 10). iPSC clones were picked around 2 weeks later P477937PC00 and reseeded for testing of germ-layer differentiation into ectoderm, mesoderm, and endoderm. Positive clones were selected for karyotyping analysis. iPSC stock cells were maintained in mTeSR plus media (Stem Cell Technologies) with P / S and passaged using EDTA (Gibco; 500 nM) and cultured on laminin-coated plates (BioLamina). Neuronal differentiation For quick neuronal differentiation we utilized expression of neurogenin-2 (NGN2) regulated by a doxycycline-inducible tet-ON system (https: / / doi.org / 10.1016 / j.scr.2020.101945). The DNA was inserted into safe-harbor locations in the genome of the iPSC using the TALEN system, andpositive clones were selected. On day 0 iPSC were treated with doxycycline (Thermo FisherScientific; 5 µg / ml) for 4 hours and following split with Accutase (Stem Cell Technologies) into alaminin-coated 60 mm NUNC petri dish in Neurobasal (50%) and Advanced DMEM-F12 medium(50%) containing B27 (Gibco; 1:50), N2 (Gibco; 1:100), Insulin-Transferrin-Selenium (ITS) (Gibco;1:100), GlutaMAXTM Supplement (Gibco; 1:100), P / S 1%, LDN 193189 (Stem Cell Technologies;100nM), SB 431542 (Stem Cell Technologies; 10 µM), doxycycline (2 µg / ml), and Rock inhibitorY-27632 (BioGems; 10 µM). The following day the media was changed without the addition ofRock inhibitor Y-27632. On day 4 the cells were split into the experimental set-up on platesprecoated with poly-ornithine and laminin in BrainPhys™ Neuronal Medium (Stem CellTechnologies) containing B27 (Gibco; 1:50), N2 (Gibco; 1:100), P / S, brain-derived neurotrophicfactor (BDNF) (Stem Cell technologies; 10 ng / ml), glia derived neurotrophic factor (GDNF) (StemCell technologies; 10ng / ml), cAMP (Stem Cell Technologies; 200 nM), and ascorbic acid (Sigma;200 µM). Every 3-4 days half the media was exchanged with fresh media. From day 12doxycycline was not included in the media. ASOs were added on day 14 and treated for 96 hours.On day 18 the cells were harvested for RNA extraction or fixation.In vitro screening A simple and very sensitive approach involves construction of a miRNA reporter plasmid that carries a single perfect match miRNA binding site in the 3’ UTR of a reporter gene, such as luciferase. This method has been extensively used in cultured cells to validate miRNA inhibition and also to compare the potency of different antimiR designs.The miR-27b reporter was generated by cloning annealed oligonucleotides corresponding tosingle perfect-match target site for human miR-27b into the 3′ UTR of the Renilla luciferase gene in the dual-luciferase psiCHECK2 plasmid (Promega). For luciferase assays, U-87 cells were seeded in 96-well Corning® CellBIND® Surface cell culture microwell plates (Sigma-Aldrich cat.no. CLS3330) at a density of 25,000 cells per well the day before transfection. The cells were transfected using lipofectamine 2000 (ThermoFischerScientific cat. no. 11668-019) at a final concentration of 0.4 µL / well in Opti-MEM™ I ReducedSerum Medium, GlutaMAX™ Supplement (ThermoFischer Scientific cat. no.51985026). A library P477937PC00of 17 antisense oligonucleotides was screened using the lucirease reporter assays by co-transfecting each antimiR-27b with the luciferase reporter plasmid and the miR-27b mimic in finalconcentrations of 0.2nM, 1 nM, 5 nM. A scrambled sequence oligonucleotide, a vector containing no miRNA match site and a mock transfection were included as controls. All samples were run intechnical duplicates. After 4 hours the cells were washed in Opti-MEM™ medium and freshcomplete cell culture medium was added to the wells. 24 hours after transfection the luciferase assay was conducted using Dual-Glo® Luciferase Assay System (Promega cat.no. E2920) as per manufacturer’s instructions. The amount of luminescence was determined on a plate reader (VarioSkan Lux, ThermoFischer Scientific) after 30 minutes incubation of reagents in the plates. The results were analysed by subtraction of background luminescence and then normalizing Renilla signal to the constitutive Firefly signal. The average of the two technical duplicates were then normalized to empty vector and expressed as percentage. For the determination of IC50 values, the cells were transfected with a wide range of antimiR-27bconcentrations ranging from 80 nM in 2-fold dilutions to 0.0049 nM.MPP+ Viability assay in PC-12 cells PC-12 cells were seeded in 96-well plates (Thermo, 150628) at a density of 40.000 cells / well in 100µL complete Ham's F-12K culture medium (see 2.1.2) and cultivated as described above (see2.1.2). Two days later, neurons were treated with medium containing 1µM of ASO and 200µMMPP+. Cells were further incubated at 37°C. After 72 hours of unassisted ASO uptake, PrestoBlue HS reagent was added on top of the cell medium at a concentration corresponding to a 1:10 dilution in the well and incubated for 1h at 37°C. The absorbance signal was read in Varioskan LUX, sample values were normalized to mock cell values in Excel (Microsoft) and statistically analyzed as well as plotted in GraphPad Prism (Version 10).Mitochondrial membrane potential Assay (TMRE) in primary cortical neuronsPrimary cortical rat neurons were seeded into black 96-well plates with clear bottom (Corning, 3904) at a density of 30.000 cells / well in 100µL of complete Neurobasal medium and cultured as described above (see SECTION). On DIV7, cells were treated with 1µM ASO and 48h later, MPP+was added at a final concentration of 50µM. 96h post ASO addition, the media was replaced byTMRE dye (Thermo, T669), which was diluted to a final concentration of 25nM in completeNeurobasal medium without phenol red (Gibco, 12348017). The mitochondrial dye was incubatedfor 30’, then Hoechst 33342 Solution (Thermo, 62249) was added in a final concentration of 8µMand further incubated for 10’ at 37°C. The medium was changed to complete Neurobasal medium without phenol red (Gibco, 12348017) and images were taken in Cytation 1 Cell Imaging Reader P477937PC00(BioTek) with 37°C and 5% CO2. TMRE Intensity per cell was measured using Cellprofiler(Version 4.2.5). Primary objects were identified using Hoechst. Cell soma (secondary object) was identified using the Distance-B method, which was further dilated. Cytoplasm (Tertiary object)was obtained by substracting the nuclei from the soma. Mean TMRE intensity was measured inthe Tertiary Object (Cytoplasm) of each cell. The sequences of the ASOs used for the in vivo studies was presented in the table below (Table 2). Sense ID Sense sequence (5'-3')NMC133 AbsGbsAbsdAsdCsTbsTbsdAsdGsCbsdCsAbsCbsTbsdGsdTsGbsAbNMC129mC AbsGbsdAsAbs5MdCsdTsTbsdAsdGsCbs5MdCsdAsCbsTbsdGsTbsGbsAbNMC133mC AbsGbsAbsdAs5MdCsTbsTbsdAsdGsCb s5MdCsAbsCbsTbsdGsdTsGbsAbTable 2: DNA / LNA sequences for ASOs used in the in vivo study. Nomenclature: DNA nucleotides (dN [dA, dC, dG, dI, dT]), LNA nucleotides (Nb [Ab, Cb, Gb, Tb]), 5-methyl-deoxy-cytidines (5MdC), Phosphorothioate backbone (s).In vivo methods (including behaviour)Animals and general procedures All experimental procedures involving animals were carried out in accordance with the European Communities Council Directive (2010 / 63 / EU) at Motac’s facility in Bordeaux.Wild-type miceThe animals used were 10-weeks old male wild-type C57bl / 6 mice bought from Janvier Labs and were acclimatized for 5 days before treatment. MPTP model To produce neurodegeneration of dopaminergic neurons in the substantia nigra, mice were treated with MPTP hydrochloride (30 mg / kg i.p. once daily for five consecutive days). MitoPark model The MitoPark mouse was created by selectively inactivating the Tfam (mitochondrial transcription factor A) gene in dopamine-producing neurons. This was achieved using the dopamine transporter (DAT) promoter to drive the expression of Cre recombinase in these neurons, leading to the deletion of Tfam specifically in dopaminergic cells. Dat Cre*Tfam Cre / + F / F: These mice express Cre under the control of the Dat promoter and had a single copy of Tfam containing Cre. Recombination occurs, leading to the inactivation of Tfam specifically in dopaminergic neurons. P477937PC00 Dat Cre*Tfam + / + F / F: These mice also express Cre under the control of the Dat promoter, but their Tfam alleles were all wild type. Therefore, there was no inactivation of Tfam, even in dopaminergic neurons. The Dat CreTfam Cre / + F / F mice exhibit impaired mitochondrial function in dopaminergic neurons, while the Dat CreTfam + / + F / F mice serve as a control. Intracerebroventricular injection All doses were given by a single 4 µl intracerebroventricular injection (Table 1) with a glass-pipette over a 3 min period. Table 3: Coordinates for ICV injection site AP anteroposterior L lateral DV Dorsoventral Site Volume / site (mm) (mm) (mm) Right lateral ventricle -0.5 1.0 -2.4 4 µl7 days after surgery, blood samples were collected under deep anaesthesia followed by euthanasia. Brain tissue was collected, and the brains were split in the midline and dissected intothe following ipsi- and contra-lateral regions: anterior cortex, posterior cortex, striatum, substantianigra, cerebellum and hippocampus. All tissues were weighed and snap-frozen. Blood was collected as much as possible into EDTA tubes, gently mixed by hand and stored on ice. Blood samples were centrifuged at 4°C (1600 g, 10 min) to obtain plasma within 30 min of sample collection. Rats Wild-type rats Rats received cisterna magna injection (6 or 12 µL) of the test item or vehicle. Metacam 1 mg / kg (10ml / kg) was given as an analgesic before and after the surgery. Each animal was placed in an anaesthetic chamber supplied with a continuous flow of oxygen (1.5 l / min) and 3% isoflurane. Following the loss of consciousness, the animal was placed in a stereotactic frame (Kopf), and its head was fixed into position using ear bars. Once the injection was completed, rats were removed from the stereotactic frame. Vetbond glue was applied to avoid CSF leak and the skin was closed with surgical suture Vicryl 6.0. All the rats were then maintained on a heating pad in an observation cage until full recovery from anaesthesia. Rats were humanely euthanised under deep anaesthesia on day 30 after ASO injection in accordance with European Veterinary Medical Association guidelines. P477937PC00 AAV-Synuclein model Male and female Sprague-Dawley rats weighing 175-200 g were grouped and housed in a temperature-controlled room under a 12 h light / dark cycle with free access to food (standard pellets, Safe) and water. Metacam 2 mg / kg (1ml / kg) was given as an analgesic before and after the surgery. Each animal was placed in an anaesthetic chamber with a continuous flow of oxygen (1.5 l / min) and 5% isoflurane. Following the loss of consciousness, the rats was placed in a Kopf stereotactic apparatus and maintained under isoflurane anaesthesia during intracerebral injections. A small craniotomy was made in the skull in the appropriate location using Bregma as a reference (Paxinos and Watson, 2007). Using a glass pipette, each rat receiveds two bilateral injections of 2µl (1µl each track, 2µl each side) of the highly concentrated vector. The coordinates used to target the region immediately above the substantia nigra be: 1st track: 1µl AP −4.95, L 2.2 and DV -7.4 2nd track: 1µl AP −5.1, L 2.0 and DV -7.4. After the injection, the pipette was left in place for 3 min before being slowly withdrawn from the brain. The viral vectors used was adeno-associated viruses (AAV) expressing the wild synuclein or a control. Behavioral assays in mice Locomotor activity The apparatus (Imetronic) used an array of infrared beams to determine the activity and mobility of a subject. The activity cage was incompletely divided into two compartments (front and back). Three parameters was recorded: activity, rearing and movement between the back and front compartments. ‘Activity’ was the total number of infrared beam breaks. ‘Rearing’ was the total number of infrared beam breaks for the upper array. ‘Back and Front’ was the number of movements from one compartment of the cage to the other. Locomotor activity was assessed for 2 hours at baseline (10 weeks), 22 weeks and before euthanasia (37 weeks). The apparatus (35.5 x 23.5 cm, Imetronic) used an array of infrared beams to determine the activity and mobility of a subject. The activity cage was incompletely divided into two compartments (front and back). Three parameters was recorded: activity, rearing and movement between the back and front compartments. ‘Activity’ was the total number of infrared beam breaks. ‘Rearing’ was the total number of infrared beam breaks for the upper array. ‘Back and Front’ was the number of movements from one compartment of the cage to the other. Locomotor activity was assessed for 2 hours at baseline (before the surgery) and at 11 and 15 (cohort 2 only) weeks after AAV injection. P477937PC00 Barnes maze The Barnes Maze was a widely used behavioural test designed to evaluate spatial learning and memory in rodents. The test was conducted on a circular platform with holes evenly spaced along its perimeter. Only one of these holes leads to an escape box, while the others were false exits. The protocol can be divided into the following stages:Training phase :The acquisition period (learning phase) consisted of three daily trials taking place on four consecutive days with an inter-trial interval of 25 minutes. To begin each trial, the test mouse wasplaced under an opaque start box in the center of the maze for 10 seconds. The box was removed,and the mouse can freely explore the maze for 3 minutes while a digital video camera records the trial from overhead. When the test mouse enters the burrow via the target hole, the hole was covered, and the mouse was left undisturbed for one minute. If the test mouse fails to enter the hole by the end of the trial, it was gently prodded into the burrow and left undisturbed for one minute. Ethovision XT tracking software was used to determine the latency to find the target hole, the path length travelled to find the target hole, and locomotor velocity during the test. Probe trial: Short-term memory was tested in a single probe trial 24 hours after the final day of the four acquisition trials. The trial lasted 90 seconds. The burrow was removed from under the maze, andthe target hole fit with an identical false bottom as the rest of the holes. The latency and pathlength traveled to find the correct target hole, velocity, and duration of the probe trial spent in proximity to the correct target hole was measured using Ethovision XT tracking software (Noldus). This test was performed at 18 weeks. Behavioral assays in rats Stepping test Stepping adjustments was measured as described by Olsson et al. (J Neurosci.1995, 15: 3863- 75). The experimenter take the rat with one hand holding both hind limbs and the other holding one of the forelimbs. The free paw was placed in contact with a flat surface. The experimenter then move the animal slowly sideways in forehand and backhand directions. The number of adjusting steps count for both paws in the backhand and forehand directions. Animals was tested in random order. The stepping test was performed at baseline (before the surgery) and at 11 and 15 (cohort 2 only) weeks after AAV injection. Locomotor activity The apparatus (35.5 x 23.5 cm, Imetronic) used an array of infrared beams to determine the activity and mobility of a subject. The activity cage was incompletely divided into two P477937PC00 compartments (front and back). Three parameters was recorded: activity, rearing and movement between the back and front compartments. ‘Activity’ was the total number of infrared beam breaks. ‘Rearing’ was the total number of infrared beam breaks for the upper array. ‘Back and Front’ was the number of movements from one compartment of the cage to the other. Locomotor activity was assessed for 2 hours at baseline (before the surgery) and at 11 and 15 (cohort 2 only) weeks after AAV injection. Histology Stereological Counting of TH Positive Neurons TH immunohistochemistry was performed as follows. 1 in every 4 sections was used for the immunohistochemistry. Tissue sections was taken from the refrigerator and left to adjust to room temperature. After thorough rinsing with PBS, endogenous peroxidase was inhibited with a peroxidase-blocking solution (Dako REAL, S2023) for 10 minutes. After further thorough rinsing with PBS, non-specific labelling was prevented by blocking antigenic sites in PBS containing 2% bovine serum albumin (BSA), 0.3% triton X-100 and 0.01% thimerosal for 30 minutes. Sections then be incubated overnight at room temperature in primary antibody (monoclonal rabbit anti-TH, EP1532Y, ab137869, Abcam) diluted at 1:5000 in PBS containing 0.2% BSA, 0.3% triton X-100 and 0.01% thimerosal. The next day, sections was given a thorough rinse with PBS and incubated with labelled polymer-horseradish peroxidase (HRP) anti-rabbit (Dako EnVision+TM Kit, K4011) for 30 minutes. After thorough rinsing with PBS, TH staining was revealed with 3,3′-diaminobenzidine (DAB) (Dako DAB Kit, K3468) for 12 seconds and stopped with several PBS washes. Sections then be mounted onto gelatine-coated glass slides, counterstained with a Nissl stain, dehydrated, cleared in xylene and cover-slipped in permanent mounting media.The slides were digitalized using a PannoramicScan II (3DHISTECH, Hungary) at an x20magnification with an extended mode in which a 5-layer focus was automatically acquired and flattened in a single-focus final picture. The SN was delineated on each picture using Visiopharm (Denmark). The TH-immunopositive (TH+) was estimated by an artificial intelligence application in Visiopharm based on the deep learning of the staining, shape, and size of the TH characteristic. The mean estimated number of neurons and SEM then be calculated for each group. Measurement of Striatal Density of TH-Positive Terminals TH immunohistochemistry was performed on one rostro-caudal series as follows. After thoroughrinsing with PBS, endogenous peroxidase was inhibited with peroxidase- blocking solution (DakoREAL, S2023) for 10 min. After thorough rinsing with PBS, non- specific labelling was preventedby blocking antigenic sites in PBS containing 2% bovine serum albumin (BSA), 0.3% triton X-100 P477937PC00 and 0.01% thimerosal, for 30 min. Sections was then incubated overnight at room temperature in primary antibody (monoclonal rabbit anti-TH, EP1532Y, ab137869, Abcam) diluted at 1:5000 in PBS containing 0.2% BSA, 0.3% triton X-100 and 0.01% thimerosal. On the next day, sections was rinsed with PBS and then incubated with labelled polymer-horseradish peroxidase (HRP) anti-rabbit (Dako EnVision+TM Kit, K4011) for 30 min. After thorough rinsing with PBS, TH revelation was obtained with 3,3′-diaminobenzidine (DAB) (Dako DAB Kit, K3468) for 12 seconds and stopped with several PBS washes. Sections then be mounted onto gelatine-coated slides and left to air dry. Thereafter, slides was scanned and analysed for levels of optical density. The threshold surface quantification of the staining was assessed on all sections stained in the same conditions. The slides was digitalized using a PannoramicScan II (3DHISTECH, Hungary) at an x20 magnification with an extended mode in which a 5-layer focus was automatically acquired and flattened in a single focus final picture. The striatum was delineated on each picture using Visiopharm software (Denmark). A threshold was defined corresponding to the antibody staining. Both the surface of the staining and the surface of the region was extracted, and the percentage of staining was finally expressed. Then, average of percentage of TH staining, and corresponding standard errors of the mean was calculated for each group. Assessment of Striatal Iba1 Immunostaining Iba1 immunohistochemistry was performed in 2 sections of the striatum per animal. On the day of staining, after thorough rinsing with PBS, endogenous peroxidase was inhibited with peroxidase-blocking solution (Dako REAL, S2023) for 10 min. After further thorough rinsing with PBS, non-specific labelling was prevented by blocking antigenic sites in PBS containing 2% BSA, 0.3% triton X-100 and 0.01% thimerosal, for 30 min, for the blockade of non-specific antigenic sites. Sections then be incubated overnight at room temperature in an antibody buffer solution of PBS -1 / 500 BSA -0.3% triton X-100 containing a goat anti-Iba-1 (Abcam, Ab5076) at a dilution of 1 / 1000. Sections then be rinsed thoroughly in PBS and incubated for 30 minutes with anti-goatDAKO Immpress vector (ImmPRESS HRP Anti-Goat Ig (Peroxidase) Polymer Detection Kit MP-7405). After thorough rinsing, Iba1 immunological staining first be revealed with SG vector kitrevelation (VECTOR SG peroxidase substrate kit SK-4700) and stopped after 90 sec with a seriesof washes in PBS solution. Coronal sections then be carefully placed onto gelatine-coated slides and left to air dry. Thereafter, slides were cover-slipped. The slides was digitalized using a PannoramicScan II (3DHISTECH, Hungary) at a x20 magnification with an extended mode in which 5-layer focus automatically be acquired and flattened in a single focus final picture. P477937PC00The striatum was delineated on each picture using Visiopharm software (Denmark). The microgliawas isolated by an artificial intelligence application in Visiopharm based on the Iba1 staining, deep learning of the shape, size of microglia arborisation characteristics. Mean estimated number of microglia, the % of Iba1 staining area and SEM was then calculated for each group. The morphology quantification was defined for each structure and each animal.In vitro methodsCell culture PC-12 cells PC-12 Adh rat cell line was purchased from ATCC® (CRL-1721.1™) and cells were routinely cultivated in Ham's F-12K (Kaighn's) Medium (Gibco, 21127022) supplemented with 2.5% Fetal Bovine Serum (FBS) (Sigma, F4135-500), 15% Horse Serum and 1% Penicillin / Streptomycin (Sigma, P4333-100). Primary cortical rat neurons Culture plates were coated 1-day prior cell plating with 50µg / mL Poly-D-Lysine (Gibco, A3890401) diluted in sterile H2O for 1h at 37°C, washed 3x in sterile H2O, and incubated with 1180µg / mL mouse laminin (Gibco, 23017015) diluted in sterile PBS at 4°C overnight. The laminin was aspirated, and wells were washed with sterile H2O twice. Primary rat cortex neurons were purchased from Thermo Fisher (A1084002) and defrosted as well as cultivated in Neurobasal complete medium supplemented with 2% B27 (Gibco, A3582801), 1% Penicillin / Streptomycin, 0.5mM GlutaMAX (Gibco, A1286001), according to manufacturer’s instructions. On DIV2 and DIV4 after defrosting, half of the cell medium was aspirated and replaced by fresh and 37°C-warm Neurobasal complete medium. iPSC-derived neurons Generation and stem-cell culture Induced pluripotent stem cells were generated from human skin biopsies. The skin biopsies were kept in DMEM-1885 media containing 10% Fetal bovine serum (FBS) and 1% Penicillin / streptomycin (P / S) for at least two weeks and dividing fibroblasts were split into new culture dishes and frozen for N2 freezer storage. Fibroblast cells were transformed into induced pluripotent stem cells (iPSC) by transfecting with the transcription factors Oct3 / 4, Klf-4, Sox-2, c- myc, also referred to as Yamanaka factors (Ref: Cell. 2006 Aug 25;126(4):663-76. doi: 10.1016 / j.cell.2006.07.024. Epub 2006 Aug 10). iPSC clones were picked around 2 weeks later and reseeded for testing of germ-layer differentiation into ectoderm, mesoderm, and endoderm. Positive clones were selected for karyotyping analysis. iPSC stock cells were maintained in P477937PC00 mTeSR plus media (Stem Cell Technologies) with P / S and passaged using EDTA (Gibco; 500 nM) and cultured on laminin-coated plates (BioLamina). Neuronal differentiation (NGN2) For quick neuronal differentiation we utilized expression of neurogenin-2 (NGN2) regulated by a doxycycline-inducible tet-ON system (https: / / doi.org / 10.1016 / j.scr.2020.101945). The DNA wasinserted into safe-harbor locations in the genome of the iPSC using the TALEN system, andpositive clones were selected. On day 0 iPSC were treated with doxycycline (Thermo Fisher Scientific; 5 µg / ml) for 4 hours and following split with Accutase (Stem Cell Technologies) into a laminin-coated 60 mm NUNC petri dish in Neurobasal (50%) and Advanced DMEM-F12 medium (50%) containing B27 (Gibco; 1:50), N2 (Gibco; 1:100), Insulin-Transferrin-Selenium (ITS) (Gibco; 1:100), GlutaMAXTM Supplement (Gibco; 1:100), P / S 1%, LDN 193189 (Stem Cell Technologies; 100nM), SB 431542 (Stem Cell Technologies; 10 µM), doxycycline (2 µg / ml), and Rock inhibitor Y-27632 (BioGems; 10 µM). The following day the media was changed without the addition of Rock inhibitor Y-27632. On day 4 the cells were split into the experimental set-up on platesprecoated with poly-ornithine and laminin in BrainPhys™ Neuronal Medium (Stem CellTechnologies) containing B27 (Gibco; 1:50), N2 (Gibco; 1:100), P / S, brain-derived neurotrophic factor (BDNF) (Stem Cell technologies; 10 ng / ml), glia derived neurotrophic factor (GDNF) (Stem Cell technologies; 10ng / ml), cAMP (Stem Cell Technologies; 200 nM), and ascorbic acid (Sigma; 200 µM). Every 3-4 days half the media was exchanged with fresh media. From day 12 doxycycline was not included in the media. ASOs were added on day 14 and treated for 96 hours. On day 18 the cells were harvested for RNA extraction or fixation. miR-27b reporter assay (luciferase) A simple and very sensitive approach involves construction of a miRNA reporter plasmid that carries a single perfect match miRNA binding site in the 3’ UTR of a reporter gene, such as luciferase. This method had been extensively used in cultured cells to validate miRNA inhibition and also to compare the potency of different antimiR designs. The miR-27b reporter was generated by cloning annealed oligonucleotides corresponding to single perfect-match target site for human miR-27b into the 3′ UTR of the Renilla luciferase gene in the dual-luciferase psiCHECK2 plasmid (Promega). For luciferase assays, U-87 cells were seeded in 96-well Corning® CellBIND® Surface cell culture microwell plates (Sigma-Aldrich cat.no. CLS3330) at a density of 25,000 cells per well the day before transfection. The cells were transfected using lipofectamine 2000 (ThermoFischerScientific cat. no. 11668-019) at a final concentration of 0.4 µL / well in Opti-MEM™ I ReducedSerum Medium, GlutaMAX™ Supplement (ThermoFischer Scientific cat. no.51985026). A libraryof 17 antisense oligonucleotides was screened using the lucirease reporter assays by co- transfecting each antimiR-27b with the luciferase reporter plasmid and the miR-27b mimic in final P477937PC00 concentrations of 0.2nM, 1 nM, 5 nM. A scrambled sequence oligonucleotide, a vector containing no miRNA match site and a mock transfection were included as controls. All samples were run intechnical duplicates. After 4 hours the cells were washed in Opti-MEM™ medium and freshcomplete cell culture medium was added to the wells. 24 hours after transfection the luciferase assay was conducted using Dual-Glo® Luciferase Assay System (Promega cat.no. E2920) per manufacturer’s instructions. The amount of luminescence was determined on a plate reader (VarioSkan Lux, ThermoFischer Scientific) after 30 minutes incubation of reagents in the plates. The results were analysed by subtraction of background luminescence and then normalizing Renilla signal to the constitutive Firefly signal. The average of the two technical duplicates were then normalized to empty vector and expressed as percentage. For the determination of IC50 values, the cells were transfected with a wide range of antimiR-27b concentrations ranging from 80 nM in 2-fold dilutions to 0.0049 nM. MPP+ viability in PC-12 cells PC-12 cells were seeded in 96-well plates (Thermo, 150628) at a density of 40.000 cells / well in 100µL complete Ham's F-12K culture medium (see 2.1.2) and cultivated as described above (see2.1.2). Two days later, neurons were treated with medium containing 1µM of ASO and 200µMMPP+. Cells were further incubated at 37°C. After 72 hours of unassisted ASO uptake, PrestoBlue HS reagent was added on top of the cell medium at a concentration corresponding to a 1:10 dilution in the well and incubated for 1h at 37°C. The absorbance signal was read in Varioskan LUX, sample values were normalized to mock cell values in Excel (Microsoft) and statistically analyzed as well as plotted in GraphPad Prism (Version 10). TMRE assay in primary cortical neurons Primary cortical rat neurons were seeded into black 96-well plates with clear bottom (Corning, 3904) at a density of 30.000 cells / well in 100µL of complete Neurobasal medium and cultured as described above (see SECTION). On DIV7, cells were treated with 1µM ASO and 48h later, MPP+ was added at a final concentration of 50µM.96h post ASO addition, the media was replaced by TMRE dye (Thermo, T669), which was diluted to a final concentration of 25nM in complete Neurobasal medium without phenol red (Gibco, 12348017). The mitochondrial dye was incubated for 30’, then Hoechst 33342 Solution (Thermo, 62249) was added in a final concentration of 8µM and further incubated for 10’ at 37°C. The medium was changed to complete Neurobasal mediumwithout phenol red (Gibco, 12348017) and images were taken in Cytation 1 Cell Imaging Reader(BioTek) with 37°C and 5% CO2. TMRE Intensity per cell was measured using Cellprofiler (Version 4.2.5). Primary objects were identified using Hoechst. Cell soma (secondary object) was identified using the Distance-B method, which was further dilated. Cytoplasm (Tertiary object) P477937PC00 was obtained by subtracting the nuclei from the soma. Mean TMRE intensity was measured in the Tertiary Object (Cytoplasm) of each cell. 3.6 Mitophagy Assay PC12 cells were seeded in dark 96-well plate at 30.000 cells / well density. After 24 hours, cells were treated with anti-miR-27b antisense oligonucleotides, NMC133 or NMC129mC, at either 0.5 or 1 µM concentration for 24 hours. To compare the effect of the ASOs, some wells were treated with MTK458 (7980, Tocris), a small molecule that activates PINK1, enhancing mitophagy downstream. Mitochondria and lysosomes were stained with 100 nM Mitotracker Deep Red (M22426, Invitrogen) and 200 nM Lysotracker Green (L7526, Invitrogen) for 30 minutes at 37°C and 5% CO2. Cell nuclei were stained using 1:20.000 dilution of Hoechst 33342. To inducemitochondrial damage, 1 µM CCCP (ab141229, Abcam) or FCCP (ab120081, Abcam) were usedas mitochondrial uncouplers. After incubation, the wells were washed three times using warm PBS. Cells were imaged using BioTek Cytation 1 at 10X at 37°C and 5% CO2. Images were analysed using CellProfiler software. Oligonucleotide preparations:The sequences of the ASOs used for the in vivo studies is presented in the table below (Table 2).Sense ID Sense sequence (5'-3')NMC133 (SEQ IDAbsGbsAbsdAsdCsTbsTbsdAsdGsCbsdCsAbsCbsTbsdGsdTsGbsAb No.20) NMC129mC (SEQ ID AbsGbsdAsAbs5MdCsdTsTbsdAsdGsCbs5MdCsdAsCbsTbsdGsTbsGbsAb No.16) NMC133mC (SEQ ID AbsGbsAbsdAs5MdCsTbsTbsdAsdGsCb s5MdCsAbsCbsTbsdGsdTsGbsAb No.20) Table 2: DNA / LNA sequences for ASOs used in the in vivo study. Nomenclature: DNA nucleotides (dN [dA, dC, dG, dI, dT]), LNA nucleotides (Nb [Ab, Cb, Gb, Tb]), 5-methyl-deoxy-cytidines (5MdC), Phosphorothioate linkage (s).RNA and protein purification: Total RNA (mRNA and miRNA) was extracted from two subregions, the ipsilateral posterior cortex (PCTX) (NT240202) and the ipsilateral substantia nigra (SN) (NT240205). The procedure was almost identical for both regions. The subregions were lysed in 600 µl RLT buffer (+ 10 µL β- mercaptoethanol per 1 mL RLT buffer) using a Bullet Blender Storm Pro (NextAdvance, New York, USA) with speed set at 8 and for 3 minutes, the samples from PCTX were then diluted to 10 mg / 350 µl RLT and the samples from SN were diluted to 6.3 mg / 350 µl RLT. Subsequently the samples were passed through QIAshredder columns (Cat no: 79656, Qiagen, Hilden, Germany) securing a thorough homogenisation of the brain tissue. RNA was extracted from 350 P477937PC00 µl (10 mg) of each sample utilizing the DNA / RNA / Protein mini kit (Cat no: 80004, Qiagen, Hilden, Germany) and the protocol provided by the manufacturer was followed. The RNA was eluted in 20 µL Nuclease-free water (NFW) and passed twice through the column and stored at -80°C. Thetotal RNA concentration in the samples was measured using a Thermo Scientific™ μDrop™ andμDrop Duo Plates (Pr. Code: 11899143). miRNA reverse transcription: Total RNA was thawed on ice and diluted to 2 ng / µL RNA with NFW. The reverse transcriptionwas conducted using the TaqMan™ MicroRNA Reverse Transcription Kit (Cat no. 4366597,Applied Biosystems™) according to manufacturer’s instructions. Reverse transcription wasconducted with three TaqMan™ MicroRNA Assay miRNA-specific primers (Cat no: 4440887,Thermo Fischer Scientific, Waltham, MA, USA) in two separate fractions. First fraction with hsa- miR-27b (Assay ID: 000409) and U6 snRNA (Assay ID: 001973) and second fraction with hsa- miR-27a (Assay ID: 000408). The cDNA libraries were stored at -20°C after preparation. mRNA reverse transcription: Total RNA samples were thawed on ice and diluted to 40 ng / µL with NFW in a total volume of 8 µL. Reverse transcription of mRNA was conducted using Superscript IV reverse transcriptase (cat. no: 18090010, Thermo Fischer Scientific, Waltham, MA, USA) as per manufacturer’s instructions. After the RT reaction, all the samples were diluted to 80 µL with NFW for a final concentration of 4 ng / µL and stored at -20°C. qPCR:qPCR was conducted with a QuantStudio 6 Flex system (Applied Biosystems, Waltham, MA,USA) using PrimeTime™ Predesigned qPCR Assays (Table 3) synthesized by Integrated DNATechnologies (Newark, NJ, USA) and PrimeTime™ Gene Expression Master Mix (cat. no:1055771, Newark, NJ, USA) as per manufacturer’s instructions. ActB was used as a house-keeping gene. Gene Forward primer Reverse Primer Probe Assay NameBach1 5’-5’- 5’- / 56- Mm.PT.58.3070 GCCACATCTA CCAATCCTCC FAM / CCGCTC 1909 CGCAGTCG-3’ GTGTCAACA- TGG / ZEN / CTC (SEQ ID. No. 3’ (SEQ ID. No.ATATCTCTCC 23) 24) AC / 3IABkFQ / -3’ Nrlp3 5’-5’- 5’- / 56- Mm.PT.58.1397 CACTCATGTT CGGTTGGTGC FAM / TGCCTC 4318 P477937PC00 GCCTGTTCTT TTAGACTTGA- ACT / ZEN / TCTA C-3’ 3’ GCTTCTGCCG (SEQ ID. No.(SEQ ID. No. / 3IABkFQ / -3’ 25) 26)Hmox1 5’-5’- 5’- / 56- Mm.PT.58.8600 ACACTCTGGA TTGTGTTCCT FAM / AGACCG 055 GATGACACCT CTGTCAGCAT CCT / ZEN / TCC -3’ C-3’ GCTCAACATT / (SEQ ID. No. (SEQ ID. No.3IABkFQ / -3’ 27) 28)Keap1 5’-5’- 5’- / 56- Mm.PT.58.1114 AGACTGACGC GAGTTTGGCA FAM / ACCGTTA 9550 AGAGCTCA -3’ GTGTAGGCA - CC / ZEN / TCTT (SEQ ID. No.3’ CAGGCAGTGT 29)(SEQ ID. No.G / 3IABkFQ / -3’ 30)Nfkb1 5’-5’- 5’- / 56- Mm.PT.58.1199 CATTCTGACC AGGTCCATCT FAM / CCTTCTC 3856 TTGCCTATCT CCTTGGTCT- TC / ZEN / TGTC ACA-3’ 3’ TGTGAGTTGC (SEQ ID. No. (SEQ ID. No.CG / 3IABkFQ / - 31) 32) 3’Sod2 5’-5’- 5’- / 56- Mm.PT.58.1067 GGACAAACCT TGTCAGCTTC FAM / CTTGATA 5177 GAGCCCTAAG TCCTTAAACT GC / ZEN / CTCC -3’ TCT-3’ AGCAACTCTC (SEQ ID. No.(SEQ ID. No.CT / 3IABkFQ / -3’ 33) 34)Nfe2l2 5’-5’- 5’- / 56- Mm.PT.58.4219 CCTTGTACTT GAGGGACTG FAM / ATGACC 0606 TGAAGACTGT GGCCTGAT-3’ ATG / ZEN / AGT ATGC-3’(SEQ ID. No.CGCTTGCCCT (SEQ ID. No. 36) / 3IABkFQ / -3’ 35)Pink1 5’-5’- 5’- / 56- Mm.PT.58.5988 CTACCATGAT GCAGCCAAAG FAM / CAGCCA 924 TCT / ZEN / GAG P477937PC00 GACCTTGCAG TCTGAGATCA- TCCCACTCCA T-3’ 3’ C / 3IABkFQ / -3’ (SEQ ID. No.(SEQ ID. No.37) 38)Tnf 5’-5’- 5’- / 56- Mm.PT.58.1257 AGACCCTCAC TCTTTGAGAT FAM / CCACGT 5861 ACTCAGATCA- CCATGCCGTT CGT / ZEN / AGC 3’ G-3’ AAACCACCAA (SEQ ID. No. (SEQ ID. No.GT / 3IABkFQ / -3’ 39) 40)Il6 5’-5’- 5’- / 56- Mm.PT.58.1000 AGCCAGAGTC TCCTTAGCCA FAM / CCTACC 5566 CTCAGAGA-3’ CTCCTTCTGT- CCA / ZEN / ATTT (SEQ ID. No.3’ CCAATGCTCT 41)(SEQ ID. No.CCT / 3IABkFQ / - 42) 3’Il-1b 5’-5’- 5’- / 56- Mm.PT.58.4161 GACCTGTTCT CTCTTGTTGA FAM / TTCCAAA 6450 TTGAAGTTGA TGTGCTGCTG CC / ZEN / TTTG CG-3’ -3’ ACCTGGGCTG (SEQ ID. No.(SEQ ID. No.T / 3IABkFQ / -3’ 43) 44)Bmi1 5’-5’- 5’- / 56- Mm.PT.58.2962 CTCCAAGATG TGTTCGATGC FAM / AACTTTC 9986 GCCGCTT-3’ ATTTCTGCTT AT / ZEN / TGTCT (SEQ ID. No.G-3’ TTTCCGCCCG 45)(SEQ ID. No.C / 3IABkFQ / -3’ 46)Paarg 5’-5’- 5’- / 56- Mm.PT.58.3116 CTGCTCCACA TGCAGGTTCT FAM / AGCTGA 1924 CTATGAAGAC ACTTGATCGC CCC / ZEN / AAT AT-3’ -3’ GGTTGCTGAT (SEQ ID. No. (SEQ ID. No.TACA / 3IABkFQ 47) 48) / -3’ActB 5’-5’- 5’- / 56- Mm.PT.58.3354 GCGAGCACA ATGCCGGAG FAM / CCGCCA 0333 GCTTCTTTG-3’ CCGTTGTC-3’ CCA / ZEN / GTT P477937PC00 (SEQ ID. No. (SEQ ID. No.CGCCATG / 3IA 49) 50) BkFQ / -3’Table 3: PrimeTime™ Predesigned qPCR AssaysThe miR-27 qPCR was done on a QuantStudio 6 Flex (Applied Biosystems, Waltham, MA, USA) using commercial TaqMan assays (Thermo Fischer Scientific, Waltham, MA, USA) andPrimeTime™ Gene Expression Master Mix (cat. no: 1055771, Newark, NJ, USA) as permanufacturer’s instructions. U6 was used as a house-keeping gene (Table 4).Gene Assay number Cat.no:miR-27a 000408 4440887miR-27b 000409 4440887U6 001973 4427975Table 4: TaqMan miRNA assays ASOs tissue concentration determined with hELISA: Analysis of ASO concentration in the tissue was done using a hybridization ELISA (hELISA) assay. Capture and detection probes, used for both NMC129mC and NMC133 were designed to align with the 5’ and 3’ end of the sequence respectively, which consisted of the same nucleotides for both ASOs (Table 4). We analyzed the concentration of ASOs in lysate from the tissue stored at -80°C in RLT buffer. The samples were thawed, vortexed briefly and then diluted to 0.002 mg / µl in NFW.55 µl of each sample, were transferred in duplicates to a 96-well plate. A 2-fold standard for the ASOs ranging from 4000 pm to 1.9 pm was included on each plate in technical duplicates, to ensure as precise determination of ASO concentration as possible. The standard was made with a NFW+RLT buffer ratio corresponding to the samples. 50 nM capture and 25 nM detection probe were diluted in 10x SCC+0.1% Tween buffer and 55 µL of the solution was added to each of the wells. Followed by incubation at 37°C at 300 rpm for 1 hour.100 µl from each of the wells were transferred to a Pierce™ Streptavidin Coated Plates (Cat no:15119, Thermo Fischer Scientific, Waltham, MA, USA) followed by 30 minutes of incubation at RT and 300 rpm. Subsequently the plate was emptied on an absorbing tissue and the wells were washed three times with 2x SCC+0.05% Tween buffer for 3 minutes at 300 rpm, between each wash, the wells were emptied completely by forcefully knocking the plate towards absorbing tissue, which were changed between each wash.100 µl of anti-Digoxigenin-POD, Fab fragments (Cat: 11207733910, Roche Diagnositcs,Mannheim, Germany) diluted 1:4000 in PBS+0.05% Tween was added to each of the wells followed by 1 hour incubation at RT and 300 rpm. The washing procedure from above wasrepeated and 100 µl of SuperSignal™ ELISA Pico Chemiluminescent Substrate (Cat no: 37069, P477937PC00 Thermo Fischer Scientific, Waltham, MA, USA) was added to each well followed by measuring ofsignal on a Thermo Scientific™ Varioskan™ LUX multimode microplate reader (Pr. Code:15350747). Data analysis:The Renilla luciferase activity was normalized to Firefly luciferase activity and plotted againstlog(M). The dose-response curves were fitted using 3-parameter non-linear fit and IC50 values calculated in nM. All qPCR results were analyzed using the ΔΔCt method13normalized to controls.All data were analyzed in GraphPadPrism ver. 10.2.2, (GraphPad, San Diego, CA, USA).Items 1) A compound comprising an inhibitor capable of inhibiting microRNA-27b expression orfunction, for use in the treatment or alleviation of Parkinsons Disease, or symptoms of Parkinsons Disease, or morbidities associated with Parkinsons Disease. 2) The compound for use according to item 1, wherein the inhibitor of microRNA-27b is anantisense oligonucleotide comprising of 8-21 consecutive nucleotides which are fully complementary to the mature microRNA-27b sequence (SEQ ID NO 1) 5'- uucacaguggcuaaguucugc-3', wherein the oligonucleotide comprises a sequence that is complementary to the seed sequence in position 2-7 of SEQ ID NO 1. 3) The compound for use according to item 2, wherein the antisense oligonucleotidecomprises at least one affinity enhancing nucleotide analogue. 4) The compound for use according to item 3, wherein the at least one affinity enhancingnucleotide analogue is selected from the list of Locked Nucleic Acid (LNA), tricyclo-DNA, 2'-fluoro, 2'-O-methyl, 2' methoxyethyl (2'-MOE), 2' cyclic ethyl (cET), UNA, and Conformationally Restricted Nucleoside (CRN). 5) The compound for use according to item 4, wherein the at least one affinity enhancingnucleotide analogue is an LNA, such as beta-D-oxy LNA. 6) The compound for use according to any of the preceding items, comprising an antisenseoligonucleotide capable of inhibiting microRNA-27b expression, wherein the antisense oligonucleotide consists of 18 or 19 consecutive nucleotides comprising (SEQ ID NO 2) 5'-agaacttagccactgtga-3’ and wherein at least one nucleotide is an LNA. P477937PC007) The compound according to any one of the preceding items comprising an antisenseoligonucleotide capable of inhibiting microRNA-27b expression, wherein the oligonucleotide is a LNA / DNA mixmer having not more than 4 contiguous DNA nucleotides, such as not more than 3 contiguous DNA nucleotides, such as not more than two contiguous DNA nucleotides.8) The compound for use according to any of items 2-7, wherein at least one internucleosidebond is a phosphorothioate bond.9) The compound for use according to any of items 2-8, wherein the oligonucleotide isselected from anyone of SEQ ID NO: 5 – 22.10) The compound for use according to any of items 2-9, wherein the oligonucleotide isselected from anyone of the sequences: a. (SEQ ID NO 22) 5’ AGAacTTaiCcACTgtGA 3’b. (SEQ ID NO 20) 5’ AGAacTTagCcACTgtGA 3’c. (SEQ ID NO 19) 5’ CAgaaCTtaGccACtgTGA 3’d. (SEQ ID NO 16) 5’ AGaActTagCcaCTgTGA 3’e. (SEQ ID NO 12) 5’ AGaaCTtAGcCaCtgTGA 3’f. (SEQ ID NO 8) 5’ AGaActTAgcCaCTGtGA 3’Wherein capital letters are LNA, small letters are DNA, capital C is 5-methylcytosine, LNA is beta-D-oxy LNA, “i” is inosine and all internucleoside bonds are phosphorothioate bonds.11) The compound for use according to item 10, wherein the oligonucleotide is selected fromanyone of the sequences: (SEQ ID NO 16) 5' AGaActTagCcaCTgTGA 3’, (SEQ ID NO 20) 5' AGAacTTagCcACTgtGA 3’, wherein capital letters depict LNA nucleotides and lowercase letters depict DNA nucleotides, and at least one internucleoside bond is a phosphorothioate linkage.12) The compound for use according to item 11, wherein capital C denotes LNA 5-methylcytosine.13) The compound for use according to item 11 or 12, wherein LNA is beta-D-oxy LNA.14) The compound for use according to any of items 11-13, wherein all internucleoside bondsare phosphorothioate bonds. P477937PC0015) The compound for use according to any of the preceding items, wherein LNA is beta-D-oxy LNA and all internucleoside bonds are phosphorothioate bonds.16) The compound for use according to any one of the preceding items, wherein theoligonucleotide is selected from anyone of the sequences: (SEQ ID NO 16) 5' AGaActTagCcaCTgTGA 3’, or (SEQ ID NO 20) 5' AGAacTTagCcACTgtGA 3’, Wherein capital letters denote LNA, and lowercase letters denote DNA, and LNA is beta- D-oxy LNA, and all internucleoside bonds are phosphorothioate bonds.17) The compound for use according to item 16, wherein capital C is LNA 5-methylcytosine.18) The compound according to item 16, wherein the oligonucleotide is selected from anyoneof SEQ ID NO: 16 5’ AbsGbsdAsAbs5MdCsdTsTbsdAsdGsCbs5MdCsdAsCbsTbsdGsTbsGbsAb and SEQ ID NO: 20 5’ AbsGbsAbsdAs5MdCsTbsTbsdAsdGsCbs5MdCsAbsCbsTbsdGsdTsGbsAb wherein DNA nucleotides are (dN [dA, dC, dG, dI, dT]), LNA nucleotides are (Nb [Ab, Cb, Gb, Tb]), 5-methyl-deoxy-cytidines are (5MdC), and Phosphorothioate backbone is denoted (s).19) The compound for use according to anyone of items 1-18, wherein the compound is aconjugate that comprises the antisense oligonucleotide of any one of claims 1-18.20) The compound for use according to items 19, wherein the compound is a bioconjugate.21) The compound according to any of the preceding items, wherein the use is for treatmentor alleviation of early-stage idiopathic Parkinson’s disease.22) The compound according to any of the preceding items, wherein the treatment oralleviation is of one or more symptoms of Parkinson’s disease.23) The compound according to any of the preceding items, wherein the use is for treatment,reducing, preventing or alleviation of unintended weight loss associated with Parkinsons disease. P477937PC00 24) The compound according to any of the preceding items, wherein the use is for treatment,prevention, reduction or alleviation of gait disturbances associated with Parkinsons disease. 25) The compound according to any of the preceding items, wherein the use is for treatment,prevention, reduction or alleviation of motor symptoms associated with Parkinsons disease. 26) The compound according to any of the preceding items, wherein the use is for treatment,rescuing or alleviation of learning and memory loss associated with Parkinsons Disease. 27) The compound according to any of the preceding items, wherein the use is for preventingneuronal death, or dopaminergic neuronal loss in Parkinsons Disease. 28) The compound according to any of the preceding items, wherein the antisenseoligonucleotide use is for inhibiting miR-27b expression in the posterior cortex, striatum or substantia nigra. 29) The compound according to any of the preceding items, wherein the use is for preventingdegeneration of the nigrostriatal system in association with Parkinsons Disease. 30) The compound according to any of the preceding items, wherein the use is for preventingor alleviating CNS inflammation, such as microgliosis. 31) The compound according to any of the preceding items, wherein the use is for inductionof mitophagy, or oxidative stress alleviation in the brain in Parkinsons Disease. 32) The compound according to any of the preceding items, wherein the use is for promotingsynuclein degradation in the brain in Parkinsons Disease. 33) The compound according to the preceding item, wherein the use is for treating oralleviating synucleinopathies, such as alpha-synucleinopathies. Litterature list P477937PC00 1. Cardo LF, Coto E, Ribacoba R, Menéndez M, Moris G, Suárez E, Alvarez V (2014) MiRNA profile in the substantia nigra of Parkinson’s disease and healthy subjects. J Mol Neurosci 54:830–836. https: / / doi.org / 10.1007 / S12031-014-0428-Y 2. Kim J, Inoue K, Ishii J, Vanti WB, Voronov S V., Murchison E, Hannon G, Abeliovich A (2007) A microRNA feedback circuit in midbrain dopamine neurons. Science (1979) 317:1220–1224. https: / / doi.org / 10.1126 / SCIENCE.1140481 / SUPPL_FILE / KIM.SOM.PDF 3. Fazeli S, Motovali-Bashi M, Peymani M, Hashemi MS, Etemadifar M, Nasr-Esfahani MH, Ghaedi K (2020) A compound downregulation of SRRM2 and miR-27a-3p with upregulation of miR-27b-3p in PBMCs of Parkinson’s patients is associated with the early stage onset of disease. PLoS One 15:e0240855. https: / / doi.org / 10.1371 / JOURNAL.PONE.0240855 4. L W, J L, Y L, Y C, W L, H Z, A Z, N L (2021) MicroRNA Candidate Biomarkers for Parkinson’s Disease and Idiopathic REM Sleep Behavior Disorder. https: / / doi.org / 10.21203 / RS.3.RS- 1163742 / V1 5. Chen L, Yang J, Lü J, Cao S, Zhao Q, Yu Z (2018) Identification of aberrant circulating miRNAs in Parkinson’s disease plasma samples. Brain Behav 8:e00941. https: / / doi.org / 10.1002 / BRB3.941 6. Poewe W, Seppi K, Tanner CM, Halliday GM, Brundin P, Volkmann J, Schrag AE, Lang AE (2017) Parkinson disease. Nat Rev Dis Primers 3:17013. https: / / doi.org / 10.1038 / nrdp.2017.13 7. Clark EH, de la Torre AV, Hoshikawa T, Briston T (2021) Targeting mitophagy in Parkinson’s disease. J Biol Chem 296:. https: / / doi.org / 10.1074 / JBC.REV120.014294 8. Fivenson EM, Lautrup S, Sun N, Scheibye-Knudsen M, Stevnsner T, Nilsen H, Bohr VA, Fang EF (2017) Mitophagy in neurodegeneration and aging. Neurochem Int 109:202–209. https: / / doi.org / 10.1016 / J.NEUINT.2017.02.007 9. Kasten M, Hartmann C, Hampf J, Schaake S, Westenberger A, Vollstedt EJ, Balck A, Domingo A, Vulinovic F, Dulovic M, Zorn I, Madoev H, Zehnle H, Lembeck CM, Schawe L, Reginold J, Huang J, Konig IR, Bertram L, Marras C, Lohmann K, Lill CM, Klein C (2018) Genotype- Phenotype Relations for the Parkinson’s Disease Genes Parkin, PINK1, DJ1: MDSGene Systematic Review. Mov Disord 33:730–741. https: / / doi.org / 10.1002 / mds.27352 10. Cen X, Chen Y, Xu X, Wu R, He F, Zhao Q, Sun Q, Yi C, Wu J, Najafov A, Xia H (2020) Pharmacological targeting of MCL-1 promotes mitophagy and improves disease pathologies in an Alzheimer’s disease mouse model. Nat Commun 11:. https: / / doi.org / 10.1038 / S41467-020- 19547-6 11. Singh F, Prescott AR, Rosewell P, Ball G, Reith AD, Ganley IG (2021) Pharmacological rescue of impaired mitophagy in Parkinson’s disease-related LRRK2 G2019S knock-in mice. Elife 10:. https: / / doi.org / 10.7554 / ELIFE.67604 12. Kim J, Fiesel FC, Belmonte KC, Hudec R, Wang WX, Kim C, Nelson PT, Springer W, Kim J (2016) miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by P477937PC00 targeting PTEN-induced putative kinase 1 (PINK1). Mol Neurodegener 11:55. https: / / doi.org / 10.1186 / S13024-016-0121-4 13. Murata H, Takamatsu H, Liu S, Kataoka K, Huh NH, Sakaguchi M (2015) NRF2 Regulates PINK1 Expression under Oxidative Stress Conditions. PLoS One 10:e0142438. https: / / doi.org / 10.1371 / JOURNAL.PONE.0142438 14. Barisciano G, Colangelo T, Rosato V, Muccillo L, Taddei ML, Ippolito L, Chiarugi P, Galgani M, Bruzzaniti S, Matarese G, Fassan M, Agostini M, Bergamo F, Pucciarelli S, Carbone A,Mazzoccoli G, Colantuoni V, Bianchi F, Sabatino L (2020) miR-27a is a master regulator ofmetabolic reprogramming and chemoresistance in colorectal cancer. British Journal of Cancer 2020122:9122:1354–1366. https: / / doi.org / 10.1038 / s41416-020-0773-2 15. Miron VE, Priller J (2020) Investigating Microglia in Health and Disease: Challenges and Opportunities. Trends Immunol 41:785–793. https: / / doi.org / 10.1016 / j.it.2020.07.002 16. Tansey MG, Wallings RL, Houser MC, Herrick MK, Keating CE, Joers V (2022) Inflammation and immune dysfunction in Parkinson disease. Nat Rev Immunol. https: / / doi.org / 10.1038 / s41577- 022-00684-6 17. Ho MS (2019) Microglia in parkinson’s disease. Adv Exp Med Biol 1175:335–353. https: / / doi.org / 10.1007 / 978-981-13-9913-8_13 / FIGURES / 3 18. Li L, Qi C, Liu Y, Shen Y, Zhao X, Qin H, Zhang Y, Yu T (2021) MicroRNA miR-27b-3p regulate microglial inflammation response and cell apoptosis by inhibiting A20 (TNF-α-induced protein 3). Bioengineered 12:9902–9913. https: / / doi.org / 10.1080 / 21655979.2021.1969195 / SUPPL_FILE / KBIE_A_1969195_SM2028.ZIP 19. Guerau-De-Arellano M, Smith KM, Godlewski J, Liu Y, Winger R, Lawler SE, Whitacre CC, Racke MK, Lovett-Racke AE (2011) Micro-RNA dysregulation in multiple sclerosis favours pro- inflammatory T-cell-mediated autoimmunity. Brain 134:3575. https: / / doi.org / 10.1093 / BRAIN / AWR262 20. Wang X, Michaelis EK (2010) Selective neuronal vulnerability to oxidative stress in the brain. Front Aging Neurosci 2:12. https: / / doi.org / 10.3389 / fnagi.2010.00012 21. Meredith GE, Rademacher DJ (2011) MPTP mouse models of Parkinson’s disease: an update. J Parkinsons Dis 1:19–33. https: / / doi.org / 10.3233 / JPD-2011-11023 22. Simola N, Morelli M, Carta AR (2007) The 6-hydroxydopamine model of Parkinson’s disease. Neurotox Res 11:151–167. https: / / doi.org / 10.1007 / BF03033565 23. Xu W, Li F, Liu Z, Xu Z, Sun B, Cao J, Liu Y, Xu W, Li F, Liu Z, Xu Z, Sun B, Cao J, Liu Y (2017) MicroRNA-27b inhibition promotes Nrf2 / ARE pathway activation and alleviates intracerebral hemorrhage-induced brain injury. Oncotarget 8:70669–70684. https: / / doi.org / 10.18632 / ONCOTARGET.19974

Claims

P477937PC00 Claims1) A compound comprising an inhibitor capable of inhibiting microRNA-27b expression orfunction, for use in the treatment or alleviation of Parkinsons Disease, or symptoms of Parkinsons Disease, or morbidities associated with Parkinsons Disease.2) The compound for use according to claim 1, wherein the inhibitor of microRNA-27b is anantisense oligonucleotide comprising of 8-21 consecutive nucleotides which are fully complementary to the mature microRNA-27b sequence (SEQ ID NO 1) 5'- uucacaguggcuaaguucugc-3', wherein the oligonucleotide comprises a sequence that iscomplementary to the seed sequence in position 2-7 of SEQ ID NO 1.3) The compound for use according to claim 2, wherein the antisense oligonucleotidecomprises at least one affinity enhancing nucleotide analogue.4) The compound for use according to claim 3, wherein the at least one affinity enhancingnucleotide analogue is selected from the list of Locked Nucleic Acid (LNA), tricyclo-DNA, 2'-fluoro, 2'-O-methyl, 2' methoxyethyl (2'-MOE), 2' cyclic ethyl (cET), UNA, andConformationally Restricted Nucleoside (CRN).5) The compound for use according to claim 4, wherein the at least one affinity enhancingnucleotide analogue is an LNA, such as beta-D-oxy LNA.6) The compound for use according to any of the preceding claims, comprising an antisenseoligonucleotide capable of inhibiting microRNA-27b expression, wherein the antisenseoligonucleotide consists of 18 or 19 consecutive nucleotides comprising (SEQ ID NO 2)5'-agaacttagccactgtga-3’ and wherein at least one nucleotide is an LNA.7) The compound according to any one of the preceding claims comprising an antisenseoligonucleotide capable of inhibiting microRNA-27b expression, wherein theoligonucleotide is a LNA / DNA mixmer having not more than 4 contiguous DNA nucleotides, such as not more than 3 contiguous DNA nucleotides, such as not more than two contiguous DNA nucleotides.8) The compound for use according to any of claims 2-7, wherein at least one internucleosidebond is a phosphorothioate bond.P477937PC009) The compound for use according to any of claims 2-8, wherein the oligonucleotide isselected from anyone of SEQ ID NO: 5 – 22.10) The compound for use according to any of claims 2-9, wherein the oligonucleotide isselected from anyone of the sequences: g. (SEQ ID NO 22) 5’ AGAacTTaiCcACTgtGA 3’h. (SEQ ID NO 20) 5’ AGAacTTagCcACTgtGA 3’i. (SEQ ID NO 19) 5’ CAgaaCTtaGccACtgTGA 3’j. (SEQ ID NO 16) 5’ AGaActTagCcaCTgTGA 3’k. (SEQ ID NO 12) 5’ AGaaCTtAGcCaCtgTGA 3’l. (SEQ ID NO 8) 5’ AGaActTAgcCaCTGtGA 3’Wherein capital letters are LNA, small letters are DNA, capital C is 5-methylcytosine, LNAis beta-D-oxy LNA, “i” is inosine and all internucleoside bonds are phosphorothioatebonds.11) The compound for use according to claim 10, wherein the oligonucleotide is selected fromanyone of the sequences: (SEQ ID NO 16) 5' AGaActTagCcaCTgTGA 3’,(SEQ ID NO 20) 5' AGAacTTagCcACTgtGA 3’,wherein capital letters depict LNA nucleotides and lowercase letters depict DNA nucleotides, and at least one internucleoside bond is a phosphorothioate linkage.12) The compound for use according to claim 11, wherein capital C denotes LNA 5-methylcytosine.13) The compound for use according to claim 11 or 12, wherein LNA is beta-D-oxy LNA.14) The compound for use according to any of claims 11-13, wherein all internucleoside bondsare phosphorothioate bonds.15) The compound for use according to any of the preceding claims, wherein LNA is beta-D-oxy LNA and all internucleoside bonds are phosphorothioate bonds.16) The compound for use according to any one of the preceding claims, wherein theoligonucleotide is selected from anyone of the sequences: (SEQ ID NO 16) 5' AGaActTagCcaCTgTGA 3’, or (SEQ ID NO 20) 5' AGAacTTagCcACTgtGA 3’,P477937PC00 Wherein capital letters denote LNA, and lowercase letters denote DNA, and LNA is beta- D-oxy LNA, and all internucleoside bonds are phosphorothioate bonds.17) The compound for use according to claim 16, wherein capital C is LNA 5-methylcytosine.18) The compound according to claim 16, wherein the oligonucleotide is selected from anyoneof SEQ ID NO: 16 5’ AbsGbsdAsAbs5MdCsdTsTbsdAsdGsCbs5MdCsdAsCbsTbsdGsTbsGbsAb and SEQ ID NO: 20 5’ AbsGbsAbsdAs5MdCsTbsTbsdAsdGsCbs5MdCsAbsCbsTbsdGsdTsGbsAb wherein DNA nucleotides are (dN [dA, dC, dG, dI, dT]), LNA nucleotides are (Nb [Ab, Cb, Gb, Tb]), 5-methyl-deoxy-cytidines are (5MdC), and Phosphorothioate backbone is denoted (s).19) The compound for use according to anyone of claims 1-18, wherein the compound is aconjugate that comprises the antisense oligonucleotide of any one of claims 1-18.20) The compound for use according to claim 19, wherein the compound is a bioconjugate.21) The compound according to any of the preceding claims, wherein the use is for treatmentor alleviation of early-stage idiopathic Parkinson’s disease.22) The compound according to any of the preceding claims, wherein the treatment oralleviation is of one or more Parkinson’s disease.23) The compound according to any of the preceding claims, wherein the use is for treatment,reducing, preventing or alleviation of unintended weight loss associated with Parkinsonsdisease.24) The compound according to any of the preceding claims, wherein the use is for treatment,prevention, reduction or alleviation of gait disturbances associated with Parkinsonsdisease.25) The compound according to any of the preceding claims, wherein the use is for treatment,prevention, reduction or alleviation of motor symptoms associated with Parkinsons disease.P477937PC0026) The compound according to any of the preceding claims, wherein the use is fortreatment, rescuing or alleviation of learning and memory loss associated withParkinsons Disease.27) The compound according to any of the preceding claims, wherein the use is forpreventing neuronal death, or dopaminergic neuronal loss in Parkinsons Disease.28) The compound according to any of the preceding claims, wherein the antisenseoligonucleotide use is for inhibiting miR-27b expression in the posterior cortex, striatumor substantia nigra.29) The compound according to any of the preceding claims, wherein the use is forpreventing degeneration of the nigrostriatal system in association with Parkinsons Disease.30) The compound according to any of the preceding claims, wherein the use is forpreventing or alleviating CNS inflammation, such as microgliosis.31) The compound according to any of the preceding claims, wherein the use is for inductionof mitophagy, or oxidative stress alleviation in the brain in Parkinsons Disease.32) The compound according to any of the preceding claims, wherein the use is forpromoting synuclein degradation in the brain in Parkinsons Disease.33) The compound according to the preceding claim, wherein the use is for treating oralleviating synucleinopathies, such as alpha-synucleinopathies.

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